Tumor-infiltrating Lymphocytes Populations Research Articles

PD-L1 Expression and Tumor-Infiltrating Lymphocytes Define Different Subsets of MAPK Inhibitor-Treated Melanoma Patients.

To evaluate the expression of tumor PD-L1 and changes in tumor-infiltrating lymphocyte (TIL) populations in patients with metastatic melanoma treated with targeted MAPK inhibitors. Ninety-three tumors were analyzed from 40 patients treated with a BRAF inhibitor alone (BRAFi; n = 28) or combination of BRAF and MEK inhibitors (Combi; n = 12). Tumors were excised before treatment (PRE), early during treatment (EDT), and at progression (PROG). Immunohistochemical staining was performed for CD4, CD8, CD68, FOXP3, LAG3, PD-1, and PD-L1 and correlated with clinical outcome. Patients' tumors that were PD-L1 positive at baseline showed a significant decrease in PD-L1 expression at PROG (P = 0.028), whereas patients' tumors that were PD-L1 negative at baseline showed a significant increase in PD-L1 expression at PROG (P = 0.008) irrespective of treatment with BRAFi or Combi. Overall PD-L1 expression highly correlated with TIL immune markers. BRAFi-treated patients showed significant increases in CD4(+), CD8(+), and PD-1(+) lymphocytes from PRE to EDT (P = 0.001, P = 0.001, P = 0.017, respectively), and Combi-treated patients showed similar increases in CD4(+) and CD8(+) lymphocytes from PRE to EDT (P = 0.017, P = 0.021). The addition of MEKi to BRAFi did not result in significant reduction in immune infiltration in EDT biopsies. This provides support for conducting trials that combine MAPKi with immune checkpoint inhibitors in the hope of improving complete and durable response rates. PD-L1 expression at PROG on MAPK inhibitors varied according to baseline expression suggesting that combining MAPKi with immunotherapies concurrently may be more effective in patients with PD-L1 expression and TILs in baseline melanoma samples.

T-cell Subsets in Peripheral Blood and Tumors of Patients Treated With Oncolytic Adenoviruses

The quality of the antitumor immune response is decisive when developing new immunotherapies for cancer. Oncolytic adenoviruses cause a potent immunogenic stimulus and arming them with costimulatory molecules reshapes the immune response further. We evaluated peripheral blood T-cell subsets of 50 patients with refractory solid tumors undergoing treatment with oncolytic adenovirus. These data were compared to changes in antiviral and antitumor T cells, treatment efficacy, overall survival, and T-cell subsets in pre- and post-treatment tumor biopsies. Treatment caused a significant (P < 0.0001) shift in T-cell subsets in blood, characterized by a proportional increase of CD8(+) cells, and decrease of CD4(+) cells. Concomitant treatment with cyclophosphamide and temozolomide resulted in less CD4(+) decrease (P = 0.041) than cyclophosphamide only. Interestingly, we saw a correlation between T-cell changes in peripheral blood and the tumor site. This correlation was positive for CD8(+) and inverse for CD4(+) cells. These findings give insight to the interconnections between peripheral blood and tumor-infiltrating lymphocyte (TIL) populations regarding oncolytic virotherapy. In particular, our data suggest that induction of T-cell response is not sufficient for clinical response in the context of immunosuppressive tumors, and that peripheral blood T cells have a complicated and potentially misleading relationship with TILs.

Focused ultrasound-induced blood-brain barrier opening to enhance interleukin-12 delivery for brain tumor immunotherapy: a preclinical feasibility study.

BackgroundInterleukin-12 (IL-12) has long been considered to be effective in triggering an anticancer immune response, however, the dosage has been limited by potential systemic immunotoxicity. Since focused ultrasound (FUS) has been confirmed to temporally and locally open the blood–brain barrier (BBB), the purpose of this study was to elucidate the possibility of combining FUS-induced BBB opening with IL-12 delivery to enhance the anticancer immunological response for glioma treatment.MethodsFUS energy combined with microbubble administration was delivered transcranially to open BBB, and C-6 glioma rats were used in this study. The efficacy in inducing BBB opening and the corresponding immunological response were primarily evaluated in normal animals. The anticancer immune-triggering chemokine, IL-12, was intraperitoneally administered during the treatment phase to evaluate the effect of immunological response on tumor progression. Glioma animals were sub-grouped to evaluate the effect of the immune response in suppressing glioma when IL-12 was combined with FUS-induced BBB opening. We performed flow cytometry to verify consequent immune cell population changes of peripheral/tissue lymphocytes as well as macrophages from the animals. Brain sections of sacrificed animals were also used for histological and immunohistochemical analysis. IL-12 level among experimental groups were measured via ELISA analysis. We also analyzed survival and followed tumor progression in vivo via T2-weighted magnetic resonance imaging.ResultsFUS-induced BBB opening had no obvious effect on the T lymphocytes population in normal animals, either in the brain or systemically. Yet, it triggered mild changes in the tumor-infiltrating lymphocyte (TIL) population, particularly in numbers of CD3+CD8+ cytotoxic T lymphocytes (CTLs) in the tumor region. IL-12 administration triggered a profound increase in all TIL populations, including CD3+CD4+ T helper cells (Th), CTL, and CD4+CD25+ regulatory T cells (Treg), but combined FUS-BBB opening with IL-12 administration produced the most significant IL-12 increase, CTL increase and CTL/Treg ratio increase, thus contributing to the most significant suppression of tumor progression and increased animal survival.ConclusionThis study provides evidence that FUS-BBB opening can enhance immune-modulating agent delivery to the brain, which improve the anticancer immune response in brain tumor treatment.

CTLA-4 Blockade Plus Adoptive T-Cell Transfer Promotes Optimal Melanoma Immunity in Mice

Immunotherapeutic approaches to the treatment of advanced melanoma have relied on strategies that augment the responsiveness of endogenous tumor-specific T-cell populations [eg, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade-mediated checkpoint inhibition] or introduce exogenously prepared tumor-specific T-cell populations [eg, adoptive cell transfer (ACT)]. Although both approaches have shown considerable promise, response rates to these therapies remain suboptimal. We hypothesized that a combinatorial approach to immunotherapy using both CTLA-4 blockade and nonlymphodepletional ACT could offer additive therapeutic benefit. C57BL/6 mice were inoculated with syngeneic B16F10 melanoma tumors transfected to express low levels of the lymphocytic choriomeningitis virus peptide GP33 (B16GP33), and treated with no immunotherapy, CTLA-4 blockade, ACT, or combination immunotherapy of CTLA-4 blockade with ACT. Combination immunotherapy resulted in optimal control of B16GP33 melanoma tumors. Combination immunotherapy promoted a stronger local immune response reflected by enhanced tumor-infiltrating lymphocyte populations, and a stronger systemic immune responses reflected by more potent tumor antigen-specific T-cell activity in splenocytes. In addition, whereas both CTLA-4 blockade and combination immunotherapy were able to promote long-term immunity against B16GP33 tumors, only combination immunotherapy was capable of promoting immunity against parental B16F10 tumors as well. Our findings suggest that a combinatorial approach using CTLA-4 blockade with nonlymphodepletional ACT may promote additive endogenous and exogenous T-cell activities that enable greater therapeutic efficacy in the treatment of melanoma.

OX40 agonist immunotherapy expands tumor reactive CD8 T cells with a unique T cell receptor repertoire and synergizes with PDL-1 blockade to promote tumor regression

Previously, the field of tumor immunology has relied on T cell receptor (TCR) transgenic mice and model tumor antigens to study the T cell:tumor cell interaction. However, these models have limitations. Namely, they do not reflect the natural affinity, repertoire, or precursor frequency of tumor antigen-specific T cells. Using a novel TCR affinity tool, the Nur77GFP mouse, we have characterized the signal strength and phenotype of naturally occurring tumor antigen-specific CD8 T cells. Our data suggest that the co-expression of Nur77GFP and PD-1 identifies a population of polyclonal CD8+ tumor infiltrating lymphocytes (TIL) that are tumor antigen-specific. This population produces more IFN-γ in situ and upon re-stimulation with syngeneic but not allogeneic tumor cells. In addition, treatment of tumor bearing mice with an OX40 agonist mAb doubled the frequency of tumor reactive T cells receiving strong TCR signals as compared to the control (~two-fold increase in CD8+Nur77GFPhi p=.0027). PD-1, PDL-1, or CTLA4 blockade also had a modest influence on the frequency of CD8+Nur77GFPhi TIL but this was not as significant as anti-OX40 mAb. To further understand the anti-tumor immune response, CD8+Nur77GFPhi and Nur77GFPlo TIL were sorted and the TCRb CDR3 region was sequenced. We observed clonal expansions of unique Vb sequences exclusively found in the TIL population after anti-OX40 immunotherapy. Unexpectedly, the CD8+ Nur77GFPlo population sorted from mice treated with the anti-OX40 mAb contained very similar Vb sequences and together these represented more than 30% of the TCR repertoire. This suggests that both weak and strong tumor antigens drive TIL expansion after anti-OX40 mAb immunotherapy. Importantly, these repertoire changes were not observed with the control antibody in TIL nor in the spleens of OX40 agonist treated mice. OX40 agonist therapy also increased the frequency of PD-1+ TIL suggesting that targeting this inhibitory molecule in combination with OX40 might improve tumor rejection. In fact, when established tumors (~50mm2) were treated with a combination of anti-OX40 and anti-PDL-1, significant tumor regression and disease free survival was observed in multiple tumor models (C57Bl/6:MCA205 50% survival p < 0.0001, Balb/c:CT26 30% survival p = 0.0105). Tumor rejection corresponded to an increase in the circulation of CD44hi, proliferating, CD8 T cells that was observed only in the combination therapy group as compared to either single agent alone. Therefore, we propose that one mechanism of OX40 agonist immunotherapy is via the priming and expansion of low and high affinity tumor antigen specific CD8 T cells.

OX40 and other immunoregulatory molecules are highly expressed on tumor infiltrating lymphocytes in oral, head and neck squamous cell carcinoma

OX40 is a potent co-stimulatory receptor on the surface of T lymphocytes. An OX40 agonist was recently tested in a Phase I clinical trial and was found to be well tolerated and enhanced both humoral and cellular immunity in patients with metastatic cancer. Both PD-1 and CTLA-4 blockade are showing therapeutic clinical activity in late stage cancer patients. Hence, we hypothesize that the immune suppressed tumor microenvironment that characterizes oral, head and neck squamous cell carcinoma (OHNSCC) can be overcome by strategies that favor antitumor inflammatory T cell responses and may be an ideal tumor site for immune modulation. This investigation aims to provide a better understanding of the expression of OX40, PD-1, CTLA-4 and CD103 in OHNSCC tumor infiltrating lymphocytes (TIL). Additionally we evaluated whether phenotypic differences existed in patients with HPV positive vs. negative status. Methods TIL and autologous blood were isolated from 29 patients undergoing surgery for OHNSCC. A sample of tissue was obtained from the resection specimen of the primary tumor and sent to the laboratory with autologous blood. Tumor was minced and subjected to digestive enzymes. Blood and tumor samples were separated over ficoll gradient to enrich for lymphocytes. Samples were frozen at -140°c and later thawed in batches for analysis. Proteins were then labeled with fluorescently conjugated antibodies and analyzed by flow cytometry. Results The percentage of FoxP3+CD25+CD4+ (Tregs) of CD4+ T cells is significantly higher in OHNSCC Tumor Infiltrating Lymphocytes (TIL) compared to blood (p < .0005), regardless of HPV status. OX40 expression is significantly higher in the TIL Treg population relative to blood Tregs (p < .0005), also independent of HPV status. PD1 expression is increased in the Tregs (p < .0005), conventional CD4 (p=.0066), and CD8 T cells (p < .0005), in TIL relative to blood. CTLA-4 expression is increased in TIL relative to blood in all subsets with the highest expression observed in the Treg population (.0056). CD103 expression is increased on CD8 T cells in TIL relative to blood (p < .0005). CD8s from HPV positive patients have higher percentage CD103 positive cells than CD8s from HPV negative patients (p=.0056). The highest percentages of PD-1, CTLA-4 and OX40 triple positive cells were found in the Treg population of TIL compared to blood (p < .0005). Due to increased expression of OX40, CTLA-4 and PD-1 in TIL of OHNSCC patients we hypothesize that future combination clinical trials that target these pathways will be therapeutically beneficial.

Clinical scale zinc finger nuclease (ZFN)-driven gene-editing of PD-1 in tumor infiltrating lymphocytes (TIL) for the potential treatment of metastatic melanoma

Multiple inhibitory pathways exist to block the immune response to cancer potentially limiting the effectiveness of adoptive cell transfer (ACT). Programmed cell death-1 (PD-1) is a member of the CD28 superfamily and is expressed on activated T cells. Its ligands, PDL-1 and PDL-2 are expressed on a variety of tumor cells, including melanoma. The binding of PD-1 to PDL-1 inhibits T cell effector function, and represents an important mechanism for PDL-1 expressing tumors to evade the host immune response to cancer. PD-1 thus represents an attractive target for gene-editing of tumor-targeted T cells prior to ACT. To this end, our aim was to eliminate PD-1 expression in tumor infiltrating lymphocytes (TIL) by genome-editing using zinc finger nucleases (ZFNs) directed against the PD-1 gene at a scale sufficient for patient treatment. Using the MaxCyte GT Flow Transfection System to deliver mRNA encoding the PD-1 ZFNs, we show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9 - 84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Importantly, the PD-1 gene-edited TIL product displayed an effector memory phenotype and expanded approximately 500 - 2000 fold during a rapid cell expansion in vitro while retaining T cell effector function. Thus further study to determine the safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

Potential immunotherapeutic role of interleukin-2 and interleukin-12 combination in patients with hepatocellular carcinoma.

BackgroundMany recent therapeutic interventions are necessary to improve the treatment of hepatocellular carcinoma (HCC), including immunotherapy, which seems to offer one of the new realistic therapeutic modalities. This study aims to investigate the optimization of immunotherapy for HCC patients by appraisal of both interferon (IFN)-γ levels and phenotyping of lymphocytes obtained from peripheral blood and fine-needle aspirates.MethodsThe isolated lymphocytes were cultured in the presence of interleukins (IL)-2, IL-4, and IL-12. Enzyme-linked immunosorbent assay and flow cytometric techniques were used for the assessment of human IFN-γ production and the studied T-cell subpopulations, respectively.ResultsMixed cell populations of peripheral blood lymphocytes and tumor infiltrating lymphocytes treated with IL-2 plus IL-12 showed a marked and significant elevation in IFN-γ levels in their culture media, a significant decrease in the percentage of cluster of differentiation (CD)4+CD25+ regulatory T-cells, and a nonsignificant increase in the percentage of CD8+ cytotoxic T-cells. Meanwhile, IL-2 plus IL-4 treatment demonstrated nonsignificant effects.ConclusionOur data suggested that IL-12 together with IL-2 caused a suppression of CD4+CD25+ regulatory T-cells and an elevation of IFN-γ levels, which play a crucial immunotherapeutic role in the management of HCC patients.

PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) can mediate regression of metastatic melanoma; however, TILs are a heterogeneous population, and there are no effective markers to specifically identify and select the repertoire of tumor-reactive and mutation-specific CD8⁺ lymphocytes. The lack of biomarkers limits the ability to study these cells and develop strategies to enhance clinical efficacy and extend this therapy to other malignancies. Here, we evaluated unique phenotypic traits of CD8⁺ TILs and TCR β chain (TCRβ) clonotypic frequency in melanoma tumors to identify patient-specific repertoires of tumor-reactive CD8⁺ lymphocytes. In all 6 tumors studied, expression of the inhibitory receptors programmed cell death 1 (PD-1; also known as CD279), lymphocyte-activation gene 3 (LAG-3; also known as CD223), and T cell immunoglobulin and mucin domain 3 (TIM-3) on CD8⁺ TILs identified the autologous tumor-reactive repertoire, including mutated neoantigen-specific CD8⁺ lymphocytes, whereas only a fraction of the tumor-reactive population expressed the costimulatory receptor 4-1BB (also known as CD137). TCRβ deep sequencing revealed oligoclonal expansion of specific TCRβ clonotypes in CD8⁺PD-1⁺ compared with CD8⁺PD-1- TIL populations. Furthermore, the most highly expanded TCRβ clonotypes in the CD8⁺ and the CD8⁺PD-1⁺ populations recognized the autologous tumor and included clonotypes targeting mutated antigens. Thus, in addition to the well-documented negative regulatory role of PD-1 in T cells, our findings demonstrate that PD-1 expression on CD8⁺ TILs also accurately identifies the repertoire of clonally expanded tumor-reactive cells and reveal a dual importance of PD-1 expression in the tumor microenvironment.

Antigen-specific TIL therapy for melanoma: a flexible platform for personalized cancer immunotherapy

Adoptive cell therapy (ACT) for melanoma has shown to be an effective treatment modality with clinical responses in approximately 50% of stage IV disease patients. However, durable complete responses are observed in only a small proportion (10-20%) of patients. Recent data have demonstrated that the tumor-infiltrating lymphocyte (TIL) infusion products used for therapy harbor only low frequencies of T cells reactive against presently known shared melanoma-associated epitopes. Furthermore, ongoing studies indicate that T cell reactivity against patient-specific mutated antigens may be common in human melanoma. Collectively, these two observations suggest that the development of a clinically applicable approach that allows the selection of defined tumor-reactive T cell populations in a patient-specific manner would be of value. Here we develop such a platform technology and demonstrate its value in a preclinical model. First, reversible (strep-tagged) HLA-A*0201 molecules for clinical use were produced with a UV-sensitive peptide ligand, and the resulting MHC class I monomers could be loaded with peptides of interest in straightforward peptide exchange reactions. To subsequently evaluate the potential of these MHC exchange streptamers for antigen-specific T cell enrichment, we performed individual or combined enrichments of melanoma-reactive T cell populations from TIL populations. Enrichment of both high (1≥%) and low frequency (<1%) tumor-specific T cell populations, either alone or combined, was shown to be feasible with a mean enrichment factor of 27 and 56. To evaluate the potential of this technology for the generation of T cell products that recognize patient-specific neo-antigens, we performed combined enrichments of two HLA-A*0201 restricted T cell responses, directed against neo-antigens in the CDK4 (2.2% of CD8+ cells) and GCN1L1 (0.58% of CD8+ cells) gene products that we identified by exome sequencing. Combined enrichment of these two patient-specific T cell populations resulted in a neo-antigen specific cell product with a purity of 92.4%. Functional analysis of the neo-antigen selected T cell product against autologous tumor in NSG mice showed superior tumor control and survival compared to non-selected ‘standard’ TIL. Together, these data demonstrate that selection of (panels of) antigen-specific T cells using the combination of UV-induced ligand exchange and reversible streptamer technology is feasible. In combination with exome-driven analysis of tumor-specific T cell reactivity in human cancer, this strategy forms a highly flexible platform for the development of antigen-specific cell products for personalized cancer immunotherapy.

Homogeneity of the adaptive immune repertoire of primary and metastatic ovarian tumors (P2205)

Abstract The presence, abundance, population, and diversity of Tumor Infiltrating Lymphocytes (TILs) have been identified as prognostic indicators in several cancers. Emerging cancer therapeutics including immunomodulators and adoptive T-cell therapy highlight the need to better understand and track this population of T cells. We have developed a multiplex PCR amplification assay for the rearranged T-cell receptor Beta (TCRB), followed by deep sequencing to characterize the adaptive immune response spatially within ovarian tumor. Specifically, we used our assay to characterize the heterogeneity of ovarian tumor TIL populations. We sequenced 10 primary and metastatic ovarian tumors collected from 5 patients. Each tumor was divided into a grid pattern with 8-22 sections (tumor size dependent). For each tumor section we collected data on the number, diversity, and the unique CDR3 sequences carried by the TILs, to characterize the intra-tumor heterogeneity of TIL count, diversity, and T cell clone overlap. In addition, we sequenced the immune repertoire from a paired peripheral blood sample to identify if the immune response was tumor specific. We found that the immune response within peripheral blood was significant and quantifiably different than the tumor repertoire. TIL repertoire within sections of a tumor showed high similarity to each other. These data indicate that the TCR repertoire of the ovarian tumor environment is tumor specific and homogeneous.

The crosstalk: Tumor-infiltrating lymphocytes rich in regulatory T cells suppressed cancer-associated fibroblasts

Background. The interactions between cancer-associated fibroblasts (CAFs) and cancer cells or tumor-infiltrating lymphocytes (TILs) and cancer cells play important roles in cancer progression and metastasis. However, studies related to the crosstalk between CAFs and TILs in tumor microenvironment (TME) are still lacking. In this study, we mainly investigated the interactions between CAFs and TILs. Material and methods. The distribution of TILs rich in regulatory T cells (Tregs) in breast cancer tissues was evaluated using hematoxylin-eosin staining and immunohistochemistry with anti-CD3, anti-Foxp3, and anti-α-smooth muscle actin antibodies. Homologous CAFs/normal fibroblasts (NFs) and TILs cultured in vitro were identified and detected using immunocytochemistry and flow cytometry (FCM). The direct interaction among these cell types was studied via a factorial design in a co-cultured system. Their indirect interaction was assayed using Transwell plates. The cell cycle and apoptosis of CAFs/NFs co-cultured with TILs was analyzed using propidium iodide staining. Results. Histochemistry demonstrated most of the TILs including Tregs, were distributed in the cancer stroma, adjoining to CAFs. This finding implies that both cell types interact closely in the TME. Identification of the cultured cells showed that CAFs maintained their activated phenotype within limited passages in vitro, and that the TILs population contained a high percentage of Tregs. Data analysis of the factorial design suggests significant interactions among CAFs, NFs, and TILs in both direct and indirect contact ways. The CAFs and NFs were suppressed signally by TILs, which are probably induced by the secretory cytokines derived from TILs or Tregs. Although apoptosis was not detected in CAFs/NFs, the cell cycle assay suggested that the CAFs/NFs were arrested in the G2/M phase by the TILs and their secretory cytokines. Conclusion. CAFs and NFs were dramatically suppressed by Tregs-rich TILs. This suggests the interaction between TILs and CAFs might modify the TME in an unknown manner.

Abstract IA18: T cell adoptive therapy for cancer: Translating the science.

Abstract A variety of immunizations, cytokines and immune checkpoint inhibitors have been used in the effort to marshal an anti-tumor T-cell response capable of rejecting a patient's tumor. Another approach is to activate and expand a population of autologous tumor-reactive T-cells in vitro and administer them back to a patient who has been optimally prepared to support T-cell survival and function. This process, adoptive cell therapy (ACT), allows the use of reagents in vitro that might not be tolerated in vivo, and benefits from manipulations (such as recipient immunosuppression) that can be applied prior to T-cell transfer, to avoid damaging the anti-tumor response. The finding that the tumor infiltrating lymphocytes (TIL) within most human melanomas are tumor reactive has provided the means for studying ACT in cancer patients. Murine models have shown that recipient immunosuppression removes endogenous suppressor cells, induces homeostatic proliferation of transfused T-cells and removes cells that compete for T-cell growth factors. So protocols transferring cultured TIL (along with supportive systemic IL2) into patients with metastatic melanoma who have received a preparative regimen of high-dose cyclophosphamide and fludarabine (Cy-Flu), with or without additional total body irradiation (TBI) have been conducted. In a group of 93 patients (over 80% of whom had visceral metastases and prior IL-2 treatment), the overall response rate was 56% with 21% achieving complete responses. More striking is the fact that with all patients having at least 5-year follow up, 19 of the 20 complete responders have maintained those responses. There were 10 complete responses in 25 patients given Cy-Flu and the highest dose of TBI. There is mounting evidence that the TIL administered can contain multiple specificities, commonly recognizing not only proteins in the pigment production pathway, but also tumor-specific mutations. This experience has shown that ACT is capable of inducing complete responses in patients with disseminated melanoma that appear to have curative potential. For patients without a tumor-reactive TIL population, it has proven possible to genetically engineer their peripheral blood lymphocytes (PBL) with receptors which recognize their cancer and administer those in lieu of a native TIL population. Retrovirally inserting a T-cell receptor (TCR) cloned from another patient (sharing the appropriate HLA allele) can confer tumor recognition. PBL modified with a TCR against melanocytic lineage antigens (such as MART-1 and gp100) or a tumor-testis antigens (such as NY-ESO-1) have both shown efficacy when given in ACT protocols. In a critical advance, it has been shown that TCR-engineered PBL against NY-ESO-1 not only have activity against metastatic melanoma, but also the NY-ESO-1 positive tumor synovial sarcoma. This conclusively showed that tumors other than melanoma can respond to ACT. In order to further broaden the scope of tumors which can be treated, chimeric antigen receptors consisting of a binding domain derived from a single chain immunoglobulin coupled to the T-cell transduction moiety CD3-zeta have been developed. One such receptor, reactive with CD19 on follicular lymphomas and CLL, has shown activity in refractory patients with those diseases. Ultimately, the real promise from T-cell gene therapy is not in merely redirecting T-cell specificity, but in the intelligent modification of T-cell functions to better effect tumor rejection. One such example in clinical testing is to engineer melanoma TIL to secrete IL-12. This cytokine enhances T-cell secretion of gamma interferon and augments tumor antigen presentation to T-cells. In murine models, this results in a 100-fold increase in T-cell efficacy and does not require the concomitant administration of supportive systemic IL2. A phase I/II protocol testing this concept is underway and early results suggest that not only can some patients respond when unmodified TIL have failed, but objective responses can be seen when many fewer TIL are given with no accompanying IL-2. A host of other functional modifications of T-cells are currently under study and hold out the promise that carefully crafted populations of T-cells can be constructed with gene engineering to induce the durable rejection of a variety of advanced cancers in patients. Citation Format: James C. Yang. T cell adoptive therapy for cancer: Translating the science. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA18.

Design of T-cell receptor libraries with diverse binding properties to examine adoptive T-cell responses

Adoptive T cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, that introduces antigen-specific T cell receptors (TCRs) into ex vivo activated T cells, is designed to overcome central tolerance mechanisms that prevent responses by endogenous T cell repertoires. Studies have suggested that use of higher affinity TCRs against class I MHC antigens could drive the activity of both CD4+ and CD8+ T cells, but the rules that govern the TCR binding optimal for in vivo activity are unknown. Here we describe a high-throughput platform of “reverse biochemistry” whereby a library of TCRs with a wide range of binding properties to the same antigen is introduced into T cells and adoptively transferred into mice with antigen-positive tumors. Extraction of RNA from tumor-infiltrating lymphocytes or lymphoid organs allowed high-throughput sequencing to determine which TCRs were selected in vivo. The results showed that CD8+ T cells expressing the highest affinity TCR variants were deleted in both the tumor infiltrating lymphocyte population and in peripheral lymphoid tissues. In contrast, these same high-affinity TCR variants were preferentially expressed within CD4+ T cells in the tumor, suggesting they played a role in antigen-specific tumor control. The findings thus revealed that the affinity of the transduced TCRs controlled the survival and tumor infiltration of the transferred T cells. Accordingly, the TCR library strategy enables rapid assessment of TCR binding properties that promote peripheral T cell survival and tumor elimination.

An exploratory study to investigate the immunomodulatory activity of BMS-936558 in patients with metastatic clear-cell renal cell carcinoma.

TPS2613 Background: With the enhanced understanding of the immune response to a tumor, new immunotherapeutic agents may expand treatment options for metastatic renal cell carcinoma (mRCC). The programmed death-1 (PD-1) receptor is upregulated by activated lymphocytes, and upon binding to its ligands, inhibits lymphocyte function. In a phase 1 study of the anti-PD-1 monoclonal antibody BMS-936558, encouraging antitumor activity has been observed in patients with previously treated mRCC. Responses occurred in extensive mRCC and were durable in many patients. Here we describe an exploratory study to investigate the immune-regulatory activity of various doses of BMS-936558 in patients with metastatic clear-cell RCC. Methods: Eighty patients with metastatic clear-cell RCC will be enrolled in this open-label, parallel group, randomized study. Patients who have received prior treatment with ≥3 therapies, including ≥1 anti-angiogenic therapy, will be enrolled in Arms 1-3 (~20 patients per arm); 20 treatment-naïve patients will be enrolled in Arm 4. BMS-936558 will be administered every 3 weeks until disease progression or unacceptable toxicity at 0.3 mg/kg (Arm 1), 2 mg/kg (Arm 2), and 10 mg/kg (Arms 3 and 4). Patients in the 0.3-mg/kg group may escalate to 2 mg/kg if not responding. Tumor assessments will occur every 6 weeks for the first 12 months then every 12 weeks for the remainder of the study. The primary objective is to evaluate the effects of BMS-936558 on the frequency of circulating immune cell subsets, levels of soluble factors associated with immune responses, and tumor infiltrating lymphocyte populations (based on pre-treatment and week 5 paired biopsies). Secondary objectives are to assess the safety, tolerability, antitumor activity, and immunogenicity of BMS-936558. Exploratory objectives include the association of clinical activity and safety with selected biomarkers in peripheral blood and tumor microenvironment and the characterization of the pharmacokinetics of BMS-936558. As of February 1, 2012, 30 patients were enrolled.

Abstract 1538: Enhanced antitumor effects of intratumoral (IT) hu14.18-IL2 immunocytokine (IC) compared to intravenous (IV) hu14.18-IL2 are distinguished by increased activated tumor infiltrating lymphocytes (TILs) and IC retention

Abstract Introduction: Hu14.18-IL2 (APN301, Apeiron Biologics) is an immunocytokine (IC) consisting of human IL2 linked to each IgG heavy chain of the hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of IV hu14.18-IL2 IC in neuroblastoma and melanoma are underway, with activity already demonstrated in neuroblastoma. We have previously shown that intratumoral IC treatment (IT-IC) results in enhanced anti-tumor activity in mouse models. These studies were designed to determine the mechanisms involved in the enhanced activity and to provide justification for future clinical testing of localized IT administration of this and other immunocytokines. Methods: We characterize tumor growth, survival outcomes, histology and phenotype of tumor infiltrating lymphocytes (TILs) by flow cytometry of IT-hu.14.18-IL2 treatment of A/J mice bearing subcutaneous NXS2 neuroblastomas. Looking at these parameters, IT-IC treated mice are compared to IV-IC treated, IT PBS treated and untreated mice. Data: Mice receiving IT-IC show significant increases [by immunohistochemistry (IHC) and by flow cytometry] of NKp46+ Natural Killer (NK) cells and CD8a+ cytotoxic T cells in TIL populations compared to control tumor-bearing mice. Improved survival and inhibition of tumor growth are observed in mice receiving IT-IC vs. untreated mice or mice receiving IV-IC. Comparisons within treatment groups, or independent of treatment groups, show that the number of NK or CD8 T cells in the tumor correlates inversely with change in tumor size. Analyses by IHC and flow-cytometry show greater IC detection in tumor after IT-IC vs. IV-IC. Moreover, IT-IC results in improved IC retention in tumors and increased NKG2D effector receptors on intratumoral NKG2A/C/E cells and on CD8 T cells when compared to control mice or mice receiving IV-IC. The augmented NKG2D seen in TILs was not seen in spleen cells, supporting the localized nature of the intratumoral changes induced by this treatment. Conclusions: In this murine neuroblastoma model, enhanced antitumor effects of IT hu14.18-IL2 compared to IV hu14.18-IL2 are distinguished by increased activated TILs and IC retention, improved survival and inhibition of tumor growth. These observations suggest that localized administration of immunocytokines in human patients may show analogous advantages over IV administration. In accordance, we have designed a phase I/II clinical trial protocol investigating the maximum tolerated dose (MTD) and efficacy endpoints of intratumorally administered hu14.18-IL2 in stage III/IV melanoma patients with recurrent or refractory disease, and are working towards approval for its activation. Supported by R01-CA-32685-27. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1538. doi:1538-7445.AM2012-1538

Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population.

Correlation between CD4+CD25+Treg cells and CCR4 in nasopharyngeal carcinoma

OBJECTIVE CD4 CD25 T regulatory (Treg) cells are a population of T cells which suppress an overactive immune system. CCR4 is a chemokine receptor involved in the recruitment of lymphocytes. Nasopharyngeal carcinoma (NPC) is resistant to immunosurveillance, owing to the increased number of tumor-infiltrating Treg cells which are recruited to the tumor by CCR4. METHODS The percentage of CD4+CD25+ Treg cells and CCR4+ cells in tissue or peripheral blood (PB) lymphocytes of patients with untreated NPC or normal subjects was analysed by flow cytometry. RESULTS In both tissue and PB lymphocytes, the percentage of CD4+CD25+ Treg cells and CCR4+cells was significantly elevated in patients with NPC in comparison with that in the normal tissue of controls. Furthermore, in the patients with NPC, a higher percentage of CD4+CD25+ Treg cells was found in the tumor-infiltrating (TI) lymphocyte population than in the PB population. In the NPC patient group, a general trend towards an increased percentage of TI Treg cells was found in the patients with advanced stage NPC. The number of CD4+CD25+ Treg cells was positively related to the number of CCR4+ cells in the tumor and in the PB of the patients with NPC, while the number of CD4+CD25+ Treg cells was negatively related to the number of CD4+CD25 － T cells. CONCLUSION Immunosuppression was observed in NPC, especially at the tumor sites. CD4+CD25+ Treg cells may suppress CD4+CD25 － T cells. CCR4 may have an important role in the recruitment of CD4+CD25+ Treg cells to tumor sites, thus causing resistance to immunosurveillance.

Effect of matrix metalloproteinase-2 on the ability of human dendritic cells to prime inflammatory TH2 cells via an IL-12 and OX40L dependent pathway.

10622 Background: Antigens expressed by melanoma cells (MAA) and recognized by T lymphocytes have been used in various immunization strategies to treat cancer patients. However, the therapeutic efficacy of these approaches remains limited. Because MMP-2 (matrix metalloproteinase-2) activity is critical for melanoma progression, this protein represents a unique antigen for vaccine therapy. Methods: CD4+ T cells were stimulated with overlapping MMP-2 peptides. After 12 days, MMP-2-specific responses were determined by intracellular staining of cytokines. Specific T cells from positive samples were enriched and cloned using limiting dilution. Cytokine production was assessed by ELISA, intracellular staining, or cytometric bead array method. Results: We identified 11 novel CD4 MMP-2-derived epitopes, and showed that the corresponding T cells, detected in the CD45RO+/CD62L- memory fraction, displayed an inflammatory TH2 profile, i.e. mainly secreting TNF, IL-4 and IL-13 and expressing the TH2-specific transcription factor GATA-3. In contrast, CD4+ T cells that recognize other associated MAAs were typically TH1. Interestingly, naïve T cells primed with active MMP-2 gave rise to TH2 specific cells, while naïve T cells primed with peptides generated IFNg-secreting specific cells, consistent with a TH1 profile. Accordingly, we showed that the MMP-2 enzyme inhibited IL-12p35 production and induced OX40L expression by DCs. Both events were responsible for the TH2 skewing of MMP-2-specific T cells and other MAA-specific T cells. Furthermore, MMP-2 specific TH2 cells were found in 13 tumor-infiltrating lymphocyte populations out of the 31 melanoma patients tested. Conclusions: The protease activity of MMP-2 influences DC phenotype, more precisely IL-12 production and OX40L expression, which are responsible for the observed TH2 lineage commitment of MMP-2-specific T cells and other MAA-specific T cells. These findings open the way to therapeutic strategies skewing these already frequent and diverse MMP-2-specific responses towards a more efficient anti-tumor TH1 phenotype to treat melanoma patients. No significant financial relationships to disclose.

Abstract 466: Does body weight influence tumor-infiltrating B cells in murine pancreatic cancer

Abstract Background: We have developed a murine model of pancreatic cancer in congenitally obese (LepOb and LepDb) mice where tumors grow larger, metastasize, and decrease survival. Obese mice also had altered splenic T and B cells as well as downregulation of B cell and immunoglobulin genes and reduced numbers of B cells in the tumors. The question remained, however, whether alterations in tumor-infiltrating lymphocytes were a reflection of body weight or leptin metabolism. Therefore, we designed an experiment to determine whether diet-induced weight gain would alter systemic and tumor-infiltrating lymphocytes. Methods: Thirty lean C57 female mice were fed either a control 10% fat (n=10) or a 60% fat diet (n=20) starting at 6 weeks of age. At 11 weeks, all mice were inoculated with 2.5x105 PAN02 murine pancreatic cancer cells. After 6 weeks tumors that developed in 18 mice, the spleen (n=12), and the sera were harvested. Mice were categorized as Lean or Overweight if they weighed less or more than the mean final weight of the 60% fat animals (23.1g). Tumors in Lean (n=10) and Overweight (n=8) mice were sectioned and stained for B and T cells. Two observers blinded to animal weight graded cell density on a scale of 0-4 in 10 high-powered fields. Flow cytometry was performed on the spleen from Lean and Overweight mice for B, T, activated T and B (ACT), natural killer (NK) cells, and macrophages (MACRO). Serum leptin was measured. ANOVA and Student's t-test were applied. Results: Tumors were larger (1.33 vs. 0.54g, p=0.03) and leptin levels were higher (3.1 vs. 1.4ng/ml, p=0.05) in Overweight mice. Mean tumor-infiltrating lymphocyte (TIL) scores and spleen lymphocyte data are presented in the table. Conclusion: The data suggest that 1) both B and T cells are present in the pancreatic cancer microenvironment and 2) TIL and splenic lymphocyte populations do not differ between lean and overweight mice. We conclude that obesity, as opposed to overweight, is required to alter systemic and tumor microenvironment lymphocyte populations. B Cell TILT Cell TILB Cell (%) SpleenT Cell (%) SpleenACT (%) SpleenNK (%) SpleenMACRO (%) SpleenLean0.7±0.12.2±0.264±244±32.3±0.72.3±0.71.6±0.2Overweight0.7±0.12.0±0.258±139±51.5±0.31.4±0.41.4±0.3 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 466.