| 1. |
- Fotaki, Grammatiki, 1988-
(författare)
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Allogeneic dendritic cells as adjuvants in cancer immunotherapy
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In recent years, immunotherapeutic approaches have achieved remarkable successes through checkpoint blockade antibodies, advances in the use of chimeric antigen receptor (CAR) T cells and new insights into the immunosuppressive role of the tumor microenvironment (TME). Through the advances, the role of cancer vaccines based on ex vivo manipulated autologous dendritic cells (DC) has been challenged. The main aim of DC-based vaccination is the induction of tumor-specific T-cell responses through presentation of tumor-associated antigens. However, this process has been found to be highly dependent on the ability of the injected vaccine-DCs to activate endogenous bystander DCs.In this work, we examined the feasibility of having an allogeneic source of vaccine-DCs (alloDCs), not for direct antigen-presentation to T cells but as an immune primer aiming to activate bystander DCs. In paper I, we treated alloDCs with a T helper cell type 1 (Th1)-promoting maturation cocktail alone or combined with a replication-deficient, infection-enhanced adenoviral vector (Ad5M) as a potential gene delivery vehicle. We found that mature pro-inflammatory alloDCs, either non-transduced or transduced, created a cytokine- and chemokine-enriched milieu in vitro, and promoted the activation of co-cultured immune cells, including cytolytic NK cells, from unrelated donors. The emerged milieu induced the maturation of bystander DCs, which cross-presented antigens from their environment to autologous antigen-specific T cells. In paper II, we found that alloDCs promoted the migration of murine immune cells both to the site of injection and to the draining lymph node. When Ad5M was used for the delivery of the melanoma-associated antigen gp100, we found that gp100-expressing alloDCs were able to control tumor growth through gp100-specific T-cell responses and alteration of the TME. In paper III, we found that co-administration of alloDCs with an adenoviral vector encoding for HPV-antigens is effective in controlling the growth of HPV-related tumors and this may depend on a cross-talk between alloDCs and NK cells which leads to further recruitment of immune cells into the TME. In paper IV, we observed that concomitant targeting of immune checkpoint receptors or co-stimulatory molecules results in synergistic therapeutic effects in a murine colorectal model.
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| 2. |
- Ma, Jing
(författare)
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Cancer Immunotherapy : Oncolytic viruses and CAR-T cells
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Various forms of cancer immunotherapy have developed rapidly with improved survival and quality of life for cancer patients. Cancer immunotherapy aims to educate the patient’s immune system to eliminate cancer cells, including immune checkpoint inhibitors (ICIs), adoptive cell transfer (mostly T cells), oncolytic viruses (OVs) and cancer vaccines. Especially ICIs have induced durable responses in patients with many different types of cancers. Chimeric antigen receptor (CAR)-T cell therapy has shown good efficacy in treating hematologic malignancies. However, there is still a significant number of patients that do not benefit from these treatments due to immune evasion. Strategies to modify cancer immunotherapies with immunomodulating agent needs to be investigated to maximize the effect of immunotherapy. Helicobacter pylori Neutrophil Activating Protein (HP-NAP) could be used as an immunomodulating agent to recruit, activate and mature immune cells, such as dendritic cells (DCs), monocytes and neutrophils, and also induce T helper type 1 (Th1)-polarized response. In this thesis, we examined to arm oncolytic virus or CAR-T cells with HP-NAP.Papers I and II investigate oncolytic viruses. In paper I, we investigated wild-type Adenovirus (Ad), Semliki forest virus (SFV) and Vaccinia virus (VV), for their ability to mediate lysis of tumor cells, which was found to be associated with the release of danger-associated molecular patterns (DAMPs) and subsequently triggered phagocytosis and maturation of DCs. However, only SFV-infected tumor cells triggered significant Th1-cytokine release by DCs and induced antigen-specific T cell activation, while VV induced immunosuppressive responses. In Paper II, we armed VV and SFV with the tumor-associated antigen GD2 and HP-NAP. We found that arming these OVs with HP-NAP resulted in distinct anti-tumor immune response and therapeutic benefit. VV-GD2m-NAP showed significantly increased therapeutic efficacy compared to VV-GD2m, associated with elevated antiGD2 antibody production. In contrast, there was no additive antitumor effect for SFV-GD2m-NAP compared with SFV-GD2m. Due to intrinsic properties of OVs, engineering OVs with immunomodulating agents needs careful consideration. Engineering SFV or similar viruses, which is very immunogenic, should focus on improving oncolysis, de-bulking tumor and release of tumor-associated antigens, while for VV or similar viruses, with immunosuppressive properties, the focus can be on arming the virus with immune modulators to improve anti-tumor immune response. Papers III and IV investigate CAR-T cells. In paper III, CAR-T cells were engineered to inducible secrete HP-NAP upon antigen recognition (CAR(NAP)-Ts). CAR(NAP)-Ts successfully reduced tumor growth and prolonged survival of mice in several solid tumor models with epitope spreading and initiated endogenous anti-tumor immune responses. Secreted HP-NAP created an immunologically hot tumor microenvironment with enhanced infiltration of immune cells (DCs, neutrophils, macrophages, and cytotoxic natural killer cells). In paper IV, we developed CAR T cells targeting CD20 (rituCD20CAR T cells). We found that rituCD20CAR T cells could efficiently kill CD20-positive lymphoma cell lines (U2932, Karpas422, DB, U698, Raji, Daudi) as well as primary mantle cell CD20-positive lymphoma (CD20+ MCL) cells accompanying with IFNγ secretion. Both rituCD20CAR and NAP-armed rituCD20CAR(NAP) T-cell treatment delayed tumor growth and prolonged mice survival in the murine lymphoma A20-hCD20 model. In summary, combing OVs and CAR-T cells with the immunomodulating agent HP-NAP is a promising way of maximizing the benefit of immunotherapy to combat cancers.
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| 3. |
- Forsberg, Ole, 1979-
(författare)
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Generation of Therapeutic T Cells for Prostate Cancer
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented herein focuses on the activation of the adaptive immune system in order to develop T cell-based immunotherapy for viral infections and cancer. The main goal was to identify and activate viral or tumoral antigen-specific T cells by using different identification, isolation and stimulation techniques. One such approach was that we modified dendritic cells (DCs) with an adenoviral vector encoding the full length pp65 antigen from cytomegalovirus (CMV). Through strategic modification techniques we demonstrate that it is possible to obtain DCs presenting antigen-specific peptides both on major histocompatibility complex (MHC) class I and MHC class II molecules for simultaneous CD8+ and CD4+ T cell activation. We also demonstrate that it is possible to generate prostate antigen-specific CD8+ T cells from a naïve repertoire of T cells by using DCs and HLA-A2-restricted peptides derived from prostate tumor-associated antigens or by using an adenoviral vector encoding the full length prostate tumor-associated antigen STEAP. We further demonstrate that CD8+ T cells directed against several prostate-specific peptide epitopes can be found in peripheral blood and in the prostate tumor area of prostate cancer patients. Furthermore, we have characterized a number of prostate-derived cell lines in terms of HLA haplotype and tumor-association antigen expression. We concluded that our methods for generating T cells restricted to CMV antigen have the ability to be applied for adoptive T cell transfer to patients with CMV disease and that prostate antigen-specific T cells can be found within prostate cancer patients, which enables future development of immunotherapeutic strategies for prostate cancer.
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| 4. |
- Anagandula, Mahesh
(författare)
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Studies of Enterovirus Infection and Induction of Innate Immunity in Human Pancreatic Cells
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Several epidemiological and clinical studies have indicated a possible role of Enterovirus (EV) infection in type 1 diabetes (T1D) development. However, the exact casual mechanism of these viruses in T1D development is not known. The aim of this thesis is to study various EVs that have been shown to differ in their immune phenotype, lytic ability, association with induction of islet autoantibodies, ability to replicate, cause islet disintegration and induce innate antiviral pathways in infected pancreatic cells in vitro. Furthermore, EV presence and pathogenic process in pancreatic tissue and isolated islets of T1D patients was also studied.Studies in this thesis for first time show the detection of EV RNA and protein in recent onset live T1D patients supporting the EV hypothesis in T1D development. Further all EV serotypes studied were able to replicate in islets, causing variable amount of islet disintegration ranging from extensive islet disintegration to not affecting islet morphology at all. However, one of the EV serotype replicated in only two out of seven donors infected, highlighting the importance of individual variation between donors. Further, this serotype impaired the insulin response to glucose stimulation without causing any visible islet disintegration, suggesting that this serotype might impaired the insulin response by inducing a functional block. Infection of human islets with the EV serotypes that are differentially associated with the development of islet autoantibodies showed the islet cell disintegration that is comparable with their degree of islet autoantibody seroconversion. Suggesting that the extent of the epidemic-associated islet autoantibody induction may depend on the ability of the viral serotypes to damage islet cells. Furthermore, one of the EV strains showed unique ability to infect and replicate both in endo and exocrine cells of the pancreas. EV replication in both endo and exocrine cells affected the genes involved in innate and antiviral pathways and induction of certain genes with important antiviral activity significantly varied between different donors. Suggesting that the same EV infection could result in different outcome in different individuals. Finally, we compared the results obtained by lytic and non lytic EV strains in vitro with the findings reported in fulminant and slowly progressing autoimmune T1D and found some similarities. In conclusion the results presented in this thesis further support the role of EV in T1D development and provide more insights regarding viral and host variation. This will improve our understanding of the possible causative mechanism by EV in T1D development.
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| 5. |
- Ramachandran, Mohanraj
(författare)
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Cancer Immunotherapy : Evolving Oncolytic viruses and CAR T-cells
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the last decade cancer immunotherapy has taken huge strides forward from bench to bedside and being approved as drugs. Cancer immunotherapy harnesses the power of patient’s own immune system to fight cancer. Approaches are diverse and include antibodies, therapeutic vaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses and immune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responses have been observed for certain cancers with checkpoint antibodies and chimeric antigen receptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to be combined for broader effective treatment responses because cancers evolve to escape immune recognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24]) was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2 agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expression of HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatory cytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activate dendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically matured and activated DCs to secrete the T-helper type-1 (Th-1) polarizing cytokine IL-12. HP-NAP-matured DCs were functional; able to migrate to draining lymph nodes and prime antigen-specific T-cell proliferation. CAR T-cells were engineered to secrete HP-NAP upon T-cell activation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus with natural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA target sequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthy CNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while it retained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneic neuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated with type-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combining immunotherapeutic strategies with HP-NAP is a promising approach to combat cancers and SFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.
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| 6. |
- Sarén, Tina, 1991-
(författare)
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CAR T cells for Immunotherapy of Cancer
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In recent years, immunotherapy has revolutionized cancer treatment by prolonging survival and even curing patients lacking other available therapies. Besides immune checkpoint inhibitors, one of the major advances is attributed to the success of chimeric antigen receptor (CAR)-T cell therapy in treating patients with B-cell malignancies. Although many patients respond, some are resistant to treatment and others will relapse after an initial response.CAR-T cell efficacy can be enhanced by adding a transgene cassette encoding an immune-stimulatory molecule that induce bystander immunity and a broader anti-cancer response. This is particularly important to improve efficacy in solid tumors where the “CAR-target-antigen” is often heterogeneously expressed. The neutrophil-activating protein (NAP), a virulence factor of Helicobacter Pylori, is an efficient immune stimulator. When secreted from CAR-T cells NAP can induce bystander immunity with killing of “CAR-target-negative” tumor cells.In my thesis, I aim at investigating resistance mechanisms to CAR-T therapy and developing new improved CAR-T therapies.In paper I, clinical response in patients with B-cell lymphoma was evaluated after treatment with two doses of third-generation CD19CAR-T cells. Response was observed in 9 out of 24 patients (37.5%) irrespectively of receiving one or two doses. We also analyzed whether the profile of the individual CAR-T infusion products could predict response. CAR-Ts from responders had high cytotoxic and low dysfunctional profile while CAR-Ts from non-responders had high dysfunction profile. Extended culture time during manufacturing was associated with dysfunction. As CAR-T products of non-responders were on average cultured longer, this may explain their dysfunctionality. In paper II, we developed CD20-targeting CAR-T cells (CAR20-T), which efficiently killed CD20-expressing human lymphoma cell lines and patient-derived lymphoma cells in vitro. By arming CAR20-T cells with NAP we could delay tumor growth in lymphoma-bearing mice compared to conventional, unarmed CAR-T cells.In paper III, we engineered five IL13Rα2-targeting CAR-T cell constructs for glioblastoma therapy. The candidates only differed in the complementary determining regions (CDRs) of the single-chain variable fragment portion of the CAR. We found that CDR-mediated CAR clustering could lead to antigen-independent tonic signaling and subsequent CAR-T cell dysfunction. We also identified one candidate which did not display any significant tonic signaling and that possessed therapeutic effect on mice with orthotopic glioblastoma. IL13Rα2 is heterogeneously expressed in glioblastoma tumors so in paper IV, we armed the best CAR-T cell construct with NAP and found that NAP-armed IL13Rα2 CAR-T cells could prolong survival of glioblastoma-bearing mice.
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| 7. |
- Yu, Di, 1985-
(författare)
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Adenovirus for Cancer Therapy : With a Focus on its Surface Modification
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. We engineered Ad5 virus with the protein transduction domain (PTD) from the HIV-1 Tat protein (Tat-PTD) inserted in the hypervariable region 5 (HVR5) of the hexon protein in the virus capsid. Tat-PTD-modified Ad5 shows a dramatically increased transduction level of CAR-negative cells and bypassed fiber-mediated transduction. It also overcomes the fiber-masking problem, which is caused by release of excess fiber proteins from infected cells. To achieve specific viral replication in neuroblastoma and neuroendocrine tumor cells, we identified the secretogranin III (SCG3) promoter and constructed an adenovirus Ad5PTD(ASH1-SCG3-E1A) wherein E1A gene expression is controlled by the SCG3 promoter and the achaete-scute complex homolog 1 (ASH1) enhancer. This virus shows selective and efficient killing of neuroblastoma cell lines in vitro, and delays human neuroblastoma xenograft tumor growth on nude mice. To further enhance the viral oncolytic efficacy, we also switched the fiber 5 to fiber 35 to generate Ad5PTDf35. This vector shows dramatically increased transduction capacity of primary human cell cultures including hematopoietic cells and their derivatives, pancreatic islets and exocrine cells, mesenchymal stem cells and primary tumor cells including primary cancer initiating cells. Ad5PTDf35-based adenovirus could be a useful platform for gene delivery and oncolytic virus development. Viral oncolysis alone cannot completely eradicate tumors. Therefore, we further armed the Ad5PTDf35-D24 virus with a secreted form of Helicobacter pylori Neutrophil Activating Protein (HP-NAP). Expression of HP-NAP recruits neutrophils to the site of infection, activates an innate immune response against tumor cells and provokes a Th1-type adaptive immune response. Established tumor on nude mice could be completely eradicated in some cases after treatment with this virus and the survival of mice was significantly prolonged.
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| 8. |
- Carlsson, Björn, 1975-
(författare)
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Adoptive T Cell Therapy of Viral Infection and Cancer : Ex vivo Expansion of Cytomegalovirus- and Prostate Antigen-specific T Cells
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The main focus of my thesis has been to develop protocols for generating antigen-specific cytotoxic T lymphocytes (CTLs) and T helper cells (TH) for adoptive transfer to treat cytomegalovirus (CMV) disease and prostate cancer. CMV viremia is a severe complication in immunocompromised stem cell transplanted patients. Prostate cancer is a leading cause of death for men in Western countries. Although different in nature, CMV-infected cells and prostate cancer cells can both be eliminated through specific activation of the adaptive immune system. To generate CMV pp65-specific T cells, I utilized dendritic cells (DCs) modified with an HLA-A*0201/pp65495-503 peptide, a recombinant adenovirus coding for pp65, in vitro transcribed pp65 mRNA and a recombinant pp65 protein. Peptide stimulation yielded large numbers of peptide-specific CD8+ T cells with high lytic activity while adenovirus or mRNA stimulation resulted in the expansion of CTLs against multiple pp65 epitopes. The recombinant protein activated primarily CD4+ TH cells. Stimulation with DCs co-modified with pp65 mRNA and pp65 protein simultaneously generated both pp65-specific CTLs and TH cells. Such T cells would cover all pp65 epitopes while avoiding potential virus related biohazards. The mRNA/protein combinatory approach can be used to stimulate T cells ex vivo from virtually all stem cell donors for adoptive T cell transfer. I have identified two immunogenic HLA-A*0201-restricted peptide epitopes from the prostate tissue antigen TARP. Repeated stimulations with TARP peptide-pulsed DCs yielded up to 20% TARP-directed CD8+ T cells even when starting from undetectable frequencies (<0.01%). The T cells could be sorted to 99% purity and expanded 1000-fold with retained specificity and activity. We also detected TARP-directed CD8+ T cells in the blood of prostate cancer patients. Therefore, TARP seems to have potential as antigen in DC vaccination or adoptive T cell therapy of prostate cancer.
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| 9. |
- Dzojic, Helena, 1978-
(författare)
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Adenovirus-mediated CD40 Ligand Immunotherapy of Prostate and Bladder Cancer
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses.We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy.In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.
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| 10. |
- Jin, Chuan, 1986-
(författare)
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Improvement of adoptive T-cell therapy for Cancer
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer immunotherapy has recently made remarkable clinical progress. Adoptive transfer of T-cells engineered with a chimeric antigen receptor (CAR) against CD19 has been successful in treatment of B-cell leukemia. Patient’s T-cells are isolated, activated, transduced with a vector encoding the CAR molecule and then expanded before being transferred back to the patient. However some obstacles restrict its success in solid tumors. This thesis explores different aspects to improve CAR T-cells therapy of cancer.Ex vivo expanded T-cells are usually sensitive to the harsh tumor microenvironment after reinfusion. We developed a novel expansion method for T-cells, named AEP, by using irradiated and preactivated allo-sensitized allogeneic lymphocytes (ASALs) and allogeneic mature dendritic cells (DCs). AEP-expanded T-cells exhibited better survival and cytotoxic efficacy under oxidative and immunosuppressive stress, compared to T-cells expanded with established procedures.Integrating retro/lentivirus (RV/LV) used for CAR expressions randomly integrate in the T-cell genome and has the potential risk of causing insertional mutagenesis. We developed a non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element (NILV-S/MAR) for T-cells transduction. NILV-S/MAR-engineered CAR T-cells display similar cytotoxicity to LV-engineered CAR T-cells with undetectable level of insertional event, which makes them safer than CAR T-cells used in the clinic today.CD19-CAR T-cells have so far been successful for B-cell leukemia but less successful for B-cell lymphomas, which present semi-solid structure with an immunosuppressive microenvironment. We have developed CAR T-cells armed with H. pylorineutrophil-activating protein (HP-NAP). HP-NAP is a major virulence factor and plays important role in T-helper type 1 (Th1) polarizing. NAP-CAR T-cells showed the ability to mature DCs, attract innate immune cells and increase secretion of Th1 cytokines and chemokines, which presumably leads to better CAR T-cell therapy for B-cell lymphoma.Allogeneic-DCs (alloDCs) were used to further alter tumor microenvironment. The premise relies on initiation of an allo-reactive immune response for cytokine and chemokines secretion, as well as stimulation of T-cell response by bringing in tumor-associated antigen. We demonstrated that alloDCs promote migration and activation of immune cells and prolong the survival of tumor-bearing mice by attracting T-cells to tumors and reverse the immune suppressive tumor microenvironment.
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