Effective Adoptive Immunotherapy Research Articles

Modulating the proliferative and cytotoxic properties of patient-derived TIL by a synthetic immune niche of immobilized CCL21 and ICAM1.

A major challenge in developing an effective adoptive cancer immunotherapy is the ex-vivo generation of tumor-reactive cells in sufficient numbers and with enhanced cytotoxic potential. It was recently demonstrated that culturing of activated murine CD8+ T-cells on a "Synthetic Immune Niche" (SIN), consisting of immobilized CCL21 and ICAM-1, enhances T-cell expansion, increases their cytotoxicity against cultured cancer cells and suppresses tumor growth in vivo. In the study reported here, we have tested the effect of the CCL21+ICAM1 SIN, on the expansion and cytotoxic phenotype of Tumor Infiltrating Lymphocytes (TIL) from melanoma patients, following activation with immobilized anti-CD3/CD28 stimulation, or commercial activation beads. The majority of TIL tested, displayed higher expansion when cultured on the coated SIN compared to cells incubated on uncoated substrate and a lower frequency of TIM-3+CD8+ cells after stimulation with anti-CD3/CD28 beads. Comparable enhancement of TIL proliferation was obtained by the CCL21+ICAM1 SIN, in a clinical setting that included a 14-day rapid expansion procedure (REP). Co-incubation of post-REP TIL with matching target cancerous cells demonstrated increased IFNγ secretion beyond baseline in most of the TIL cultures, as well as a significant increase in granzyme B levels following activation on SIN. The SIN did not significantly alter the relative frequency of CD8/CD4 populations, as well as the expression of CD28, CD25, several exhaustion markers and the differentiation status of the expanded cells. These results demonstrate the potential capacity of the CCL21+ICAM1 SIN to reinforce TIL-based immunotherapy for cancer patients.

Homeostatic cytokines tune naivety and stemness of cord blood-derived transgenic T cells.

Engineered T-cell therapies have proven to be successful in cancer and their clinical effectiveness is directly correlated with the infused T-cell differentiation profile. Indeed, stem cell memory and central memory T cells proliferate and persist longer in vivo compared with more-differentiated T cells, while conferring enhanced antitumor activity. Here, we propose an optimized process using cord blood (CB) to generate minimally differentiated T-cell products in terms of phenotype, function, gene expression, and metabolism, using peripheral blood (PB)-derived T cells cultured with IL-2 as a standard. Phenotypically, CB-derived T cells, particularly CD4 T cells, are less differentiated than their PB counterparts when cultured with IL-2 or with IL-7 and IL-15. Furthermore, culture with IL-7 and IL-15 enables better preservation of less-differentiated CB-derived T cells compared with IL-2. In addition, transcriptomic and metabolic assessments of CB-derived transgenic T cells cultured with IL-7 and IL-15 point out their naivety and stemness signature. These relatively quiescent transgenic T cells are nevertheless primed for secondary stimulation and cytokine production. In conclusion, our study indicates that CB may be used as a source of early differentiated T cells to develop more effective adoptive cancer immunotherapy.

In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL.

Combining synthetic biology with adoptive T-cell transfer has led to promising advances in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). Chimeric antigen receptors (CARs) are synthetic receptors that redirect T-cell specificity against cancer. CARs include "built-in" signaling domains that reprogram T-cell metabolism, enhance effector function, and support long-term persistence. Despite their success in blood-based malignancies, relapse can occur in CD19-redirected CAR T-cell therapies for several reasons, including poor engraftment, impaired in vivo proliferation, and T-cell senescence. Herein, we explain how subtle alterations in CAR design may overcome barriers to effective adoptive immunotherapy. We also discuss how the physiochemical properties of the single-chain variable fragment (scFv) affect differentiation and persistence. Moreover, we describe innovative advances in CAR engineering and provide insight into the development of humanized scFvs whose proposed benefits include increased persistence and improved clinical outcomes. Tumor cells can evade CAR T-cell-mediated detection and elimination due to the emergence or presence of CD19-negative leukemic cell subpopulations. We also discuss the opportunities and challenges in targeting other B-ALL-associated antigens. Identifying alternate targets is fundamentally necessary to restore the success of CAR T-cell therapies in CD19-negative patients with B-ALL.

Abstract 2315: Alteration of tumor metabolism by CD4+ T cells leads to TNF-α-dependent intensification of oxidative stress and tumor cell death

Abstract The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal tumors. By conducting metabolic profiling on tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathionedepletion and accumulation of reactive oxygen species (ROS) in tumor cells. We further demonstrate that T cell-derived tumor necrosis factor alpha (TNF-α) can synergize with chemotherapy to intensify oxidative stress and tumor cell death in an NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase-dependent manner. Reduction of oxidative stress, by preventing TNF-α-signaling in tumor cells or scavenging ROS, antagonized the therapeutic effects of adoptive immunotherapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effects of T cell transfer. These findings imply that reinforcing tumor oxidative stress represents an important mechanism underlying the efficacy of adoptive immunotherapy. Citation Format: Zhichun Ding, Tsadik Habtetsion, Gang Zhou. Alteration of tumor metabolism by CD4+ T cells leads to TNF-α-dependent intensification of oxidative stress and tumor cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2315.

Partial CD25 Antagonism Enables Dominance of Antigen-Inducible CD25high FOXP3+ Regulatory T Cells As a Basis for a Regulatory T Cell-Based Adoptive Immunotherapy.

FOXP3+ regulatory T cells (Tregs) represent a promising platform for effective adoptive immunotherapy of chronic inflammatory disease, including autoimmune diseases such as multiple sclerosis. Successful Treg immunotherapy however requires new technologies to enable long-term expansion of stable, antigen-specific FOXP3+ Tregs in cell culture. Antigen-specific activation of naïve T cells in the presence of TGF-β elicits the initial differentiation of the FOXP3+ lineage, but these Treg lines lack phenotypic stability and rapidly transition to a conventional T cell (Tcon) phenotype during in vitro propagation. Because Tregs and Tcons differentially express CD25, we hypothesized that anti-CD25 monoclonal antibodies (mAbs) would only partially block IL-2 signaling in CD25high FOXP3+ Tregs while completely blocking IL-2 responses of CD25low-intermediate Tcons to enable preferential outgrowth of Tregs during in vitro propagation. Indeed, murine TGF-β-induced MOG-specific Treg lines from 2D2 transgenic mice that were maintained in IL-2 with the anti-CD25 PC61 mAb rapidly acquired and indefinitely maintained a FOXP3high phenotype during long-term in vitro propagation (>90% FOXP3+ Tregs), whereas parallel cultures lacking PC61 rapidly lost FOXP3. These results pertained to TGF-β-inducible “iTregs” because Tregs from 2D2-FIG Rag1−/− mice, which lack thymic or natural Tregs, were stabilized by continuous culture in IL-2 and PC61. MOG-specific and polyclonal Tregs upregulated the Treg-associated markers Neuropilin-1 (NRP1) and Helios (IKZF2). Just as PC61 stabilized FOXP3+ Tregs during expansion in IL-2, TGF-β fully stabilized FOXP3+ Tregs during cellular activation in the presence of dendritic cells and antigen/mitogen. Adoptive transfer of blastogenic CD25high FOXP3+ Tregs from MOG35-55-specific 2D2 TCR transgenic mice suppressed experimental autoimmune encephalomyelitis in pretreatment and therapeutic protocols. In conclusion, low IL-2 concentrations coupled with high PC61 concentrations constrained IL-2 signaling to a low-intensity range that enabled dominant stable outgrowth of suppressive CD25high FOXP3+ Tregs. The ability to indefinitely expand stable Treg lines will provide insight into FOXP3+ Treg physiology and will be foundational for Treg-based immunotherapy.

Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens.

Effective adoptive immunotherapy has proved elusive for many types of human cancer, often due to difficulties achieving robust expansion of natural tumor-specific T-cells from peripheral blood. We hypothesized that antigen-driven T-cell expansion might best be triggered in vitro by acute activation of innate immunity to mimic a life-threatening infection. Unfractionated peripheral blood mononuclear cells (PBMC) were subjected to a two-step culture, first synchronizing their exposure to exogenous antigens with aggressive surrogate activation of innate immunity, followed by ?-chain cytokine-modulated T-cell hyperexpansion. Step 1 exposure to GM-CSF plus paired Toll-like receptor agonists (resiquimod and LPS), stimulated abundant IL-12 and IL-23 secretion, as well as upregulated co-stimulatory molecules and CD11c expression within the myeloid (CD33+) subpopulation. Added synthetic long peptides (>20aa) derived from widely expressed oncoproteins (MUC1, HER2/neu and CMVpp65), were reliably presented to CD4+ T-cells and cross-presented to CD8+ T-cells. Both presentation and cross-presentation demonstrated proteasomal and Sec61 dependence that could bypass the endoplasmic reticulum. Step 2 exposure to exogenous IL-7 or IL-7+IL-2 produced selective and sustained expansion of both CD4+ and CD8+ peptide-specific T-cells with a predominant interferon-?-producing T1-type, as well as the antigen-specific ability to lyse tumor targets. Other ?-chain cytokines and/or combinations were initially proliferogenic, but followed by a contractile phase not observed with IL-7 or IL-7+IL-2. Regulatory T-cells were minimally propagated under these culture conditions. This mechanistically rational culture sequence, effective even for unvaccinated donors, enables rapid preparation of T-cells recognizing tumor-associated antigens expressed by the majority of human cancers, including pancreatic cancers, breast cancers and glioblastomas.

Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level.

BackgroundThe poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC) have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα) may represent an ideal tumor-associated marker for immunotherapy for TNBC.MethodsThe aim of the present study was to apply a chimeric antigen receptor (CAR) approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo.ResultsWe found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα.ConclusionsTaken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-016-0285-y) contains supplementary material, which is available to authorized users.

396. Development of GD2-Specific Immunoliposomes for Immunotherapy of Neuroblastoma

Tumor microenvironment (TME) impairs immune effector cell localization, persistence, or the execution of their anti-tumor function. This represents a major and as yet unsolved critical challenge to the development of effective adoptive immunotherapy of solid tumors. To selectively target TME in neuroblastoma and make it permissible for survival and function of tumor-specific effector cells, we have developed a novel nanoparticle (NP) delivery platform which consists of 150 nm immunoliposomes rendered specific for neuroblastoma cells using a single-chain variable fragment (scFv) from the anti-GD2 14g2a mAb. GD2-specific but not control NPs could specifically bind GD2-positive but not GD2-negative neuroblastoma cells in vitro as determined by FACS. To examine the in vivo biodistribution of NPs, DiR-labeled GD2-specific or non-specific NPs were injected to NSG mice implanted with human neuroblastoma xenografts. Tumor tissues and normal organs were analyzed after 72 hours using ex vivo fluorescence imaging. Up to 58% of GD2-specific NPs accumulated at the tumor sites. NPs loaded with human IL-15 effectively delivered cytokine to the tumor site. A combined immunotherapy using human NKT cells expressing a GD2-specific chimeric antigen receptor (CAR) and IL-15-containing NPs resulted in potent in vivo expansion of CAR NKT cells and tumor eradication. In contrast, soluble IL-15 failed to support long-term CAR NKT cell persistence that resulted in tumor recurrence. These results inform design of cancer immunotherapy in combination with TME-modifying NPs.

Identification of Melanoma-reactive CD4+ T-Cell Subsets From Human Melanoma Draining Lymph Nodes.

Our laboratory has previously demonstrated that melanoma draining lymph node (MDLN) samples from stage III patients contained both CD4 and CD8 T cells that can be readily expanded to mediate tumor cell apoptosis in vitro and improve survival in mice bearing human melanoma xenografts. In this study, we investigated whether MDLN T cells contain melanoma-reactive CD4 T-cell compartment and what they are. To test this, we performed multiparametric (11-color and 6-color) fluorescence-activated cell sorting analyses to monitor phenotypic and functional property of CD4 T cells in response to melanoma cell antigen reexposure. Our results have demonstrated that the antigen reexposure could result in a generation of CD4CCR7CD62LCD27 T-cell subsets with various effector cell-like properties. Within the CD4CCR7CD62LCD27 T-cell compartment, in response to antigen reexposure, some of the cells expressed significantly upregulated CD40L and/or CXCR5, and some of them expressed significantly upregulated interleukin-2 and/or tumor necrosis factor-α. This may suggest the existence of melanoma-reactive CD4 "effector-precursor" cells within the expanded MDLN cells and their differentiation into various effector lineages in response to antigen restimulation. Recent clinical trials have demonstrated that effective adoptive cellular immunotherapy maybe enhanced by antigen-specific CD4 T cells. Therefore, results of this study may significantly benefit innovative design of +adoptive cellular immunotherapy that can potentially mediate enhanced and durable clinical responses.

Immunoenhancing properties of the anti-tumor effects of adoptively transferred T cells with chemotherapeutic cyclophosphamide by co-administration of bone marrow cells

In this study we aimed to determine the anti-tumor efficacy of co-treatment of adoptively transferred T cells with bone marrow either harvested from naïve mice or G-CSF activated after treatment with the anti-cancer drug cyclophosphamide (CTX) as a source enriched in stem cells. CTX-treated Swiss Albino (CD-1) mice were injected with 2×105 Ehrlich ascetic carcinoma (EAC) cell line and then adoptively transferred with in vitro co-activated T cells with or without bone marrow one day post CTX treatment. All mice were vaccinated with tumor lysate and Hiltonol®. The results showed that co-transfer of activated T cells with bone marrow provided the highest antitumor effect and induced marked increase in numbers of splenocytes, leucocytes and bone marrow cells. Interestingly, T cells derived from EAC tumor-bearing host induced higher effects than those from normal mice. In sum, our data suggest that combination of CTX and activated transferred T cells with bone marrow induces proliferation and expansion of immune cells, which are functional and can be exploited in vivo to foster more effective antitumor adoptive immunotherapy strategies.

412. Development of GD2-Specific Immunoliposomes for Immunotherapy of Neuroblastoma

Tumor growth creates a highly immunosuppressive tumor microenvironment (TME) that impairs T-cell localization, persistence, or the execution of their effector function. This represents a major and as yet unsolved critical challenge to the development of effective adoptive immunotherapy of solid tumors. To selectively target TME in neuroblastoma and make it permissible for survival and function of tumor-specific T cells, we have developed a novel nanoparticle (NP) delivery platform which consists of 150 nm immunoliposomes rendered specific for neuroblastoma cells using the Fab fragment obtained from the anti-GD2 mAb clone 14g2a. To ensure high density surface coverage and correct orientation of anti-GD2 Fab on the NPs, we synthesized a fusion protein consisting of 14g2a Fab and folate receptor (Fab14g2a-FR) and attached it on the outer layer of the immunoliposomes enriched with folic acid. Rhodamine-labeled GD2-specific but not control NPs could specifically bind GD2-positive CHLA-255 neuroblastoma cells but not GD2-negative LA-N-6 neuroblastoma cells in vitro as determined by FACS. To examine the in vivo biodistribution of NPs, DiR-labeled GD2-specific or non-specific NPs were injected to NOD/SCID mice implanted with CHLA-255 cells. Tumor tissues and normal organs were imaged after 72 hours using ex vivo fluorescence imaging. Up to 58% of GD2-specific NPs accumulated at the tumor sites. The only other organ with significant accumulation of NPs was the liver. Minor traceable portions were detected in the spleen and lung. To utilize the observed targeting capabilities of GD2-specific NPs to achieve antitumor effects, we are now loading NPs with recombinant human (rhIL-7) and will test whether the preferential delivery of rhIL-7 to the tumor site and liver, a major site of NB metastasis, will increase the survival and anti-tumor activity of T and NKT cells engineered to express a GD2-specific chimeric antigen receptor with IL-7Rα. The results of this study will inform design of immunotherapy of neuroblastoma and other tumors in combination with TME-modifying NPs.

Effect of adoptive immunotherapy with CIK cells combined with interleukin-2 on immunologic function and life qualities of patients with non-small cell lung cancer

Objective To investigate the changes of immunologic functions and life qualities after the auto-cytokine induced killer (CIK) cells combined with interleukin-2(IL-2) being transfused back to the patients with non-small cell lung cancer (NSCLC). Methods Thirty-eight NSCLC patients were enrolled, and 19 patients received not only chemotherapy and radiotherapy but also adoptive immunotherapy with CIK cells combined with IL-2 compared with the other 19 patients who only accepted radiotherapy and chemotherapy. The changes of immunologic functions and life qualities after the CIK cells combined with IL-2 being transfused back to the patients with NSCLC were observed. Immune targets of the treatment group and control group, such as peripheral blood CD3+, CD4+, CD8+ T cell percentage, ratio of CD4+ /CD8+ and Th1/Th2, were observed at the end and after 3 month of the radiation and chemotherapy. Results Both of the two groups were neither having obvious adverse reactions. After CIK cells combined with IL-2 treatment, the life qualities of patients improved, such as 18 cases of mental improvement, 15 cases of appetite improvement, 12 cases of sleep improvement, and 3 cases of fatigue symptoms improvement. The percentage of CD3+, CD4+ cells in the treatment group increased [(66.39±9.22)% vs (42.98±7.23)%, t=5.45, P=0.00; (32.27±3.75)% vs (26.38±2.51)%, t=5.73, P=0.00], the percentage of CD8+ cells declined [(17.51±1.85)% vs (20.90±2.31)%, t=5.21, P=0.00], the rate of CD4+ /CD8+ increased and the rate of Th1/Th2 obversed [(1.86±0.32) vs (1.27±0.19), t=7.13, P=0.00; (1.15±0.48) vs (0.91±0.30), t=2.42, P=0.02]. There were significant differences between the two groups. Conclusion Adoptive immunotherapy with CIK cells combined with IL-2 is safe in clinical, which can improve the immunologic functions and life qualities of the patients with NSCLC. Key words: Carcinoma, non-small-cell lung; Immunotherapy; Cytokine induced killer cells

Upregulation of Immune Checkpoint Blockade Molecules Post Allogeneic Stem Cell Transplantation Is Necessary but Insufficient to Prevent GvHD

Donor T-lymphocytes are effective adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), but life threatening complications related to GVHD limits its clinical application. Recent advancement in the field of immunotherapy has directed our interest to enhancing the anti-tumor response of donor T cells by modulating expression of checkpoint blockade molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and foxp3, the transcription factor associated with regulatory T cells. The two ligands of PD-1, PD-L1 or PD-L2 are highly expressed in the presence of inflammatory signal induced by infection or cancer and PD-1/PD-L1 interaction negatively regulates T-cell antigen receptor (TCR) signaling and dampen T cell cytotoxic activity. Herein, we studied the role of PD-1, CTLA-4 and transcription factor foxp3 expressing donor CD4+ and CD8+ T cells in the development of GVHD.Methods: We have used two established allo-HSCT murine GvHD models. Lethally irradiated wild type (WT) B6, PD-L1 knock out (KO) B6 and PD-L2 KO B6 mice were transplanted with 2 x 106 splenic T cells and 2 x 106 T cell depleted bone marrow (TCD BM) cells from H-2Kdonors. Lethally irradiated CB6F1 recipients were similarly transplanted with splenocytes and TCD BM cells from B6 donors. Acute GvHD scores were determined by combining scores obtained from histological tissue sections and weight-loss, posture, activity, fur texture and skin integrity following standard published procedures. The activation status of donor T-cells and BM and host-derived non-T cells in GvHD target organs was analyzed by flow cytometry. Data from allo-HSCT recipients were compared with the respective data obtained from B6 à B6 syngenic HSCT (syn-HSCT) recipients. Serum cytokines were determined by Luminex assay.Results: PD-L1 KO B6 allo-HSCT recipients had significantly increased acute GvHD scores compared with WT B6 allo-HSCT recipients (p<0.0005) and B6 PD-L2 KO allo-HSCT recipients (p<0.0005) measured on day 8 after transplant. All PD-L1 KO allo-HSCT recipients died within 10 days post transplant while WT B6 and PD-L2 KO allo-HSCT recipients had 20% mortality until 36 days post transplant. Increased acute GvHD was associated with increased amount of serum inflammatory cytokines and increased numbers of activated PD-1+CD69+CD4+ donor T cells. Interestingly, PD-1 expression on donor CD4+ T cells significantly increased in the spleen of transplant recipients but not in BM, while PD-1 expression was significantly increased on donor CD8+ T cells in both spleen and BM compartments of allo-HSCT recipients compared with the syn-HSCT recipients. CTLA-4 expression on CD4+ and CD8+ donor T cells were significantly increased in spleen in the first two weeks post transplant but decreased at later time points compared with syn-HSCT. Again, CTLA-4 expression on CD4+ donor T cells in the BM remained significantly higher measured on 100+ days post transplant in allo-HSCT recipients compared with the syn-HSCT but similar levels of CTLA-4 expression on CD8+ T cells were measured in BM between these two HSCT recipients. Foxp3 expression on donor T cells and the numbers of CD4+CD25+foxp3+ regulatory T (Tregs) were markedly suppressed in donor T cells on day 4 post HSCT of allo-HSCT recipients compared with the syn-HSCT recipients. Although total numbers of donor T cells in the spleen of allo-HSCT recipients remained low over time, the percentage of PD-L1-expressing donor T cells in spleen were significantly higher (p<0.005) at early time points (day 4) in allo-HSCT recipients compared with the syn-HSCT. While total numbers of host-derived cells in spleen decreased over time in mice that developed GvHD, host-derived PD-L1 expressing CD3+ T cells persisted at higher levels through day 36 post transplant. Additionally, PD-L1 expression was also increased in donor BM-derived T cells and non-T cells populations over time.Collectively, these data indicate that severe GvHD occurs in allo-HSCT recipients in spite of increased numbers of PD-1, CTLA-4 and PD-L1 expressing donor and host cells. The occurrence of severe GvHD in these allo-HSCT models systems was associated with markedly reduced levels of CTLA-4 and foxp3 transcription factor expressing Tregs indicating that these pathways may be more relevant to controlling GvHD than PD-1:PD-L1 expression. DisclosuresNo relevant conflicts of interest to declare.

S78. Proffered paper: High-affinity CD20-specific TCRs suitable for adoptive immunotherapy can be readily isolated from the allo-repertoire using reverse immunology

Meeting abstracts Studies using T-cell receptor (TCR) or chimeric antigen receptor (CAR) transduced T-cells have shown the effectiveness of adoptive immunotherapy to treat different malignancies. The efficacy and safety of such interventions greatly depends on good target selection to prevent on-

Anti-tumor effects of adoptive transfer inactivated MHC-haploidentical tumor specific lymphocytes in mouse model (P2082)

Abstract In this study, we evaluated effect of tumor-antigen-primed MHC-haploidentical lymphocytes in a TC-1 (H-2b) murine lung cancer model. The haploidentical effector cells prepared from splenocytes stimulated with the tumor cell lysates pulsed-DCs from F1 (C57BL/6 × Balb/C, H-2bd) mice, or immunized from F1 mice vaccinated with inactivated tumor cells. Haploidentical lymphocytes immunized with TC-1 could specifically kill the TC-1 cells, as well as up-regulate the NK activity. The effector cells inactivated with Mitomycin C in vitro kept the killing capacity, and migrated to tumor tissue. Compared with the cells from non-inactivated, or the syngeneic mice, the cells from Mitomycin C inactivated mice with haploidentical showed enhanced tumor inhibition. The survival time increased in tumor bearing mice and some showed tumor free, which developed no tumor after rechallenged with TC-1.Th1 cytokines including IL-2, IFN-γwere significantly increased. These studies demonstrate that the tumor antigen immunized MHC-haploidentical lymphocytes could specificity target to the tumor and kill them. MHC-mismatched alloantigens could be an effective adjuvant to break the immune tolerance and to activate innate and adoptive immunity in the tumor bearing host. The infusion of the inactivated MHC-haploidentical lymphocytes immunized with tumor antigen could be a safe and effective adoptive immunotherapy against tumor.

Current Adoptive Immunotherapy in Non-Small Cell Lung Cancer and Potential Influence of Therapy Outcome

Non-small cell lung cancer (NSCLC) is found worldwide with high incidence and poor prognoses. Nowadays, insights in the interaction between tumors and immune system have led to the development of immunotherapy as a fundamentally new concept for the treatment of NSCLC. Adoptive cell transfer represents an important advancement in cancer immunotherapy such as cytokine-induced killer and γδ T-cells. Recent clinical research studies provide evidence for the positive effects of adoptive immunotherapy, which is probably associated with levels of cytokines, cell doses, and immune microenvironment. This review summarizes the current condition of adoptive immunotherapy in NSCLC and the long-standing confusion in this field.

Chemokine Receptor Expression, Migration, and Survival of NK Cells Expanded with Mbil-15 or Mbil-21

AbstractAbstract 4683Natural killer (NK) cells can kill malignant or virus-infected cells through the interaction of activating and inhibitory receptors without needing specific antigen recognition of target cells, and therefor have broad therapeutic applications for treatment of human malignancies. However, due to their limited life-span in vivo and poor expansion in vitro, production of sufficient numbers of NK cells for effective adoptive immunotherapy poses an obstacle. Genetically engineered artificial antigen presenting cells (aAPCs) consisting of K562 modified 4-1BBL and membrane bound IL-15 or IL-21 have been reported for their ability to support ex vivo NK cell proliferation. aAPCs with mbIL-21 were shown to promote increased proliferation of NK cells with shorter telomeres, but differences in in vivo survival or tumor or tissue migration have not been assessed. Tumor and/or tissue migration is primarily mediated by the expression of chemokine receptors. Using aAPCs bearing mbIL15 or mbIL21, we expanded NK cells for 3 weeks and assessed their expression of chemokine receptors, organ migration, and in vivo survival in a xenograft model. Propagated NK cells showed relatively similar levels of low to modest expression of CCR2, CCR7, CXCR4 and CXCR5, and high expression levels of CXCR3. Mean CCR5 expression levels were similar on cells that were positive, but CCR5 was expressed on a higher percentage of NK cells expanded with mbIL-15 than those expanded with mbIL-21. In contrast, about 20% of mbIL-21 expanded NK cells expressed CX3CR1 expression whereas mbIL-15 NK cells showed almost no expression of this receptor. Results from ongoing migration and survival experiments will also be presented. Disclosures:No relevant conflicts of interest to declare.

Genomic analysis of CD8 T cells during the early phases of the immune response (110.25)

Abstract The generation of long-lived memory CD8 T cells is critical for the development of effective vaccines and adoptive immunotherapy strategies. However, the molecular mechanisms by which a T cell transitions from naïve to memory remains poorly understood. In an effort to better understand the molecular pathways involved in the differentiation of CD8 T cells, we have analyzed ovalbumin-specific OTI CD8 T cells during the very early phases of the immune response at the genomic level. Following infection with Listeria monocytogenes expressing ovalbumin, OTI CD8 T cells from infected mice were sorted and subjected to microarray analysis. We show that while cell surface phenotype of OTI T cells remained largely unchanged shortly after activation, large scale changes occurred at the level of transcription. Expression levels of many families of genes involved in signaling networks in T cells, in addition to multiple immune response factors, were differentially expressed shortly after T cells were activated, but prior to the first division. Notably, expression of IL-21, a cytokine implicated in T cell memory, was transiently expressed with peak expression occurring at 12 hours post-stimulation. This was confirmed by intracellular staining for the IL-21 protein. Similarly, the peak of STAT3 expression coincided with IL-21, a known activator of STAT3. We are currently investigating autocrine IL-21 signaling in CD8 T cells and its role in memory development.

The Capacity of CMV-Specific T-Cells to Prevent Outgrowth of Clonogenic Human Colon Carcinoma Cells Co-Expressing CMVpp65 in-Vivo and Durably Suppress Established CMVpp65+ Tumor Xenografts Is Dose Dependant and Can Be Augmented by IL-15/IL-15Rα

Abstract 4307Adoptively transferred CMV-specific T cells (CMV-CTL) can effectively prevent CMV disease in HSCT recipients. However, data regarding the doses required to eradicate disease and the relative potency of T-cells (TC) specific for different epitopes are lacking. To test the capacity of T-cells specific for different CMV antigenic peptides to target and eliminate CMV+ cells in-vivo, and establish TC doses required for treatment we have developed an in-vivo model to assess specificity and efficacy of CMV-CTLs using human colon carcinoma cells transduced with CMVpp65 as a surrogate system.HLA A0201+ colon carcinoma cells transduced to express CMVpp65 and a GFP-firefly luciferase transgene (cocapp65) were injected subcutaneously (s.c.) into NOD/Scid-IL2Rgc-KO/J mice (NSG) at doses ranging from 104 – 106 (4 mice /group) and tumor growth followed for 4 weeks using bio-luminescent imaging and tumor measurements. Consistent engraftment was observed with 3 × 105 cells. Groups of NSG mice were then injected s.c with coca-pp65 (3 × 105) mixed with HLA A0201-restricted CMVpp65 specific T-cells (A2-CMV-CTLs) at E:T ratios of 50:1, 10:1, 2:1, 0.1:1, 0.01:1 and 0.001:1. To ascertain antigen specificity of A2-CMV-CTLs, groups of 4 NSG mice were subsequently co-injected s.c. with 3 tumor-TC combinations (E:T= 1:1) as follows: (i) A2-CMV- CTLs + 3 × 105 cocapp65 (ii) A2-CMV- CTLs + A2 tumor without CMVpp65 (iii) A2-Flu-CTLs + 3 × 105 cocapp65. Control animals were injected with the cocapp65 without TC. IL-2 (2000 U i.p) was given 2 × a week to all groups. Tumor growth was measured by bioluminescence. No growth of cocapp65 was detected in any animal co-injected with A2-CMV-CTLs at E:T ratios ≥ 1:1. At E:T ratios of 0.1:1, 0.01:1 and 0.001:1, 1/4, 2/4 and 3/4 animals demonstrated tumor growth respectively. In contrast, consistent tumor growth was seen in all control animals over 4 weeks. Thus, tumors in condition (i) were completely inhibited while (ii) and (iii) continued to grow similar to controls, thus demonstrating antigen specific activity of the infused CMV-CTLs.In subsequent experiments we assessed (1) the efficacy of titrated doses of intravenously administered CMV-CTLs in animals with established tumors using 20 × 106, 7× 106 or 2× 106 T-cells per mouse (2) the contribution of IL-15/15Rα complex in augmenting the efficacy of CMV-CTLs by injecting each mouse i.p. with irradiated Baf-3 cells transduced to express human IL-15/15Rα complex.Infusion of CMV-CTLs significantly suppressed growth of cocapp65 tumors at all doses tested. However after a period of 4 weeks, the tumors in animals receiving doses of 7 × 106 and 2 × 106 CMV-CTLs began to re-grow, while tumors in animals injected with 20 × 106 remained suppressed till the end of the experiment at 6–8 weeks. At autopsy, human CD45[+] CD3[+] T-cells were detected in tumors expressing CMVpp65 as documented by IHC staining, but not in tumors lacking CMVpp65 or HLA A0201. In mice injected with the Baf-3 cells expressing IL-15/IL-15Rα, the anti-tumor activity of the transferred T-cells was sustained even in animals treated with only 2×106 CMVpp65 specific CTLs. Differences in cytolytic capacity of CMV-CTLs reactive against dominant and subdominant epitopes are currently under study.These studies demonstrate that (1) CMV-CTLs can prevent outgrowth of clonogenic human carcinoma cells co-expressing an immunogenic viral antigen in-vivo at E:T ratios that are significantly lower than those required in-vitro (2) These CMV-CTLs can also induce sustained suppression of established tumor xenografts following intravenous transfer (3) Lower doses of intravenously transferred CMV-CTLs are effective, but may require additional dosing to maintain disease control, and (4) In contrast, IL-15/IL-15Rα supplementation can augment and sustain the in-vivo activity of low doses of CMVpp65-specific CTLs. This model thus provides an in vivo system for estimating the concentrations of antigen-specific T-cells over time required for effective adoptive immunotherapy, for comparative evaluations of virus-specific T-cells differing in phenotype or specificity, and for testing the potential of cytokines and other agents to increase or sustain virus-specific T-cell activity. Disclosures:No relevant conflicts of interest to declare.

T lymphocytes derived from human cord blood provide effective antitumor immunotherapy against a human tumor

BackgroundAlthough the graft-versus-tumor (GVT) effect of donor-derived T cells after allogeneic hematopoietic stem cell transplantation has been used as an effective adoptive immunotherapy, the antitumor effects of cord blood (CB) transplantation have not been well studied.MethodsWe established the animal model by transplantation of CB mononuclear cells and/or tumor cells into NOD/SCID mice. The presence of CB derived T cells in NOD/SCID mice or tumor tissues were determined by flow cytometric and immunohistochemical analysis. The anti-tumor effects of CB derived T cells against tumor was determined by tumor size and weight, and by the cytotoxicity assay and ELISPOT assay of T cells.ResultsWe found dramatic tumor remission following transfer of CB mononuclear cells into NOD/SCID mice with human cervical tumors with a high infiltration of CD3+ T cells in tumors. NOD/SCID mice that receive neonatal CB transplants have reconstituted T cells with significant antitumor effects against human cervical and lung tumors, with a high infiltration of CD3+ T cells showing dramatic induction of apoptotic cell death. We also confirmed that T cells showed tumor specific antigen cytotoxicity in vitro. In adoptive transfer of CD3+ T cells into mice with pre-established tumors, we observed much higher antitumor effects of HPV-specific T cells by ELISPOT assays.ConclusionsOur results show that CB derived T lymphocytes will be useful for novel immunotherapeutic candidate cells for therapy of several tumors in clinic.