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Characterization and Comparison of ‘Standard’ and ‘Young’ Tumour‐Infiltrating Lymphocytes for Adoptive Cell Therapy at a Danish Translational Research Institution
Abstract
Adoptive cell therapy (ACT) with ex vivo expanded tumour-infiltrating lymphocytes (TILs) in combination with IL-2 is an effective treatment for metastatic melanoma. Modified protocols of cell expansion may allow the treatment of most enrolled patients and improve the efficacy of adoptively transferred cells. The aims of this study were to establish and validate the novel ‘Young TIL’ method at our institution and perform a head-to-head comparison of clinical-grade products generated with this protocol opposed to the conventional ‘Standard TIL’, which we are currently using in a pilot ACT trial for patients with melanoma. Our results confirm that ‘Young TILs’ display an earlier differentiation state, with higher CD27 and lower CD56 expression. In addition, CD8+ TILs expressing CD27 had longer telomeres compared with the CD27−. A recently described subset of NK cells, endowed with a high expression of CD56 (CD56bright), was detected for the first time in both types of cultures but at a higher frequency on Young TILs. Young and Standard TILs’ reactivity against autologous tumours was similar, with significant expression of TNF-α/IFN-γ/CD107a by CD8+ TILs detected in all cultures analysed. However, either slow expansion with high-dose IL-2 only or large numerical expansion with a rapid expansion protocol, which is required for current therapeutic protocols, significantly modified TIL phenotype by reducing the frequency of less differentiated, cancer-specific TILs. These studies further support the adoption of the Young TIL method in our current ACT trial and highlight the importance of continuous quality control of expansion protocols.
... Clinical trials employing autologous TIL adoptive transfer for the treatment of OvCa have progressed with optimization of TILs protocols yielding higher cell amounts after expansion, and implementation of pre-conditioning chemotherapy for improved clinical response [7,9,[11][12][13]. However, clinical benefits are sporadic and short-term stable disease seems to be the most frequent outcome [14]. ...
... The generation of TILs used in the ex vivo adoptive cell therapy co-cultures and in the in vivo animal experiment followed the adapted "young" TILs expansion protocol [12]. Briefly, ovarian cancer surgical specimens were cut into smaller fragments and put into culture in six-well G-Rex plates (Wilson Wolf Manufacturing, MN, USA) with TILs media [12,33]. ...
... The generation of TILs used in the ex vivo adoptive cell therapy co-cultures and in the in vivo animal experiment followed the adapted "young" TILs expansion protocol [12]. Briefly, ovarian cancer surgical specimens were cut into smaller fragments and put into culture in six-well G-Rex plates (Wilson Wolf Manufacturing, MN, USA) with TILs media [12,33]. After seven days, the culture content was passed through 70 µm and 40 µm strainers, and the cells were pelleted and stored at −140°C. ...
While the presence of tumor-infiltrating lymphocytes (TILs) associates with improved survival prognosis in ovarian cancer (OvCa) patients, TIL therapy benefit is limited. Here, we evaluated an oncolytic adenovirus coding for a human variant IL-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, as an immunotherapeutic strategy to enhance TIL responsiveness towards advanced stage OvCa tumors. Fragments of resected human OvCa tumors were processed into single-cell suspensions, and autologous TILs were expanded from said samples. OvCa tumor specimens were co-cultured with TILs plus vIL-2 virus, and cell killing was assessed in real time through cell impedance measurement. Combination therapy was further evaluated in vivo through a patient-derived xenograft (PDX) ovarian cancer murine model. The combination of vIL-2 virus plus TILs had best cancer cell killing ex vivo compared to TILs monotherapy. These results were supported by an in vivo experiment, where the best OvCa tumor control was obtained when vIL-2 virus was added to TIL therapy. Furthermore, the proposed therapy induced a highly cytotoxic phenotype demonstrated by increased granzyme B intensity in NK cells, CD4+ T, and CD8+ T cells in treated tumors. Our results demonstrate that Ad5/3-E2F-d24-vIL2 therapy consistently improved TILs therapy cytotoxicity in treated human OvCa tumors.
... TILs were propagated following the minimally cultured or "young TIL" method as previously described. 12 Initial expansion was successful when pooled cultures reached >50×10 6 total cells. The pooled TILs were either cryopreserved or directly propagated following the rapid expansion protocol (REP). ...
... Assessment of anti-tumor reactivity was performed in a flow cytometry based assay staining for CD107a, intracellular interferon-γ (IFNγ) and tumor necrosis factor (TNF) as previously described. 12 Briefly, in the presence of GolgiPlug (BD Biosciences, dilution 1:1000) and CD107a detection antibody, TILs were cocultured at 37°C for 5 hours with autologous TD at an effector to target ratio of 3:1, or alone (unstimulated sample). After coculture, cells were stained with surface antibodies and intracellular antibodies as described for phenotype analyses. ...
... 45 46 In patient 09, the myeloid lineage displayed upregulation in the relapse while in patient 13, a general downregulation of all immune cell subsets in the relapse was found (figure 5B,C). TCR sequencing showed an increase in the number of clonotypes from baseline tumor (12,818) to relapse sample (19,443) with a decrease in evenness (from 0.84 to 0.59) in patient 09, and decrease (from 19 143 to 11 313 clonotypes, with unchanged evenness) in patient 13. The baseline and the relapse tumors from each patient shared few clonotypes, with most of the clonotypes being unique to the particular tumor ( figure 5D). ...
Purpose
Despite impressive response rates following adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, improvement is needed to increase the efficacy and broaden the applicability of this treatment. We evaluated the use of vemurafenib, a small-molecule BRAF inhibitor with immunomodulatory properties, as priming before TIL harvest and adoptive T cell therapy in a phase I/II clinical trial.
Methods
12 patients were treated with vemurafenib for 7 days before tumor excision and during the following weeks until TIL infusion. TILs were grown from tumor fragments, expanded in vitro and reinfused to the patient preceded by a lymphodepleting chemotherapy regimen and followed by interleukin-2 infusion. Extensive immune monitoring, tumor profiling and T cell receptor sequencing were performed.
Results
No unexpected toxicity was observed, and treatment was well tolerated. Of 12 patients, 1 achieved a complete response, 8 achieved partial response and 3 achieved stable disease. A PR and the CR are ongoing for 23 and 43 months, respectively. In vitro anti-tumor reactivity was found in TILs from 10 patients, including all patients achieving objective response. Serum and tumor biomarker analyses indicate that baseline cytokine levels and the number of T cell clones may predict response to TIL therapy. Further, TCR sequencing suggested skewing of TCR repertoire during in vitro expansion, promoting certain low frequency clonotypes.
Conclusions
Priming with vemurafenib before infusion of TILs was safe and feasible, and induced objective clinical responses in this cohort of patients with checkpoint inhibitor-resistant metastatic melanoma. In this trial, vemurafenib treatment seemed to decrease attrition and could be considered to bridge the waiting time while TILs are prepared.
... Hamster HapT1 pancreatic tumor cell line was kindly provided by Dr Hernandez-Alcoceba (Pamplona, Spain) and maintained in RPMI supplemented with 10% fetal bovine serum (FBS), 1% L-Glutamine, and 1% Pen/Strep solution (Sigma-Aldrich, St. Louis, MO, USA) at 37 • C and 5% CO 2 . Ad5/3-Luc1 [17], Ad5/3-E2F-d24-hTNFa, Ad5/3-E2F-d24-, Ad5/3-E2F-d24-hTNFa-IRES-(also known as TILT-123) have been previously described [18]. ...
... Human ovarian cancer TILs were generated using an adaptation of the "young TILs" protocol [18,19]. In brief, ovarian cancer tumor samples were diced into small fragments and put into a 6-well plate with TIL medium (TM) containing RPMI 1640, supplemented with 20% FBS, 1% L-glutamine, 1% Pen/strep, 15 mM HEPES, 1 mM Na-pyruvate, 50 µm b-Mercaptoethanol (Sigma-Aldrich) and 3000 IU/mL recombinant human IL-2 (Peprotech, Stockholm, Sweden). ...
... Overall, our approach resulted in some degree of possible tumor reduction, which may derive directly from the combination of antitumor activity and changes in the immune cell composition. Indeed, we have previously found that the combination of TILT-123 and TILs in tumor-bearing immunocompetent Syrian hamster modulates the composition of immune cells [18] and induces the infiltrations of TILs [34,35] in the tumor microenvironment of this animal model. The increased infiltration of TILs (and possibly other subsets) coupled with the reduction of tumor volumes (even at early time points), is indicative of increased antitumor activity in this model. ...
Immunotherapy with tumor-infiltrating lymphocytes (TIL) or oncolytic adenoviruses, have shown promising results in cancer treatment, when used as separate therapies. When used in combination, the antitumor effect is synergistically potentiated due oncolytic adenovirus infection and its immune stimulating effects on T cells. Indeed, studies in hamsters have shown a 100% complete response rate when animals were treated with oncolytic adenovirus coding for TNFa and IL-2 (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) and TIL therapy. In humans, one caveat with oncolytic virus therapy is that intratumoral injection has been traditionally preferred over systemic administration, for achieving sufficient virus concentrations in tumors, especially when neutralizing antibodies emerge. We have previously shown that 5/3 chimeric oncolytic adenovirus can bind to human lymphocytes for avoidance of neutralization. In this study, we hypothesized that incubation of oncolytic adenovirus (TILT-123) with TILs prior to systemic injection would allow delivery of virus to tumors. This approach would deliver both components in one self-amplifying product. TILs would help deliver TILT-123, whose replication will recruit more TILs and increase their cytotoxicity. In vitro, TILT-123 was seen binding efficiently to lymphocytes, supporting the idea of dual administration. We show in vivo in different models that virus could be delivered to tumors with TILs as carriers.
... Young TILs were further expanded for 14 days in a small scale REP, as previously described. 19 Briefly, 100,000 TILs were mixed with 20 × 10 6 allogenic feeder cells, OKT3 antibody and master mix media containing 50% CM + 50% RM with 10% inactivated human AB serum. Young TILs generated in parallel from tumour specimens of metastatic melanoma (MM) were used as a comparison. ...
... At our centre, we have considerable experience in manufacturing of TILs from MM, 19,24 head and neck cancer 25 and primary renal cell carcinoma (pRCC). 26 In general, resected tumour specimens, from MM and pRCC, contains a well-defined area with tumour tissue which can easily be dissected from surrounding healthy tissues. ...
... 24 We investigated the phenotype of the TILs before and after the REP with a broad panel of surface markers and found an abundance of effector memory T cells. This is consistent with phenotype analyses performed in other solid tumours, e.g., MM. 19 The distribution of major T cell subpopulations, including CD4 + and CD8 + T cells, did not change dramatically during the REP. In general, we found a higher frequency of CD4 + T cells than CD8 + T cells in the expanded OC-TIL products, which was also true in the original TME. ...
Background:
Solid malignancies are frequently infiltrated with T cells. The success of adoptive cell transfer (ACT) with expanded tumour-infiltrating lymphocytes (TILs) in melanoma warrants its testing in other cancer types. In this preclinical study, we investigated whether clinical-grade TILs could be manufactured from ovarian cancer (OC) tumour specimens.
Methods:
Thirty-four tumour specimens were obtained from 33 individual patients with OC. TILs were analysed for phenotype, antigen specificity and functionality.
Results:
Minimally expanded TILs (Young TILs) were successfully established from all patients. Young TILs contained a high frequency of CD3+ cells with a variable CD4/CD8 ratio. TILs could be expanded to clinical numbers. Importantly, recognition of autologous tumour cells was demonstrated in TILs in >50% of the patients. We confirmed with mass spectrometry the presentation of multiple tumour antigens, including peptides derived from the cancer-testis antigen GAGE, which could be recognised by antigen-specific TILs. Antigen-specific TILs could be isolated and further expanded in vitro.
Conclusion:
These findings support the hypothesis that patients with OC can benefit from ACT with TILs and led to the initiation of a pilot clinical trial at our institution .
Trial registration:
clinicaltrials.gov: NCT02482090.
... After 3-6, pooled SKILs were cryopreserved or further expanded with Rapid Expansion Protocol (REP). 20 Tumor-infiltrating lymphocytes and tumor cell lines As previously described, 21 fresh tumor tissue was collected and transported to the laboratory for tumor-infiltrating lymphocytes (TILs) culture. In short, to obtain minimally expanded TILs, the tumor biopsies were divided into 1-3 Two intradermal injections of the EVX-01 peptides and one control (DSMO) injection were administered on day 0 (approximately 2 weeks from the last vaccination). ...
... 21 Autologous tumor cell lines (TCLs) were established via serial passage of adherent cells from the same tumor biopsies. 20 The TCLs were routinely tested negative for mycoplasma (AppliChem; Darmstadt, Germany), and the number of passages between collection and use in the described experiments was <10. ...
Background
Neoantigens can serve as targets for T cell-mediated antitumor immunity via personalized neopeptide vaccines. Interim data from our clinical study NCT03715985 showed that the personalized peptide-based neoantigen vaccine EVX-01, formulated in the liposomal adjuvant, CAF09b, was safe and able to elicit EVX-01-specific T cell responses in patients with metastatic melanoma. Here, we present results from the dose-escalation part of the study, evaluating the feasibility, safety, efficacy, and immunogenicity of EVX-01 in addition to anti-PD-1 therapy.
Methods
Patients with metastatic melanoma on anti-PD-1 therapy were treated in three cohorts with increasing vaccine dosages (twofold and fourfold). Tumor-derived neoantigens were selected by the AI platform PIONEER and used in personalized therapeutic cancer peptide vaccines EVX-01. Vaccines were administered at 2-week intervals for a total of three intraperitoneal and three intramuscular injections. The study’s primary endpoint was safety and tolerability. Additional endpoints were immunological responses, survival, and objective response rates.
Results
Compared with the base dose level previously reported, no new vaccine-related serious adverse events were observed during dose escalation of EVX-01 in combination with an anti-PD-1 agent given according to local guidelines. Two patients at the third dose level (fourfold dose) developed grade 3 toxicity, most likely related to pembrolizumab. Overall, 8 out of the 12 patients had objective clinical responses (6 partial response (PR) and 2 CR), with all 4 patients at the highest dose level having a CR (1 CR, 3 PR). EVX-01 induced peptide-specific CD4+ and/or CD8+T cell responses in all treated patients, with CD4+T cells as the dominating responses. The magnitude of immune responses measured by IFN-γ ELISpot assay correlated with individual peptide doses. A significant correlation between the PIONEER quality score and induced T cell immunogenicity was detected, while better CRs correlated with both the number of immunogenic EVX-01 peptides and the PIONEER quality score.
Conclusion
Immunization with EVX-01-CAF09b in addition to anti-PD-1 therapy was shown to be safe and well tolerated and elicit vaccine neoantigen-specific CD4+and CD8+ T cell responses at all dose levels. In addition, objective tumor responses were observed in 67% of patients. The results encourage further assessment of the antitumor efficacy of EVX-01 in combination with anti-PD-1 therapy.
... For the in vivo humanization of immunodeficient mouse, blood from the same patient was collected and PBMCs isolated with Lymphoprep gradient density separation. Due to limited cell availability, patient PBMCs were expanded for 14 days using the "young" TILs protocol [24,25]. Expanded PBMCs were stored at −140°C, and 24 h prior to animal injection PBMCs were rested in 6-well G-Rex plates with TILs media [22]. ...
... Of note, differences on proportion of detected CD4 + T and CD8+ T cells in the ex vivo and in vivo studies can be partially explained by the PBMCs expansion protocol utilised in the latter case prior mice injection. Addition of exogenous wt IL-2 cytokine to cell cultures can condition T cells response more promptly to further cytokine exposure [25,42]. While in the OvCa ex vivo co-cultures, no cytokine stimulation was done prior the treatment with the combination therapy. ...
Despite good results in the treatment of hematological malignancies, Natural killer (NK) cells have shown limited effectiveness in solid tumors, such as ovarian cancer (OvCa). Here, we assessed the potential of an oncolytic adenovirus expressing a variant interleukin-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, to enhance NK cell therapy efficacy in human OvCa ex vivo. Human OvCa surgical specimens were processed into single-cell suspensions and NK cells were expanded from healthy blood donors. OvCa sample digests were co-cultured ex vivo with NK cells and vIL-2 virus and cancer cell killing potential assessed in real time through cell impedance measurement. Proposed therapeutic combination was evaluated in vivo with an OvCa patient-derived xenograft (PDX) in mice. Addition of vIL-2 virus significantly enhanced NK cell therapy killing potential in treated OvCa co-cultures. Similarly, vIL-2 virus in combination with NK cell therapy promoted the best in vivo OvCa tumor control. Mechanistically, vIL-2 virus induced higher percentages of granzyme B in NK cells, and CD8+ T cells, while T regulatory cell proportions remained comparable to NK cell monotherapy in vivo. Ad5/3-E2F-d24-vIL2 virus treatment represents a promising strategy to boost adoptive NK cell therapeutic effect in human OvCa.
... PBMCs for the feeder cells were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare, Buckingham, UK). The complete pre-REP TIL and REP TIL protocols have been previously described in detail [27] . ...
... Some studies have shown that the infiltration of T-cells is associated with good prognosis in RCC [7][8][9] , whereas others have reported contradictory results in which T-cell infiltration has been associated with poor prognosis [4][5][6]76] . In addition, although the REP TILs are mostly used as the final infusion product in the adoptive TIL therapies, there are only a few studies that have explored the differences in the phenotype between the original, minimally cultured pre-REP, and REP TILs [27,77,78] . Therefore, we performed in-depth analyses of the pre-REP TILs and REP TILs that were expanded using a clinical expansion protocol from treatment-naïve RCC patients. ...
The successful use of expanded tumor-infiltrating lymphocytes (TIL) in adoptive TIL therapies has been reported, but the effects of the TIL expansion, immunophenotype, function, and T cell receptor (TCR) repertoire of the infused products relative to the tumor microenvironment (TME) are not well understood. In this study, we analyzed the tumor samples (n = 58) from treatment-naïve patients with renal cell carcinoma (RCC), “pre-rapidly expanded” TILs (pre-REP TIL, n = 15) and “rapidly expanded” TILs (REP TIL, n = 25) according to a clinical-grade TIL production protocol, with single-cell RNA (scRNA)+TCRαβ-seq (TCRαβ sequencing), TCRβ-sequencing (TCRβ-seq), and flow cytometry. REP TILs encompassed a greater abundance of CD4+ than CD8+ T cells, with increased LAG-3 and low PD-1 expressions in both CD4+ and CD8+ T cell compartments compared with the pre-REP TIL and tumor T cells. The REP protocol preferentially expanded small clones of the CD4+ phenotype (CD4, IL7R, KLRB1) in the TME, indicating that the largest exhausted T cell clones in the tumor do not expand during the expansion protocol. In addition, by generating a catalog of RCC-associated TCR motifs from >1,000 scRNA+TCRαβ-seq and TCRβ-seq RCC, healthy and other cancer sample cohorts, we quantified the RCC-associated TCRs from the expansion protocol. Unlike the low-remaining amount of anti-viral TCRs throughout the expansion, the quantity of the RCC-associated TCRs was high in the tumors and pre-REP TILs but decreased in the REP TILs. Our results provide an in-depth understanding of the origin, phenotype, and TCR specificity of RCC TIL products, paving the way for a more rationalized production of TILs.
Significance
TILs are a heterogenous group of immune cells that recognize and attack the tumor, thus are utilized in various clinical trials. In our study, we explored the TILs in patients with kidney cancer by expanding the TILs using a clinical-grade protocol, as well as observed their characteristics and ability to recognize the tumor using in-depth experimental and computational tools.
... Autologous in vitro cultured tumor cell lines (TCLs) were established via serial passage of adherent cells from tumor fragments deriving from the same lesions from which TILs were cultured. 81 TCLs were authenticated via in vitro growth patterns, morphology (light microscopy), and lineage antigen expression (PCR) where necessary. Mycoplasma testing (cat. ...
... The full REP protocol has previously been described in detail. 26,81 Phenotyping All flow cytometry samples were acquired on a NovoCyte Quanteon (ACEA Biosciences, San Diego, CA) or FACSCanto II (BD Biosciences, Franklin Lakes, NJ). See supplemental methods for detailed description of flow cytometry antibodies utilized. ...
Adoptive T-cell therapy with expanded tumour-infiltrating lymphocytes (TIL-based ACT) can induce durable responses in cancer patients from multiple histologies, with response rates of up to 50%. Antibodies blocking the engagement of the inhibitory receptor programmed cell death protein 1 (PD-1) have been successful across a variety of cancer diagnoses. We hypothesized that these approaches could be combined by using CRISPR-Cas9 gene editing to knockout PD-1 in TILs from metastatic melanoma, head-and-neck, thyroid, and colorectal cancer. Non-viral/plasmid-based PD-1 knockout was carried out immediately prior to the traditional 14-day TIL-based ACT rapid expansion protocol. A median 87.53% reduction in cell surface PD-1 expression was observed post-expansion and confirmed at the genomic level. No off-target editing was detected and PD-1 knockout had no effect on final fold expansion. Edited cells exhibited few phenotypic differences and matched control functionality. Pre-clinical-scale results were confirmed at a clinical-scale by generating a PD-1 deficient TIL product using the good manufacturing practice facilities, equipment, procedures, and starting material used for standard patient treatment. Our results demonstrate that simple, non-viral CRISPR-Cas9 methods can be feasibly adopted into a TIL-based ACT protocol to produce treatment products deficient in molecules such as PD-1, without any evident negative effects.
... TILs were isolated and expanded in vitro from tumor fragments with a two-step process in previous studies (19)(20)(21)(22)(23). Young TILs were obtained after minimal-culture, whereas rapid-expansion protocol (REP) TILs were obtained after massive expansion (25). All TILs not otherwise enriched or purified were named "bulk" TILs. ...
... Autologous short-term in vitro cultured TCLs were established via serial passage of adherent cells from tumor fragments derived from the same tumor lesion from which the TILs were generated, as described previously (25). All TCLs were generated internally and authenticated via in vitro patterns of growth, morphology (light microscopy), and when in doubt, expression of lineage antigens by PCR. ...
Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8⁺ and CD4⁺ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8⁺ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4⁺ and CD8⁺ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories.
... During REP, T-cells were activated via anti-CD3 binding and co-cultured with irradiated feeder cells (either autologous or allogeneic) (208,209). Challenges with this process include poor expansion during the pre-REP phase if no T-cells are present, lack of tumor-reactive T-cells to undergo rapid expansion, and reduction in T-cell health associated with rapid expansion and prolonged ex vivo culture (210). Therefore, methods to augment T-cell infiltration into the tumor microenvironment prior to T-cell isolation could therefore have positive effects on improving TIL manufacturing success. ...
... It has been well characterized that rapid expansion of TILs with IL-2 reduces T-cell surface CD27 and CD28 expression and results in an increased fraction of terminally differentiated T-cells in the final product (223,224). In more recent years, cells expanded via the young TIL method has mitigated the adverse effects of rapid expansion, with young TIL products demonstrating longer telomeres and higher expression of CD27 and CD28 (210,225). Pharmacologically achieving similar results with PI3Ki, however, may be less technically challenging. Already Dwyer et al. has shown that mesoCART cultured with eganelisib demonstrate reduced T-cell differentiation and increased CD27/CD28+ surface expression (113). ...
PI3K-δ and PI3K-γ are critical regulators of T-cell differentiation, senescence, and metabolism. PI3K-δ and PI3K-γ signaling can contribute to T-cell inhibition via intrinsic mechanisms and regulation of suppressor cell populations, including regulatory T-cells and myeloid derived suppressor cells in the tumor. We examine an exciting new role for using selective inhibitors of the PI3K δ- and γ-isoforms as modulators of T-cell phenotype and function in immunotherapy. Herein we review the current literature on the implications of PI3K-δ and -γ inhibition in T-cell biology, discuss existing challenges in adoptive T-cell therapies and checkpoint blockade inhibitors, and highlight ongoing efforts and future directions to incorporate PI3K-δ and PI3K-γ as synergistic T-cell modulators in immunotherapy.
... yTILs were generated by cutting tumour tissue obtained directly from surgery into 1-2 mm 2 pieces that were placed in separate wells in a 24-well plate (Sarstedt, Newton, NC, USA) with 2 mL of culture medium (90% RPMI 1640 (Invitrogen, Waltham, MA, USA), 10% heat-inactivated human AB serum (HS, Sigma-Aldrich), and 6000 IU/mL recombinant human IL-2 (Peprotech) and gentamicin (Invitrogen). Young TIL (yTIL) cultures were obtained by pooling TILs from each fragment as previously described [29][30][31], before being cryopreserved. To generate REP-TILs, yTILs were expanded using a standard small-scale REP [31]. ...
... Young TIL (yTIL) cultures were obtained by pooling TILs from each fragment as previously described [29][30][31], before being cryopreserved. To generate REP-TILs, yTILs were expanded using a standard small-scale REP [31]. In short, irradiated (40 Gy) allogeneic feeder cells (5 × 10 6 ), 30 ng/mL anti-CD3 antibody (Miltenyi, Bergisch Gladbach, Germany; OKT3), 5 mL culture medium, 5 mL REP medium (AIM-V, Invitrogen) supplemented with 10% HS, and 6000 IU/mL IL-2 and yTILs (5 × 10 4 ) were mixed in a 25 cm 2 tissue culture flask. ...
Traditionally, immune evasion and immunotherapy have been studied in cancers with a high mutational load such as melanoma or lung cancer. In contrast, small intestinal neuroendocrine tumours (SINETs) present a low frequency of somatic mutations and are described as genetically stable tumours, rendering immunotherapies largely unchartered waters for SINET patients. SINETs frequently metastasise to the regional lymph nodes and liver at the time of diagnosis, and no curative treatments are currently available for patients with disseminated disease. Here, we characterised the immune landscape of SINET and demonstrated that tumour-infiltrating lymphocytes (TILs) can be expanded and activated during autologous tumour challenge. The composition of lymphocyte subsets was determined by immunophenotyping of the SINET microenvironment in one hepatic and six lymph node metastases. TILs from these metastases were successfully grown out, enabling immunophenotyping and assessment of PD-1 expression. Expansion of the TILs and exposure to autologous tumour cells in vitro resulted in increased T lymphocyte degranulation. This study provides insights into the largely unknown SINET immune landscape and reveals the anti-tumour reactivity of TILs, which might merit adoptive T cell transfer as a feasible treatment option for patients with SINET.
... "Young TIL method" [11,13]. In the following the term, "Young TILs" refers to TILs following this initial expansion. ...
... Young TIL and REP TIL populations mainly comprised T effector memory cells (T EM ), defined as being CD3 + CD45RO + and CCR7 − (or CCR7-low), which corresponds to previous findings [13,19]. A smaller fraction of TILs were more differentiated T-effector cells (CD3 + CD45RO − CCR7 − ), while very few were T central memory cells (CD3 + CD45RO + CCR7 + ) (Supplementary files, figure S2). ...
Tumor-specific tumor-infiltrating lymphocytes (TILs) can be in vitro expanded and have the ability to induce complete and durable tumor regression in some patients with melanoma following adoptive cell therapy (ACT). In this preclinical study, we investigated the feasibility of expanding TIL from sarcomas, as well as performing functional in vitro analyses on these. TILs were expanded in vitro by the use of IL2 stimulation with or without the addition of 4-1BB and CD3 antibodies. Phenotypical and functional analyses were mainly performed by flow cytometry. TILs were expanded from 25 of 28 (89%) tumor samples from patients with 9 different sarcoma subtypes. TILs were predominantly αβ T-cells of effector memory subtype with CD4+ dominance. In particular, CD8+ TIL highly expressed LAG3 and to a lesser degree PD-1 and BTLA. In total, 10 of 20 TIL cultures demonstrated in vitro recognition of autologous tumor. In some cases, the fraction of tumor-reactive T cells was more than 20%. 4-1BB stimulation augmented expansion kinetics and favored CD8+ occurrence. In conclusion, TIL expansion from sarcoma is feasible and expanded TILs highly express LAG3 and comprise multifunctional tumor-reactive T-cells.
... Median days in culture were 29 (range days) with a median number of 2.87 × 10 6 (range [0-22.5 × 10 6 ]) TILs recovered per fragment. Next, we evaluated whether TILs could be expanded to clinically relevant numbers using a two-week rapid expansion protocol (REP) 29 . If TIL cultures could not be established, TILs previously established in the clinical studies (Trial Number NCT02482090 and NCT03287674) were used. ...
... In order to limit differences in culture time of initial TILs under different conditions from the same patient, all initial TILs from one patient were harvested within three days. TILs were further expanded in a small-scale version of the 14-day rapid expansion protocol, as previously described 29 . Here, freshly generated TILs or cryopreserved TILs rested in CM for two days were used. ...
Adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) can induce durable complete tumor regression in patients with advanced melanoma. Efforts are currently underway to expand this treatment modality to other cancer types. In the microenvironment of ovarian cancer, the engagement of co-inhibitory immune checkpoint molecules such as CTLA-4 can lead to the inactivation of TILs. Thus, approaches that directly manipulate co-inhibitory pathways within the tumor microenvironment might improve the expansion of tumor-reactive TILs. The initial expansion of TILs for ACT from tumor fragments provides a window of opportunity to manipulate an intact tumor microenvironment and improve CD8⁺ T-cell output and TIL tumor reactivity. To exploit this, we used a CTLA-4-blocking antibody, added during the initial TIL culture, and found that the blockade of CTLA-4 favored the propagation of CD8⁺ TILs from ovarian tumor fragments. Interestingly, adding the CTLA-4 blocking antibody in the initial phase of the TIL culture resulted in more potent anti-tumor TILs in comparison to standard TIL cultures. This phenotype was preserved during the rapid expansion phase. Thus, targeting CTLA-4 within the intact tumor microenvironment of tumor fragments enriches tumor-reactive TILs and may improve clinical outcome of TIL-based ACT in ovarian cancer.
... The manufacturing procedure was performed in cleanrooms according to GMP EU Annex 1. TILs were expanded in vitro according to the "Young TIL" method. 25,26 Within hours from surgical removal of the tumor, the tumor specimen was cut into small fragments and placed in individual wells with culture medium containing RPMI 1640 (Thermo Fisher Scientific), 10% human serum (Sigma-Aldrich), IL-2 6000 IU/ mL (Novartis), and Penicillin, Streptomycin and Fungizone (Thermo Fisher Scientific). During expansion, TIL cultures were pooled together to generate a bulk TIL product. ...
LTX-315 is an oncolytic peptide that elicits both local and systemic immune responses upon intratumoral injection. In the present pilot trial, we treated patients with metastatic soft tissue sarcoma with the combination of LTX-315 and adoptive T-cell therapy using in vitro expanded tumor-infiltrating lymphocytes. Six heavily pretreated patients were included in the trial and treated with LTX-315 of which four patients proceeded to adoptive T-cell therapy. Overall, the treatment was considered safe with only expected and manageable toxicity. The best overall clinical response was stable disease for 208 days, and in this patient, we detected tumor-reactive T cells in the blood that lasted until disease progression. In three patients T-cell reactivity against in silico predicted neoantigens was demonstrated. Additionally, de novo T-cell clones were generated and expanded in the blood following LTX-315 injections. In conclusion, this pilot study provides proof that it is feasible to combine LTX-315 and adoptive T-cell therapy, and that this treatment can induce systemic immune responses that resulted in stabilization of the disease in sarcoma patients with otherwise progressive disease. Further optimization of the treatment protocol is warranted to increase clinical activity. ClinicalTrials.gov Identifier: NCT03725605
... Based on this research, young TIL therapy was developed in 2010, which is to gather lymphocytes from multiple tumor sites to obtain the number of cells required for rapid expansion. In this way, young TIL therapy shortened the culturing time, so that TILs with high CD27 and CD28 expression and long telomeres were enriched (46). ...
Cancer cellular immunotherapy has made inspiring therapeutic effects in clinical practices, which brings new hope for the cure of cervical cancer. CD8+T cells are the effective cytotoxic effector cells against cancer in antitumor immunity, and T cells-based immunotherapy plays a crucial role in cellular immunotherapy. Tumor infiltrated Lymphocytes (TIL), the natural T cells, is approved for cervical cancer immunotherapy, and Engineered T cells therapy also has impressive progress. T cells with natural or engineered tumor antigen binding sites (CAR-T, TCR-T) are expanded in vitro, and re-infused back into the patients to eradicate tumor cells. This review summarizes the preclinical research and clinical applications of T cell-based immunotherapy for cervical cancer, and the challenges for cervical cancer immunotherapy.
... Naïve T cells proved to be the most resilient to DNA toxicity and most susceptible to SB transfection. Naïve T cells are the least differentiated and therefore might be the most efficacious T cells in vivo (Donia et al., 2012;Dudley et al., 2010;Gattinoni et al., 2012). In order to avoid in vitro T cell activation, the transfected T cells could be transferred back to the patient directly after transfection. ...
Sleeping Beauty (SB) Transposon-basierte Vektoren werden als Alternative zu viralen Vektoren für T-Zell-Gentherapie erforscht und ermöglichen eine schnelle und kostengünstige Genmanipulation von T-Zellen. Die Verwendung von Transposon-Vektoren erfordert jedoch die DNA-Elektroporation von T-Zellen, die sich schädlich auf T-Zellen auswirkt. DNA-elektroporierte T-Zellen weisen eine verringerte Lebensfähigkeit und eine verzögerte Aktivierung nach Stimulation des T-Zell-Rezeptors (TCR) auf. Um die Nachteile der Transposon-basierten T-Zell-Genmanipulation zu überwinden, haben wir neuartige SB-Vektoren entwickelt. Durch die Kombination von SB Transposon-basierten Minicircle-Vektoren mit SB100X Transposase-mRNA konnten T-Zellen effizient genmodifiziert werden. Unser Ansatz reduzierte die T-Zell-Mortalität und steigerte gleichzeitig die Transfektionseffizienz. Mit diesen neuartigen Vektoren wurde die stabile Expression verschiedener TCRs und CARs in über 50% der eingesetzten T-Zellen erreicht. Gentechnisch manipulierte T-Zellen konnten Antigen-spezifisch stimuliert werden und zeigten effiziente Zytokin-Sekretion und Tumorzell-Lyse. Weiterhin haben wir miRNAs entwickelt, die die Expression der endogenen TCR-Ketten unterdrücken. Der Einbau dieser miRNAs in die TCR-Expressionskassette erhöhte die Oberflächenexpression des therapeutischen TCRs, verringerte die Fehlpaarung mit endogenen TCR-Ketten und erhöhte die T-Zell-Funktionalität. Ein direkter Vergleich von SB- und Virus-modifizierten T-Zellen zeigte sowohl in vitro als auch in vivo eine vergleichbare Wirksamkeit der modifizierten T-Zellen hinsichtlich Zytokin-Sekretion, Tumorzell-Lyse und Tumorkontrolle. In dieser Arbeit konnte gezeigt werden, dass SB Minicircle-Vektoren die Herstellung von genetisch modifizierten T-Zellen ermöglichen und diese Tumor-spezifische Wirksamkeit aufweisen. Dieser Ansatz könnte die Herstellung therapeutischer T-Zellen für die personalisierte T-Zell-Gentherapie vereinfachen und beschleunigen.
... In order to simplify the generation process of TILs and potentially improve the properties of TIL cultures, a modified TIL production protocol which uses "unselected" TILs to start the REP protocol was tested [32,33]. These unselected bulk TILs, called "young" TILs, have no pre-selection step for tumor reactivity based on IFN-γ ( Figure 1). ...
Over the past decade, immunotherapy, especially cell-based immunotherapy, has provided new strategies for cancer therapy. Recent clinical studies demonstrated that adopting cell transfer of tumor-infiltrating lymphocytes (TILs) for advanced solid tumors showed good efficacy. TIL therapy is a type of cell-based immunotherapy using the patient’s own immune cells from the microenvironment of the solid tumor to kill tumor cells. In this review, we provide a comprehensive summary of the current strategies and challenges in TIL isolation and generation. Moreover, the current clinical experience of TIL therapy is summarized and discussed, with an emphasis on lymphodepletion regimen, the use of interleukin-2, and related toxicity. Furthermore, we highlight the clinical trials where TIL therapy is used independently and in combination with other types of therapy for solid cancers. Finally, the limitations, future potential, and directions of TIL therapy for solid tumor treatment are also discussed.
... TILs were isolated and expanded in vitro from tumor fragments using a two-step process previously described (14). TILs were initially isolated and minimally expanded from tumor fragments in standard RPMI1640 plus GlutaMAX and 25-mmol/L HEPES (72400-021, Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated human AB serum (H4522-100ML, Sigma-Aldrich/Merck KGaA, Darmstadt, Germany), 100 U/mL penicillin and 100-mg/mL streptomycin (15140122, Gibco, Thermo Fisher Scientific, Waltham, MA), 1.25-mg/mL Fungizone (Bristol Myers Squibb, New York, NY) and 6,000 IU/mL of rhIL-2 (004184, Novartis, Basel, Switzerland) and subsequently were massively expanded according to the Rapid Expansion Protocol (REP). ...
Responses to immunotherapy can be very durable but acquired resistance leading to tumor progression often occurs. We investigated a patient with melanoma resistant to anti–PD-1 who participated in the CA224-020 clinical trial (NCT01968109) and had further progression after an initial objective response to anti–PD-1 plus anti–LAG-3. We found consecutive acquisition of beta-2 microglobulin (B2M) loss and impaired Janus Kinase 1 (JAK1) signaling that co-existed in progressing tumor cells. Functional analyses revealed a pan T cell immune escape phenotype, where distinct alterations mediated independent immune resistance to tumor killing by autologous CD8+ tumor-infiltrating lymphocytes (TILs) (B2M loss) and CD4+ TILs (impaired JAK1 signaling). These findings shed light on the complexity of acquired resistance to immunotherapy in the post anti–PD-1 setting, indicating that co-existing altered pathways can lead to pan T cell immune escape.
... In addition, YTILs have similar or superior clinical efficacy and enable treatment of most enrolled patients. As a result, multiple institutions use minimally cultured YTILs for expansion without further selection in current clinical settings [16,17]. ...
Introduction:
Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a highly personalized type of cancer immunotherapy. TIL-based ACT exploits naturally occurring TILs, derived from the patients' tumor. This treatment has shown consistent clinical responses in melanoma, and recent results point toward a potential use in multiple cancer diagnoses. However, several limitations have restricted the clinical development and adaptation of TIL-based ACT.
Areas covered:
In this review, we present the principles of TIL-based ACT and discuss the most significant limitations for therapeutic efficacy and its widespread application. The topics of therapeutic resistance (both innate and acquired), treatment-related toxicity, and the novel research topic of metabolic barriers in the tumor microenvironment (TME) are covered.
Expert opinion:
There are many ongoing areas of research focusing on improving clinical efficacy and optimizing TIL-based ACT. Many strategies have shown great potential, particularly strategies advancing TIL efficacy (such as increasing and harnessing ex vivo the sub-population of tumor-reactive TILs) and manufacturing processes. Novel approaches can help overcome current limitations and potentially result in TIL-based ACT entering the mainstream of cancer therapy across tumor types.
... Young SKILs from samples derived from IDO peptide, PD-L1 peptide and the mixture injection sites were further expanded in a small-scale version of the 14-d rapid expansion protocol as previously described 51 . ...
Anti-programmed death (PD)-1 (aPD1) therapy is an effective treatment for metastatic melanoma (MM); however, over 50% of patients progress due to resistance. We tested a first-in-class immune-modulatory vaccine (IO102/IO103) against indoleamine 2,3-dioxygenase (IDO) and PD ligand 1 (PD-L1), targeting immunosuppressive cells and tumor cells expressing IDO and/or PD-L1 (IDO/PD-L1), combined with nivolumab. Thirty aPD1 therapy-naive patients with MM were treated in a phase 1/2 study (https://clinicaltrials.gov/, NCT03047928). The primary endpoint was feasibility and safety; the systemic toxicity profile was comparable to that of nivolumab monotherapy. Secondary endpoints were efficacy and immunogenicity; an objective response rate (ORR) of 80% (confidence interval (CI), 62.7–90.5%) was reached, with 43% (CI, 27.4–60.8%) complete responses. After a median follow-up of 22.9 months, the median progression-free survival (PFS) was 26 months (CI, 15.4–69 months). Median overall survival (OS) was not reached. Vaccine-specific responses assessed in vitro were detected in the blood of >93% of patients during vaccination. Vaccine-reactive T cells comprised CD4+ and CD8+ T cells with activity against IDO- and PD-L1-expressing cancer and immune cells. T cell influx of peripherally expanded T cells into tumor sites was observed in responding patients, and general enrichment of IDO- and PD-L1-specific clones after treatment was documented. These clinical efficacy and favorable safety data support further validation in a larger randomized trial to confirm the clinical potential of this immunomodulating approach. A first-in-class immune-modulating IDO/PD-L1-targeting vaccine combined with nivolumab in a phase 1/2 trial achieved very promising clinical activity and long-lasting immune responses in patients with advanced melanoma.
... By shortening the culture time, TILs with high expression of CD27 and CD28 and longer telomeres are enriched. 54 This faster expansion method also brings significant clinical benefit, reducing the number of patients who do not meet the treatment conditions due to rapid disease progression and clinical decline during TIL production. Weber and the Moffitt Cancer Center enhanced TIL production by targeting 4-1BB, which is a costimulatory molecule on activated T cells, involved in T cell proliferation and antigen-specific cytolytic activity. ...
Cervical cancer is a top lethal cancer for women worldwide. Although screening and vaccination programs are available in many countries, resulting in the decline of new cases, this is not true for developing countries where there are many new cases and related deaths. Cancer immunotherapy through adaptive cell therapy (ACT) has been applied in clinics, but now much attention is focused on autogenic tumor-infiltrating lymphocyte (TIL)-based therapy, which has shown more specificity and better ability to inhibit tumor growth. Data from melanoma and cervical cancers confirm that tumor-specific T cells in TILs can be expanded for more specific and effective ACT. Moreover, TILs are derived from individual patients and are ready to home back to kill tumor cells after patient infusion, aligning well with personalized and precision medicine. In addition to therapy, TIL cell types and numbers are good indicators of host immune response to the tumor, and thus they have significant values in prognosis. Because of the special relationship with human papillomavirus (HPV) infection, cervical cancer has some specialties in TIL-based prognosis and therapy. In this review, we summarize the recent advances in the prognostic significance of TILs and TIL-based therapy for cervical cancer and discuss related perspectives.
... The resulting consequences are detrimental in the context of TIL therapy, and include cellular senescence, loss of function, and reduced expression of CD27/CD28 costimulatory proteins important for T cell activation [48,50]. Young TIL trials have repeatedly shown that this method results in longer persistence of TIL in the patient and better clinical outcomes than the aforementioned standard method, and these benefits have been attributed to curtailing telomer shortening [45][46][47][51][52][53]. The overwhelming evidence of the inverse relation of culture time and TIL efficacy has become influential in the clinical use of the therapy. ...
The past decades of cancer immunotherapy research have provided profound evidence that the immune system is capable of inducing durable tumor regression. Although many commercialized anti-cancer immunotherapies are available to patients, these treatment options only scrape the surface of the potential immune-related treatment possibilities for cancer. Additionally, many individuals are ineligible for established immunotherapies due to their cancer type. The adoptive cell transfer of autologous tumor-infiltrating lymphocytes has been used in humans for over 30 years to treat metastatic melanoma, and continued modifications are making it increasingly more effective against other types of cancer. This comprehensive review outlines this therapy from its infancy through to the present day, bringing to light modifications and optimizations to the traditional workflow, as well as highlighting the influence of new methods and technologies.
... To this end, Dr. Rosenberg and others developed a "Young TIL" approach that rapidly expands TIL for administration without in vitro selection for tumor reactivity [8], which markedly improves the timeliness of TIL production as well as its survival and efficacy in vivo (Fig. 1). Later on, "Young TIL" has shown comparable clinical outcome with traditional TIL in melanoma patients [9,10]. Besides, the effects of different T cell functional modulators and cytokines on TIL manufacture have also been examined. ...
Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cellular therapy by harvesting infiltrated lymphocytes from tumors, culturing and amplifying them in vitro and then infusing back to treat patients. Its diverse TCR clonality, superior tumor-homing ability, and low off-target toxicity endow TIL therapy unique advantages in treating solid tumors compared with other adoptive cellular therapies. Nevertheless, the successful application of TIL therapy currently is still limited to several types of tumors. Herein in this review, we summarize the fundamental work in the field of TIL therapy and the current landscape and advances of TIL clinical trials worldwide. Moreover, the limitations of the current TIL regimen have been discussed and the opportunities and challenges in the development of next-generation TIL are highlighted. Finally, the future directions of TIL therapy towards a broader clinical application have been proposed.
... In addition to the manufacturing obstacles derived from TIL therapy, -it is an extremely personalized therapy which required specialized personal to manage pre-and REP stages as well as highly controlled conditions that guarantee their clinical use-, TIL expansion is a time-consuming protocol that in some cases is not viable due to the rapid clinical deterioration of some melanoma patients from which those TIL were initially isolated. Due to the limitations of the selected TIL method, a modified TIL production protocol was developed and tested in clinical trials both at the Surgery Branch, NCI and the Sheba Medical Center, Israel (47)(48)(49). With this modified method, named the "young TIL" protocol, all TIL micro-cultures generated from individual fragments are pooled together as one single bulk TIL culture, eliminating the tumor-reactivity assay (50). ...
Immunotherapy has emerged as an effective and life-changing approach for several types of cancers, both liquid and solid tumors. In combination with traditional treatments such as radiotherapy and/or chemotherapy, immune checkpoints inhibitors have improved prognosis and overall survival of patients with advanced melanoma and many other cancers. Among adoptive cell therapies (ACT), while chimeric antigen receptor T cell therapies have demonstrated remarkable efficacy in some hematologic malignancies, such as B cell leukemias, their success in solid tumors remains scarce due to the characteristics of the tumor microenvironment. On the other hand, ACT using tumor-infiltrating lymphocytes (TILs) is arguably the most effective treatment for metastatic melanoma patients, but even if their isolation has been achieved in epithelial tumors, their success beyond melanoma remains limited. Here, we review several aspects impacting TIL- and gene-modified “synthetic” TIL-based therapies and discuss future challenges that must be addressed with these approaches.
... To define the bulk antitumor reactivity in a given sample, only the highest value obtained from Y TILs or REP TILs tested against TCLs, TCLs + IFNγ or FTDs was reported. For the SAR cohort only, effector-target pairs (only TILs vs TCLs) were pre-tested with co-culture followed by IFNγ ELISPOT, as described previously [46,47]. Further testing with flow cytometry, as described in supplementary methods, was conducted only in those samples with suspected or confirmed ELISPOT reactivity. ...
Simple Summary
Human tumors are often infiltrated by T cells; however, it remains unclear what proportion of T cells infiltrating tumors are bystander and non-tumor specific. We have investigated qualitative characteristics of these tumor-infiltrating lymphocytes (TILs) based on their gene-expression in the tumor-microenvironment or on their response to autologous tumor cells in vitro. Despite a considerable inter-sample variability, we found the overall proportion of bystander (non-tumor reactive) TILs to be remarkably high. Importantly, we observed a higher proportion of bystander TILs in non-melanoma tumors, compared to melanoma. This study suggests that immunotherapeutic strategies, especially when applied to non-melanoma tumors, should be tailored to reinvigorate the small proportion of tumor-reactive T cells infiltrating the tumor-microenvironment.
Abstract
Background: Human intratumoral T cell infiltrates can be defined by quantitative or qualitative features, such as their ability to recognize autologous tumor antigens. In this study, we reproduced the tumor-T cell interactions of individual patients to determine and compared the qualitative characteristics of intratumoral T cell infiltrates across multiple tumor types. Methods: We employed 187 pairs of unselected tumor-infiltrating lymphocytes (TILs) and autologous tumor cells from patients with melanoma, renal-, ovarian-cancer or sarcoma, and single-cell RNA sequencing data from a pooled cohort of 93 patients with melanoma or epithelial cancers. Measures of TIL quality including the proportion of tumor-reactive CD8⁺ and CD4⁺ TILs, and TIL response polyfunctionality were determined. Results: Tumor-specific CD8⁺ and CD4⁺ TIL responses were detected in over half of the patients in vitro, and greater CD8⁺ TIL responses were observed in melanoma, regardless of previous anti-PD-1 treatment, compared to renal cancer, ovarian cancer and sarcoma. The proportion of tumor-reactive CD4⁺ TILs was on average lower and the differences less pronounced across tumor types. Overall, the proportion of tumor-reactive TILs in vitro was remarkably low, implying a high fraction of TILs to be bystanders, and highly variable within the same tumor type. In situ analyses, based on eight single-cell RNA-sequencing datasets encompassing melanoma and five epithelial cancers types, corroborated the results obtained in vitro. Strikingly, no strong correlation between the proportion of CD8⁺ and CD4⁺ tumor-reactive TILs was detected, suggesting the accumulation of these responses in the tumor microenvironment to follow non-overlapping biological pathways. Additionally, no strong correlation between TIL responses and tumor mutational burden (TMB) in melanoma was observed, indicating that TMB was not a major driving force of response. No substantial differences in polyfunctionality across tumor types were observed. Conclusions: These analyses shed light on the functional features defining the quality of TIL infiltrates in cancer. A significant proportion of TILs across tumor types, especially non-melanoma, are bystander T cells. These results highlight the need to develop strategies focused on the tumor-reactive TIL subpopulation.
... In other clinical trials, other costimulatory domains including the CD27, OX40, ICOS and CD28 have been shown to promote T-cell survival, activate downstream signals and enhance long-term persistence [105]. During the in vivo expansion protocol, the persistence of TILs was achieved by exposure to IL-7, IL-15 and IL-21 [106][107][108]. The current strategy focused to enhance the in vivo persistence of CAR-T cells by coculturing T-cell with Th17-polarizing cytokines to enhance the CD4 T cells persistence and induce potent antitumor effects [84]. ...
Taking advantage of the cellular immune system is the mainstay of the adoptive cell therapy, to induce recognition and destruction of cancer cells. The impressive demonstration of this principle is chimeric antigen receptor modified T (CAR-T) cell therapy which had a major impact on treating relapsed and refractory hematological malignancies. Despite the great results of the CAR-T cell therapy, many tumors are still able to avoid immune detection and further elimination, as well as, the possible associated adverse events. Herein, we highlighted the recent advances in CAR-T cell therapy, discussing their applications beneficial functions, and side effects in hematological malignancies, illustrating the underlying challenges and opportunities. Furthermore, provide an overview to overcome different obstacles using potential manufacture and treatment strategies.
... Often the transferred cells are T cells, and these cells may either be genetically engineered during the expansion phase in order to generate tumorspecific cells or naturally elicited tumor-specific cells are selectively expanded. Prior to re-introduction into patients, the patients may or may not undergo lymphodepletion, though IL-2 supplementation along with other cytokines is typically administered to better the odds of transfer cell survival [18,[36][37][38]. During the expansion phase, genetic engineering approaches to overexpress chemokine receptors whose cognate chemokines are abundant in the tumor is an exciting opportunity to enhance the recruitment of the adoptive T cells to the tumor. ...
Immunotherapeutic treatment strategies greatly extend patient survival following malignant disease across a wide range of tumor types, including even those with metastatic disease. While diverse in approach, adoptive cell therapy, introduction of T cells that express chimeric antigen receptors, and checkpoint inhibitors all aim to re-invigorate the immune system to promote tumor cell identification and elimination. This review will focus on immune cell infiltration into tumors as well as a cellular organization within the tumor microenvironment as directed by the cell-specific expression patterns of chemokines and chemokine receptors. Through better understanding the chemokine network within tumors, we can uncover mechanisms to promote beneficial immune cell infiltration that can be combined with checkpoint inhibition. Conversely, chemokine expression is not limited to cells of the immune system, and it is understood that tumor cells also express chemokines and chemokine receptors. Tumor cells can hijack the chemokine networks to promote immune suppression and metastatic tumor cell trafficking. We will discuss the ways in which the chemokine network lies at the crossroad of immune evasion and tumor regression. Overall, this review will summarize key publications in the field of immune cell recruitment to tumors, highlight the dichotomous nature of chemokine interventions into cancer, and aims to identify therapeutic pathways forward.
... TILs were expanded to reach >50 × 10 6 total cells originating from ∼48 individual fragments, which had expanded to confluent growth in 2 ml wells and eliminated adherent tumor cells (average of ∼2 × 10 6 cells per well from each TF). TIL cultures were further expanded using a standard rapid expansion protocol (REP) as previously described (24). Briefly, TILs were stimulated with 30 ng/ml anti-CD3 antibody (OKT-3, Ortho Biotech) and 6,000 U/ml IL-2 in the presence of irradiated (40 Gy) allogeneic feeder cells (healthy donor PBMCs) at a feeder/TIL ratio of 200:1. ...
Mutation-derived neoantigens are important targets for T cell-mediated reactivity toward tumors and, due to their unique tumor expression, an attractive target for immunotherapy. Neoepitope-specific T cells have been detected across a number of solid cancers with high mutational burden tumors, but neoepitopes have been mostly selected from single nucleotide variations (SNVs), and little focus has been given to neoepitopes derived from in-frame and frameshift indels, which might be equally important and potentially highly immunogenic. Clear cell renal cell carcinomas (ccRCCs) are medium-range mutational burden tumors with a high pan-cancer proportion of frameshift mutations. In this study, the mutational landscape of tumors from six RCC patients was analyzed by whole-exome sequencing (WES) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs), and tumor-infiltrating lymphocytes (TILs, germline reference). Neopeptides were predicted using MuPeXI, and patient-specific peptide–MHC (pMHC) libraries were created for all neopeptides with a rank score < 2 for binding to the patient's HLAs. T cell recognition toward neoepitopes in TILs was evaluated using the high-throughput technology of DNA barcode-labeled pMHC multimers. The patient-specific libraries consisted of, on average, 258 putative neopeptides (range, 103–397, n = 6). In four patients, WES was performed on two different sources (TF and TCL), whereas in two patients, WES was performed only on TF. Most of the peptides were predicted from both sources. However, a fraction was predicted from one source only. Among the total predicted neopeptides, 16% were derived from frameshift indels. T cell recognition of 52 neoepitopes was detected across all patients (range, 4–18, n = 6) and spanning two to five HLA restrictions per patient. On average, 21% of the recognized neoepitopes were derived from frameshift indels (range, 0–43%, n = 6). Thus, frameshift indels are equally represented in the pool of immunogenic neoepitopes as SNV-derived neoepitopes. This suggests the importance of a broad neopeptide prediction strategy covering multiple sources of tumor material, and including different genetic alterations. This study, for the first time, describes the T cell recognition of frameshift-derived neoepitopes in RCC and determines their immunogenic profile.
... However, poor in vivo persistence of transferred cells remains a major limitation. TIL culture conditions can be optimized to obtain cell products with longer in vivo persistence (21)(22)(23). However, these protocols bypass selection of tumor-specific T cells, limiting the anti-tumor capacity of expanded T cells. ...
In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo. PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve in vivo persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in Slamf6 and lack Entpd1 and Havcr2 expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39– Tim3– Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39– Tim3– Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and in vivo persistence of transferred cells in the context of ACT.
... generation and expansion of tILs TILs were propagated from patient samples using a variation of the 'young TILs' protocol described elsewhere. 30 Tumor samples were excised into 1-5 mm 3 fragments which were placed into six-well G-rex culturing plates (Wilson Wolf, Minnesota, USA) filled with 30 mL of TIL medium (TM) containing RPMI 1640 supplemented with 20% FBS, 1% L-glutamine, 1% Pen/strep, 15 mM HEPES, 1 mM Na-pyruvate, 50 μm b-mercaptoethanol (Sigma-Aldrich) and 3000 IU/mL recombinant human IL-2 (PeproTech, Sweden). Subsequently, the cells were incubated for 5 days in a humidified incubator at +37°C. ...
Background
Ovarian cancers often contain significant numbers of tumor-infiltrating lymphocytes (TILs) that can be readily harnessed for adoptive T-cell therapy (ACT). However, the immunosuppressive ovarian tumor microenvironment and lack of tumor reactivity in TILs can limit the effectiveness of the therapy. We hypothesized that by using an oncolytic adenovirus (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) to deliver tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2), we could counteract immunosuppression, and enhance antitumor TIL responses in ovarian cancer (OVCA).
Methods
We established ex vivo tumor cultures freshly derived from patients with advanced OVCA and evaluated the effects of Ad5/3-E2F-D24-hTNFa-IRES-hIL2 or Ad5/3-E2F-D24 (the control virus without TNFa and IL-2) on TILs, cytokine response and tumor viability. Tumor reactivity was assessed by determining interferon gamma (IFNg) response of clinically relevant TILs towards autologous T-cell-depleted ex vivo tumor cultures pretreated with or without the aforementioned oncolytic adenoviruses.
Results
Treatment of ex vivo tumor cultures with Ad5/3-E2F-D24-hTNFa-IRES-hIL2 caused a substantial rise in proinflammatory signals: increased secretion of IFNg, CXCL10, TNFa and IL-2, and concomitant activation of CD4+ and CD8+ TILs. Potent tumor reactivity was seen, as clinically relevant TIL secreted high levels of IFNg in response to autologous T-cell-depleted ovarian ex vivo tumor cultures treated with Ad5/3-E2F-D24-hTNFa-IRES-hIL2. This phenomenon was independent of PD-L1 expression in tumor cells, a factor that determined the variability of IFNg responses seen in different patient samples.
Conclusions
Overall, oncolytic adenovirus Ad5/3-E2F-D24-hTNFa-IRES-hIL2 was able to rewire the ovarian tumor microenvironment to accommodate heightened antitumor TIL reactivity. Such effects may improve the clinical effectiveness of ACT with TILs in patients with advanced OVCA.
... TILs were expanded to reach >50 × 10 6 total cells that originated from ∼48 individual fragments, each of which had expanded to confluent growth in 2-ml wells and eliminated adherent tumor cells (average of ∼2 × 10 6 cells per well from each tumor fragment). TIL cultures were further expanded using a standard rapid expansion protocol (REP) as previously described (36). Briefly, TILs were stimulated with anti-CD3 antibody (30 ng/ml) (OKT-3; Ortho Biotech) and IL-2 (6000 U/ml) in the presence of irradiated (40 grays) allogenic feeder cells (healthy donor PBMCs) at a feeder/ TIL ratio of 200:1. ...
The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use in peptide-MHC multimer–based approaches to comprehensively analyze T cell immunity. To overcome this challenge, we demonstrate the use of functionally empty MHC class I molecules stabilized by a disulfide bond to link the α1 and α2 helices close to the F pocket. Peptide-loaded disulfide-stabilized HLA-A*02:01 shows complete structural overlap with wild-type HLA-A*02:01. Peptide-MHC multimers prepared using disulfide-stabilized HLA-A*02:01, HLA-A*24:02, and H-2Kb can be used to identify antigen-specific T cells, and they provide a better staining index for antigen-specific T cell detection compared with multimers prepared with wild-type MHC class I molecules. Disulfide-stabilized MHC class I molecules can be loaded with peptide in the multimerized form without affecting their capacity to stain T cells. We demonstrate the value of empty-loadable tetramers that are converted to antigen-specific tetramers by a single-step peptide addition through their use to identify T cells specific for mutation-derived neoantigens and other cancer-associated antigens in human melanoma.
... cancer cell line was a kind gift from Dr. Robin L. Anderson, University of Melbourne. MM33 melanoma cells were isolated from a resected tumor, which had also been used for establishment of a T cell culture as previously described (1). Cell lines were tested negative for mycoplasma using the PCR Mycoplasma Test Kit (AppliChem) less than a year before the use for experiments. ...
... When a minimum of 50 × 10 6 TILs were obtained (typically about one to 3 weeks after surgical resection), further expansion was carried out via a standard 14-day REP protocol described previously. 15 REP TILs were used for all TIL-based analyses. Autologous tumor cell lines were established, as described elsewhere, 16 using fragments from the same tumor lesion from which the TILs were generated. ...
Up to 60% of patients treated with cancer immunotherapy develop severe or life threatening immune‐related adverse events (irAEs). Immunosuppression with high dose corticosteroids, or tumor necrosis factor (TNF) antagonists in refractory cases, is the mainstay of treatment for irAEs. It is currently unknown what impact corticosteroids and anti‐TNF have on the activity of antitumor T cells. In our study, the influence of clinically relevant doses of dexamethasone (corresponding to an oral dose of 10–125 mg prednisolone) and infliximab (anti‐TNF) on the activation and killing ability of tumor‐infiltrating lymphocytes (TILs) was tested in vitro. Overall, dexamethasone at low or intermediate/high doses impaired the activation (−46 and −62%, respectively) and tumor‐killing ability (−48 and −53%, respectively) of tumor‐specific TILs. In contrast, a standard clinical dose of infliximab only had a minor effect on T cell activation (−20%) and tumor killing (−10%). A 72‐hr resting period after withdrawal of dexamethasone was sufficient to rescue the in vitro activity of TILs, while a short withdrawal did not result in a full rescue. In conclusion, clinically relevant doses of infliximab only had a minor influence on the activity of tumor‐specific TILs in vitro, whereas even low doses of corticosteroids markedly impaired the antitumor activity of TILs. However, the activity of TILs could be restored after withdrawal of steroids. These data indirectly support steroid‐sparing strategies and early initiation of anti‐TNF therapy for the treatment of irAEs in immuno‐oncology.
... Current approaches focus on shortening the expansion protocols and optimizing them to include cytokines or agents able to delay T cell differentiation. Rapidexpansion protocols used to expand TILs induce rapid T cell differentiation, while TILs that undergo shorter expansion ex vivo persist longer (123,124). A recent work proposes to shorten the expansion protocol for CAR-T cells from day 9 to day 3, which would limit T cell differentiation while simplifying production methods and increasing access (125). ...
Genetically engineered T cells are powerful new medicines, offering hope for curative responses in patients with cancer. Chimeric antigen receptor (CAR) T cells were recently approved by the US Food and Drug Administration and are poised to enter the practice of medicine for leukemia and lymphoma, demonstrating that engineered immune cells can serve as a powerful new class of cancer therapeutics. The emergence of synthetic biology approaches for cellular engineering provides a broadly expanded set of tools for programming immune cells for enhanced function. Advances in T cell engineering, genetic editing, the selection of optimal lymphocytes, and cell manufacturing have the potential to broaden T cell–based therapies and foster new applications beyond oncology, in infectious diseases, organ transplantation, and autoimmunity. Expected final online publication date for the Annual Review of Immunology Volume 37 is April 26, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown remarkable results in melanoma, but only modest clinical benefit in other cancers, even after TIL have been genetically modified to improve their tumor homing, cytotoxic potential or overcoming cell exhaustion. The required ex vivo TIL expansion process may induce changes in the T cell clonal composition, which could likely compromise the tumor reactivity of TIL preparations and ultimately the success of TIL therapy. A promising approach based on the production of bispecific T cell engagers (TCE) by engineered T cells (STAb-T therapy) improves the efficacy of current T cell redirection strategies against tumor-associated antigens in hematological tumors. We studied the TCRβ repertoire in non-small cell lung cancer (NSCLC) tumors and in ex vivo expanded TIL from two unrelated patients. We generated TIL secreting anti-epidermal growth factor receptor (EGFR) x anti-CD3 TCE (TIL STAb ) and tested their antitumor efficacy in vitro and in vivo using a NSCLC patient-derived xenograft (PDX) model in which tumor fragments and TIL from the same patient were transplanted into hIL-2 NOG mice. We confirmed that the standard TIL expansion protocol promotes the loss of tumor-dominant T cell clones and the overgrowth of virus-reactive TCR clonotypes that were marginally detectable in primary tumors. We demonstrated the antitumor activity of TIL STAb both in vitro and in vivo when administered intratumorally and systemically in an autologous immune-humanized PDX EGFR ⁺ NSCLC mouse model, where tumor regression was mediated by TCE-redirected CD4 ⁺ TIL bearing non-tumor dominant clonotypes.
SIGNIFICANCE
Epithelial tumor-derived TIL can be engineered to secrete TCE capable of redirecting T cells bearing non-tumor-dominant clonotypes regardless of their phenotype, which could have broad applications in immunotherapy for solid tumors.
Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) has demonstrated durable clinical responses in patients with metastatic melanoma, substantiated by recent positive results of the first phase III trial on TIL therapy. Being a demanding and logistically complex treatment, extensive preclinical and clinical effort is required to optimize patient selection by identifying predictive biomarkers of response. This review aims to comprehensively summarize the current evidence regarding the potential impact of tumor-related factors (such as mutational burden, neoantigen load, immune infiltration, status of oncogenic driver genes, and epigenetic modifications), patient characteristics (including disease burden and location, baseline cytokines and lactate dehydrogenase serum levels, human leucocyte antigen haplotype, or prior exposure to immune checkpoint inhibitors and other anticancer therapies), phenotypic features of the transferred T cells (mainly the total cell count, CD8:CD4 ratio, ex vivo culture time, expression of exhaustion markers, costimulatory signals, antitumor reactivity, and scope of target tumor-associated antigens), and other treatment-related factors (such as lymphodepleting chemotherapy and postinfusion administration of interleukin-2).
The incidence of hepatocellular carcinoma (HCC) ranks first among primary liver cancers, and its mortality rate exhibits a consistent annual increase. The treatment of HCC has witnessed a significant surge in recent years, with the emergence of targeted immune therapy as an adjunct to early surgical resection. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has shown promising results in other types of solid tumors. This article aims to provide a comprehensive overview of the intricate interactions between different types of TILs and their impact on HCC, elucidate strategies for targeting neoantigens through TILs, and address the challenges encountered in TIL therapies along with potential solutions. Furthermore, this article specifically examines the impact of oncogenic signaling pathways activation within the HCC tumor microenvironment on the infiltration dynamics of TILs. Additionally, a concise overview is provided regarding TIL preparation techniques and an update on clinical trials investigating TIL-based immunotherapy in solid tumors.
The use of a patient’s own immune or tumor cells, manipulated ex vivo, enables Ag- or patient-specific immunotherapy. Despite some clinical successes, there remain significant barriers to efficacy, broad patient population applicability, and safety. Immunotherapies that target specific tumor Ags, such as chimeric Ag receptor T cells and some dendritic cell vaccines, can mount robust immune responses against immunodominant Ags, but evolving tumor heterogeneity and antigenic downregulation can drive resistance. In contrast, whole tumor cell vaccines and tumor lysate-loaded dendritic cell vaccines target the patient’s unique tumor antigenic repertoire without prior neoantigen selection; however, efficacy can be weak when lower-affinity clones dominate the T cell pool. Chimeric Ag receptor T cell and tumor-infiltrating lymphocyte therapies additionally face challenges related to genetic modification, T cell exhaustion, and immunotoxicity. In this review, we highlight some engineering approaches and opportunities to these challenges among four classes of autologous cell therapies.
Simple Summary
Adoptive cell therapy (ACT) is an innovative approach to combat cancer and infectious diseases using specialised immune cells, such as chimeric antigen receptor T-cells (CAR-Ts), tumour-infiltrating lymphocytes (TILs) and virus-specific T-cells (VSTs). Such therapies are manufactured individually for each patient and can be negatively affected by poor cell quality, often impaired by prior treatments, age, and complex manufacturing. To overcome this, this field is assessing the potential of creating cell therapies from “fit” donors to provide off-the-shelf treatment options. Induced pluripotent cells (iPSCs) have renewable characteristics and offer a solution towards off-the-shelf therapy. iPSCs can be used as an unlimited source to derive different immune cells, including natural killer (NK) cells and T-cells. iPSCs can be further genetically modified and used create different ACTs. In this review, we describe the methodologies for generating such cell therapies from iPSCs and discuss the current advances and challenges with a focus on CAR-T/NK-, TIL- and VST-based therapies.
Abstract
Adoptive cell therapy (ACT) has transformed the treatment landscape for cancer and infectious disease through the investigational use of chimeric antigen receptor T-cells (CAR-Ts), tumour-infiltrating lymphocytes (TILs) and viral-specific T-cells (VSTs). Whilst these represent breakthrough treatments, there are subsets of patients who fail to respond to autologous ACT products. This is frequently due to impaired patient T-cell function or “fitness” as a consequence of prior treatments and age, and can be exacerbated by complex manufacturing protocols. Further, the manufacture of autologous, patient-specific products is time-consuming, expensive and non-standardised. Induced pluripotent stem cells (iPSCs) as an allogeneic alternative to patient-specific products can potentially overcome the issues outlined above. iPSC technology provides an unlimited source of rejuvenated iPSC-derived T-cells (T-iPSCs) or natural killer (NK) cells (NK-iPSCs), and in the context of the growing field of allogeneic ACT, iPSCs have enormous potential as a platform for generating off-the-shelf, standardised, “fit” therapeutics for patients. In this review, we evaluate current and future applications of iPSC technology in the CAR-T/NK, TIL and VST space. We discuss current and next-generation iPSC manufacturing protocols, and report on current iPSC-based adoptive therapy clinical trials to elucidate the potential of this technology as the future of ACT.
Differentiation of naive CD4⁺ T cells into effector T cells is a dynamic process in which the cells are polarized into T helper (Th) subsets. The subsets largely consist of four fundamental categories: Th1, Th2, Th17, and regulatory T cells. We show that human memory CD4⁺ T cells can produce hepatocyte growth factor (HGF), a pleiotropic cytokine which can affect several tissue types through signaling by its receptor, c-Met. In vitro differentiation of T cells into Th-like subsets revealed that HGF producing T cells increase under Th1 conditions. Enrichment of HGF producing cells was possible by targeting cells with surface CD30 expression, a marker discovered through single-cell RNA-sequencing. Furthermore, pharmacological inhibition of PI3K or mTOR was found to inhibit HGF mRNA and protein, while an Akt inhibitor was found to increase these levels. The findings suggest that HGF producing T cells could play a role in disease where Th1 are present.
Cancer is a leading cause of death worldwide and, despite new targeted therapies and immunotherapies, a large group of patients fail to respond to therapy or progress after initial response, which brings the need for additional treatment options. Manipulating the immune system using a variety of approaches has been explored for the past years with successful results. Sustained progress has been made to understand the T cell-mediated anti-tumor responses counteracting the tumorigenesis process. The T-lymphocyte pool, especially its capacity for antigen-directed cytotoxicity, has become a central focus for engaging the immune system in defeating cancer. The adoptive cell transfer of autologous tumor-infiltrating lymphocytes has been used in humans for over 30 years to treat metastatic melanoma. In this review, we provide a brief history of ACT-TIL and discuss the current state of ACT-TIL clinical development in solid tumors. We also discuss how key advances in understanding genetic intratumor heterogeneity, to accurately identify neoantigens, and new strategies designed to overcome T-cell exhaustion and tumor immunosuppression have improved the efficacy of the TIL-therapy infusion. Characteristics of the TIL products will be discussed, as well as new strategies, including the selective expansion of specific fractions from the cell product or the genetic manipulation of T cells for improving the in-vivo survival and functionality.
In summary, this review outlines the potential of ACT-TIL as a personalized approach for epithelial tumors and continued discoveries are making it increasingly more effective against other types of cancers.
What in the World is Immunotherapy for Cancer? explores the ever-lasting problem of cancer and how it can be defeated utilizing one's own immune system. Gone are the days of chemotherapy and radiation, which can still provide a chance of one re-developing the disease. What in the World is Immunotherapy for Cancer? explores the history of cancer, the history of immunotherapy, insights into how it all works, and what might be the future of immunotherapy.
There is a pressing need for novel immunotherapeutic targets in colorectal cancer (CRC). Cytotoxic T cell infiltration is well established as a key prognostic indicator in CRC, and it is known that these tumor infiltrating lymphocytes (TILs) target and kill tumor cells. However, the specific antigens that drive these CD8+ T cell responses have not been well characterized. Recently, phosphopeptides have emerged as strong candidates for tumor-specific antigens, as dysregulated signaling in cancer leads to increased and aberrant protein phosphorylation. Here, we identify 120 HLA-I phosphopeptides from primary CRC tumors, CRC liver metastases and CRC cell lines using mass spectrometry and assess the tumor-resident immunity against these posttranslationally modified tumor antigens. Several CRC tumor-specific phosphopeptides were presented by multiple patients’ tumors in our cohort (21% to 40%), and many have previously been identified on other malignancies (58% of HLA-A*02 CRC phosphopeptides). These shared antigens derived from mitogenic signaling pathways, including p53, Wnt and MAPK, and are therefore markers of malignancy. The identification of public tumor antigens will allow for the development of broadly applicable targeted therapeutics. Through analysis of TIL cytokine responses to these phosphopeptides, we have established that they are already playing a key role in tumor-resident immunity. Multifunctional CD8+ TILs from primary and metastatic tumors recognized the HLA-I phosphopeptides presented by their originating tumor. Furthermore, TILs taken from other CRC patients’ tumors targeted two of these phosphopeptides. In another cohort of CRC patients, the same HLA-I phosphopeptides induced higher peripheral T cell responses than they did in healthy donors, suggesting that these immune responses are specifically activated in CRC patients. Collectively, these results establish HLA-I phosphopeptides as targets of the tumor-resident immunity in CRC, and highlight their potential as candidates for future immunotherapeutic strategies.
Ex vivo expansion followed by reinfusion of tumor-infiltrating leukocytes (TILs) has been used successfully for the treatment of multiple malignancies. Most protocols rely on the use of the cytokine IL-2 to expand TILs prior to reinfusion. In addition, TIL administration relies on systemic administration of IL-2 after reinfusion to support transferred cell survival. The use of IL-2, however, can be problematic because of its preferential expansion of regulatory T and myeloid cells as well as its systemic side effects. In this study, we describe the use of a novel IL-2 mutant retargeted to NKG2D rather than the high-affinity IL-2R for TIL-mediated immunotherapy in a murine model of malignant melanoma. We demonstrate that the NKG2D-retargeted IL-2 (called OMCPmutIL-2) preferentially expands TIL-resident CTLs, such as CD8+ T cells, NK cells, and γδT cells, whereas wild-type IL-2 provides a growth advantage for CD4+Foxp3+ T cells as well as myeloid cells. OMCPmutIL-2-expanded CTLs express higher levels of tumor-homing receptors, such as LFA-1, CD49a, and CXCR3, which correlate with TIL localization to the tumor bed after i.v. injection. Consistent with this, OMCPmutIL-2-expanded TILs provided superior tumor control compared with those expanded in wild-type IL-2. Our data demonstrate that adoptive transfer immunotherapy can be improved by rational retargeting of cytokine signaling to NKG2D-expressing CTLs rather than indiscriminate expansion of all TILs.
T cell-engagers are a rapidly evolving novel group of treatments that have in common the concurrent engagement of a T cell-surface molecule and a tumoral cell antigen. Bispecific antibodies and genetically engineered adoptive cell therapies, as chimeric antigen receptor or T cell receptor T cells, have similarities and differences among their mechanisms of action, toxicity profiles, and resistance pathways. Nevertheless, the success observed in the hematological field has not been obtained with solid tumors yet, as they are biologically more complex and have few truly tumor-specific cell surface antigens that can be targeted with high avidity T cells. Different strategies are under study to improve their short-term perspective, such as new generations of more active T cell-engagers, multi-target or combination of different treatments approaches, or to improve the manufacturing processes. A comprehensive review of T cell-engagers as a grouped treatment class, current status, and research directions in their application to solid tumors therapeutics are discussed here.
Patients with resectable tumor, either in the body or the tail of the pancreas, and cancer patients with a primary tumor adjacent to the splenic vasculature frequently undergo a splenectomy as standard of care during resection. The spleen provides an unutilized source of lymphocytes with potential utility for adoptive cellular therapy (ACT). In this report, spleen and peripheral blood (PB) cells from cancer patients were compared to one another and normal PB by flow cytometry with a focus on CD8⁺ T-cells, memory phenotype, and their relative expression of checkpoint proteins including program death ligand-1 (PD1). PD1 is both an activation marker for T-cells including antigen (Ag) specific responses, as well as a marker of T-cell exhaustion associated with co-expression of other checkpoint molecules such as lymphocyte activating gene-3 (LAG-3) and T-cell immunoglobulin and mucin domain containing-3 (TIM-3). In summary, the spleen is a rich source of CD8⁺PD1⁺ T-cells, with an 8-fold higher frequency compared to the PB. These CD8+ T-cells are predominantly central and transitional memory T-cells with associated effector phenotypes and low expression of TIM-3 and LAG-3 with potential utility for ACT”.
Adoptive cell therapy (ACT) with autologous tumor infiltrating lymphocytes (TIL) has been studied for patients with advanced metastatic cancers (primarily melanoma) for decades and has changed significantly during that period. Treatment with TIL includes ex vivo cell activation and expansion followed by re-infusion of these cells into the patient. After cell infusion, patients receive Interleukin-2 (IL-2). Objective response rates up to 52% have been seen in patients with metastatic melanoma. Efforts to improve TIL therapy include better selection and expansion of tumor-reactive lymphocytes, optimization of IL-2 or other T cell activating cytokine dosing, and, potentially, genetic manipulation of the immune cell product. Here we describe methods involved in the collection, expansion, and treatment with TIL for patients with metastatic melanoma.
Clinical trials of adoptive transfer of autologous tumor infiltrating lymphocytes (TILs) to patients with advanced malignant melanoma have shown remarkable results with objective clinical responses in 50% of the treated patients. In order to initiate a clinical trial in melanoma, we have established a method for expanding TILs to clinical relevant quantities in two steps with in 8 weeks. Further characterization of expanded TILs revealed an oligoclonal composition of T-cells with an effector memory like phenotype. When autologous tumor was available, TILs showed specific activity in all patients tested. TIL cultures contained specificity towards tumor cells as well as peptides derived from tumor-associated antigens (TAAs) during expansion procedures.
Adoptive cell therapy can be an effective treatment for some patients with advanced cancer. This report summarizes clinical trial results from the Surgery Branch, NCI, investigating tumor infiltrating lymphocytes (TIL) and gene engineered peripheral blood T cells for the therapy of patients with melanoma and other solid tumors.
Clinical trials on adoptive T-cell therapy (ACT) using expanded tumor-infiltrating lymphocytes (TIL) have shown response rates of over 50% in refractory melanoma. However, little is known how clinical and pathologic features impact TIL outgrowth isolated from metastatic melanoma tumors.
We analyzed the impact of clinical and pathologic features on initial TIL outgrowth in 226 consecutive patients undergoing tumor resection. Successful initial TIL outgrowth was defined as ≥40 million viable lymphocytes harvested from all tumor fragments in a 5-week culture. To normalize for the different size of resected tumors and thus available tumor fragments, we divided the number of expanded TIL by the starting number of tumor fragments (TIL/fragment).
Overall, initial TIL outgrowth was successful in 62% of patients, with patients ≤30 years of age (94%; P = 0.01) and female patients (71% vs. 57% for males; P = 0.04) having the highest rate of success. Systemic therapy 30 days before tumor harvest negatively impacted initial TIL outgrowth compared to patients who never received systemic therapy (47% vs. 71%, P = 0.02). Biochemotherapy within 0 to 60 days of tumor harvest negatively impacted the initial TIL outgrowth with a success rate of only 16% (P < 0.0001).
Parameters such as age, sex, and the type and timing of prior systemic therapy significantly affect the success rate of the initial TIL outgrowth from tumor fragments for ACT; these parameters may be helpful in selecting patients for melanoma ACT.
Most treatments for patients with metastatic melanoma have a low rate of complete regression and thus overall survival in these patients is poor. We investigated the ability of adoptive cell transfer utilizing autologous tumor-infiltrating lymphocytes (TIL) to mediate durable complete regressions in heavily pretreated patients with metastatic melanoma.
Ninety-three patients with measurable metastatic melanoma were treated with the adoptive transfer of autologous TILs administered in conjunction with interleukin-2 following a lymphodepleting preparative regimen on three sequential clinical trials. Ninety-five percent of these patients had progressive disease following a prior systemic treatment. Median potential follow-up was 62 months.
Objective response rates by Response Evaluation Criteria in Solid Tumors (RECIST) in the 3 trials using lymphodepleting preparative regimens (chemotherapy alone or with 2 or 12 Gy irradiation) were 49%, 52%, and 72%, respectively. Twenty of the 93 patients (22%) achieved a complete tumor regression, and 19 have ongoing complete regressions beyond 3 years. The actuarial 3- and 5-year survival rates for the entire group were 36% and 29%, respectively, but for the 20 complete responders were 100% and 93%. The likelihood of achieving a complete response was similar regardless of prior therapy. Factors associated with objective response included longer telomeres of the infused cells, the number of CD8(+)CD27(+) cells infused, and the persistence of the infused cells in the circulation at 1 month (all P(2) < 0.001).
Cell transfer therapy with autologous TILs can mediate durable complete responses in patients with metastatic melanoma and has similar efficacy irrespective of prior treatment. Clin Cancer Res; 17(13); 4550-7. ©2011 AACR.
Altered natural killer (NK) cell function is a component of the global immune dysregulation that occurs in advanced malignancies. Another condition associated with altered NK homeostasis is normal pregnancy, where robust infiltration with CD16- CD9+ NK cells can be identified in decidual tissues, along with a concomitant expansion of CD16- NK cells in the maternal peripheral blood. In metastatic melanoma, we identified a similar expansion of peripheral blood CD16- NK cells (median 7.4% in 41 patients with melanoma compared with 3.0% in 29 controls, P < .001). A subset of NK cells in melanoma patients also expresses CD9, which is characteristically expressed only on NK cells within the female reproductive tract. Expansion of CD16- NK cells was associated with elevated plasma transforming growth factor-beta (TGF-β levels (median 20 ng/ml, Spearman's ρ = 0.81, P = .015)). These findings suggest the possibility of exploring anti-TGF-β therapy to restore NK function in melanoma.
Adoptive T-cell therapy (ACT) using expanded autologous tumor-infiltrating lymphocytes (TIL) and tumor antigen-specific T cell expanded from peripheral blood are complex but powerful immunotherapies directed against metastatic melanoma. A number of nonrandomized clinical trials using TIL combined with high-dose interleukin-2 (IL-2) have consistently found clinical response rates of 50% or more in metastatic melanoma patients accompanied by long progression-free survival. Recent studies have also established practical methods for the expansion of TIL from melanoma tumors with high success rates. These results have set the stage for randomized phase II/III clinical trials to determine whether ACT provides benefit in stage IV melanoma. Here, we provide an overview of the current state-of-the art in T-cell-based therapies for melanoma focusing on ACT using expanded TIL and address some of the key unanswered biological and clinical questions in the field. Different phase II/III randomized clinical trial scenarios comparing the efficacy of TIL therapy to high-dose IL-2 alone are described. Finally, we provide a roadmap describing the critical steps required to test TIL therapy in a randomized multicenter setting. We suggest an approach using centralized cell expansion facilities that will receive specimens and ship expanded TIL infusion products to participating centers to ensure maximal yield and product consistency. If successful, this approach will definitively answer the question of whether ACT can enter mainstream treatment for cancer.
Adoptive Cell Transfer (ACT) of Tumor-Infiltrating Lymphocytes (TIL) in combination with lymphodepletion has proven to be an effective treatment for metastatic melanoma patients, with an objective response rate in 50%-70% of the patients. It is based on the ex vivo expansion and activation of tumor-specific T lymphocytes extracted from the tumor and their administration back to the patient. Various TIL-ACT trials, which differ in their TIL generation procedures and patient preconditioning, have been reported. In the latest clinical studies, genetically engineered peripheral T cells were utilized instead of TIL. Further improvement of adoptive T cell transfer depends on new investigations which seek higher TIL quality, increased durable response rates, and aim to treat more patients. Simplifying this therapy may encourage cancer centers worldwide to adopt this promising technology. This paper focuses on the latest progress regarding adoptive T cell transfer, comparing the currently available protocols and discussing their advantages, disadvantages, and implication in the future.
Various immunotherapeutic strategies for cancer are aimed at augmenting the T cell response against tumor cells. Adoptive cell therapy (ACT), where T cells are manipulated ex vivo and subsequently re-infused in an autologous manner, has been performed using T cells from various sources. Some of the highest clinical response rates for metastatic melanoma have been reported in trials using tumor-infiltrating lymphocytes (TILs). These protocols still have room for improvement and furthermore are currently only performed at a limited number of institutions. The goal of this work was to develop TILs as a therapeutic product at our institution.
TILs from 40 melanoma tissue specimens were expanded and characterized. Under optimized culture conditions, 72% of specimens yielded rapidly proliferating TILs as defined as at least one culture reaching ≥3×10(7) TILs within 4 weeks. Flow cytometric analyses showed that cultures were predominantly CD3+ T cells, with highly variable CD4+:CD8+ T cell ratios. In total, 148 independent bulk TIL cultures were assayed for tumor reactivity. Thirty-four percent (50/148) exhibited tumor reactivity based on IFN-γ production and/or cytotoxic activity. Thirteen percent (19/148) showed specific cytotoxic activity but not IFN-γ production and only 1% (2/148) showed specific IFN-γ production but not cytotoxic activity. Further expansion of TILs using a 14-day "rapid expansion protocol" (REP) is required to induce a 500- to 2000-fold expansion of TILs in order to generate sufficient numbers of cells for current ACT protocols. Thirty-eight consecutive test REPs were performed with an average 1865-fold expansion (+/- 1034-fold) after 14 days.
TILs generally expanded efficiently and tumor reactivity could be detected in vitro. These preclinical data from melanoma TILs lay the groundwork for clinical trials of ACT.
Tumor-infiltrating lymphocytes (TIL) and interleukin (IL)-2 administered following lymphodepletion can cause the durable complete regression of bulky metastatic melanoma in patients refractory to approved treatments. However, the generation of a unique tumor-reactive TIL culture for each patient may be prohibitively difficult. We therefore investigated the clinical and immunologic impact of unscreened, CD8+ enriched "young" TIL.
Methods were developed for generating TIL that minimized the time in culture and eliminated the individualized tumor-reactivity screening step. Thirty-three patients were treated with these CD8+ enriched young TIL and IL-2 following nonmyeloablative lymphodepletion (NMA). Twenty-three additional patients were treated with CD8+ enriched young TIL and IL-2 after lymphodepletion with NMA and 6 Gy of total body irradiation.
Young TIL cultures for therapy were successfully established from 83% of 122 consecutive melanoma patients. Nineteen of 33 patients (58%) treated with CD8+ enriched young TIL and NMA had an objective response (Response Evaluation Criteria in Solid Tumors) including 3 complete responders. Eleven of 23 patients (48%) treated with TIL and 6 Gy total body irradiation had an objective response including 2 complete responders. At 1 month after TIL infusion the absolute CD8+ cell numbers in the periphery were highly correlated with response.
This study shows that a rapid and simplified method can be used to reliably generate CD8+ enriched young TIL for administration as an individualized therapy for advanced melanoma, and may allow this potentially effective treatment to be applied at other institutions and to reach additional patients.
Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) has shown promising results in metastatic melanoma patients. Although objective response rates of over 50% have been reported, disadvantages of this approach are the labor-intensive TIL production and a very high drop-out rate of enrolled patients, limiting its widespread applicability. Previous studies showed a clear correlation between short TIL culture periods and clinical response. Therefore, we used a new TIL production technique using unselected, minimally cultured, bulk TIL (Young-TIL). The use of Young-TIL is not restricted to human leukocyte antigen (HLA)-A2 patients. The purpose of this study is to explore the efficacy and toxicity of adoptively transferred Young-TIL following lympho-depleting chemotherapy in metastatic melanoma patients, refractory to interleukin-2 and chemotherapy.
Young-TIL cultures for 90% of the patients were successfully generated, enabling the treatment of most enrolled patients. We report here the results of 20 evaluated patients.
Fifty percent of the patients achieved an objective clinical response according to the Response Evaluation Criteria in Solid Tumors, including two ongoing complete remissions (20+, 4+ months) and eight partial responses (progression-free survival: 18+, 13+, 10+, 9, 6+, 4, 3+, and 3 months). All responders are currently alive. Four additional patients showed disease stabilization. Side effects were transient and manageable.
We showed that lympho-depleting chemotherapy followed by transfer of short-term cultured TIL can mediate tumor regression in 50% of metastatic melanoma with manageable toxicity. The convincing clinical results combined with the simplification of the process may thus have a major effect on cell therapy of cancer.
The hallmark of adaptive immunity is its ability to recognise a wide range of antigens and technologies that capture this diversity are therefore of substantial interest. New methods have recently been developed that allow the parallel analysis of T cell reactivity against vast numbers of different epitopes in limited biological material. These technologies are based on the joint binding of differentially labelled MHC multimers on the T cell surface, thereby providing each antigen-specific T cell population with a unique multicolour code. This strategy of 'combinatorial encoding' enables detection of many (at least 25) different T cell populations per sample and should be of broad value for both T cell epitope identification and immunomonitoring.
Lymphodepletion before adoptive cell transfer (ACT)-based immunotherapies can enhance anti-tumor responses by augmenting innate immunity, by increasing access to homeostatic cytokines, and by depressing the numbers of regulatory T cells and myeloid-derived suppressor cells. Although it is clear that high-dose total body irradiation given together with hematopoietic stem cell (HSC) transplantation effectively enhances ACT, the relationship between the intensity of lymphodepletion and tumor treatment efficacy has not been systematically studied. Using the pmel-1 mouse model of self/tumor-reactive CD8 T cells, we observed a strong correlation between the intensity of the conditioning regimen and the efficacy of ACT-based treatments using linear regression analysis. This was the case for preparative total body irradiation administered either as a single dose (R=0.97, P<0.001) or in fractionated doses (R=0.94, P<0.001). Increased amounts of preparative total body irradiation were directly correlated with progressively more favorable ratios of transferred tumor-reactive CD8 T cells toward endogenous cells with the potential for inhibitory activity including: CD4 cells (potentially T regulatory cells); Gr1 cells (which are capable of functioning as myeloid-derived suppressor cells); and endogenous CD8 and natural killer 1.1 cells (that can act as "sinks" for homeostatic cytokines in the postablative setting). With increasing ablation, we also observed elevated lipopolysaccharide levels in the sera and heightened levels of systemic inflammatory cytokines. Thus, increased intensity lymphodepletion triggers enhanced tumor treatment efficacy and the benefits of high-dose total body irradiation must be titrated against its risks.
We determined how CD8(+) melanoma tumor-infiltrating lymphocytes (TILs) isolated from two distinct phases of expansion in preparation for adoptive T cell therapy respond to melanoma Ag restimulation. We found that TILs isolated after the rapid expansion protocol (REP) phase, used to generate the final patient TIL infusion product, were hyporesponsive to restimulation with MART-1 peptide-pulsed dendritic cells, with many CD8(+) T cells undergoing apoptosis. Telomere length was shorter post-REP, but of sufficient length to support further cell division. Phenotypic analysis revealed that cell-surface CD28 expression was significantly reduced in post-REP TILs, whereas CD27 levels remained unchanged. Tracking post-REP TIL proliferation by CFSE dilution, as well as sorting for CD8(+)CD28(+) and CD8(+)CD28(-) post-REP subsets, revealed that the few CD28(+) TILs remaining post-REP had superior survival capacity and proliferated after restimulation with MART-1 peptide. An analysis of different supportive cytokine mixtures during the REP found that a combination of IL-15 and IL-21 facilitated comparable expansion of CD8(+) TILs as IL-2, but prevented the loss of CD28 expression with improved responsiveness to antigenic restimulation post-REP. These results suggest that current expansion protocols using IL-2 for melanoma adoptive T cell therapy yields largely CD8(+) T cells unable to persist and divide in vivo following Ag contact. The few CD8(+)CD28(+) T cells that remain may be the only CD8(+) TILs that ultimately survive to repopulate the host and mediate long-term tumor control. A REP protocol using IL-15 and IL-21 may greatly increase the number of CD28(+) TILs capable of long-term persistence.
The use of fluorescently labeled major histocompatibility complex multimers has become an essential technique for analyzing disease- and therapy-induced T-cell immunity. Whereas classical major histocompatibility complex multimer analyses are well-suited for the detection of immune responses to a few epitopes, limitations on human-subject sample size preclude a comprehensive analysis of T-cell immunity. To address this issue, we developed a combinatorial encoding strategy that allows the parallel detection of a multitude of different T-cell populations in a single sample. Detection of T cells from peripheral blood by combinatorial encoding is as efficient as detection with conventionally labeled multimers but results in a substantially increased sensitivity and, most notably, allows comprehensive screens to be performed. We obtained proof of principle for the feasibility of large-scale screening of human material by analysis of human leukocyte antigen A3-restricted T-cell responses to known and potential melanoma-associated antigens in peripheral blood from individuals with melanoma.
Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) and high-dose interleukin-2 (IL-2), after nonmyeloablative chemotherapy, has been shown to result in tumor regression in half of refractory metastatic melanoma patients. In the present study, we describe 2 separate clinical protocols. Twelve patients were treated with "Selected"-TIL, as previously reported and 8 patients with the modified version of "Young"-TIL. Selected-TIL protocol required the establishment of multiple T-cell cultures from 1 patient and in vitro selection of cultures secreting interferon-gamma upon antigenic stimulation. In contrast, Young-TIL are minimally cultured T cells with superior in vitro features that do not require further selection. Two of 12 Selected-TIL patients experienced objective clinical responses (1 complete response, 1 partial response). Out of 8 treated Young-TIL patients, 1 experienced complete response, 2 partial response, and 4 patients had disease stabilization. Twenty-one of 33 enrolled Selected-TIL patients were excluded from the protocol, mainly as cultures failed the interferon-gamma selection criteria or due to clinical deterioration, compared with only 3 Young-TIL patients. Expected bone marrow suppression and high-dose IL-2 toxicity were transient. There was no treatment-related mortality. This study vindicates the feasibility and effectiveness of TIL technology and calls for further efforts to implement and enhance this modality. The use of minimally cultured, unselected Young-TIL enables the treatment of most enrolled patients. Although the cohort of Young-TIL patients treated so far is rather small and the follow-up short, the response rate is encouraging.
The two approved treatments for patients with metastatic melanoma, interleukin (IL)-2 and dacarbazine, mediate objective response rates of 12% to 15%. We previously reported that adoptive cell therapy (ACT) with autologous antitumor lymphocytes in lymphodepleted hosts mediated objective responses in 51% of 35 patients. Here, we update that study and evaluate the safety and efficacy of two increased-intensity myeloablative lymphodepleting regimens.
We performed two additional sequential trials of ACT with autologous tumor-infiltrating lymphocytes (TIL) in patients with metastatic melanoma. Increasing intensity of host preparative lymphodepletion consisting of cyclophosphamide and fludarabine with either 2 (25 patients) or 12 Gy (25 patients) of total-body irradiation (TBI) was administered before cell transfer. Objective response rates by Response Evaluation Criteria in Solid Tumors (RECIST) and survival were evaluated. Immunologic correlates of effective treatment were studied.
Although nonmyeloablative chemotherapy alone showed an objective response rate of 49%, when 2 or 12 Gy of TBI was added, the response rates were 52% and 72% respectively. Responses were seen in all visceral sites including brain. There was one treatment-related death in the 93 patients. Host lymphodepletion was associated with increased serum levels of the lymphocyte homeostatic cytokines IL-7 and IL-15. Objective responses were correlated with the telomere length of the transferred cells.
Host lymphodepletion followed by autologous TIL transfer and IL-2 results in objective response rates of 50% to 70% in patients with metastatic melanoma refractory to standard therapies.
Recent data suggest that human effector CD8+ T cells express a distinct CD27-CD45RAhigh (CD57+CD28-CD11ahigh) phenotype. Here, we propose that CTL effector function correlates better with CD56 (neuronal cell adhesion molecule (NCAM)) surface expression. CD56 was absent on cord blood CD8+ T cells, but was expressed by 4-30% of freshly isolated circulating CD8+ T cells from 15 adults. Dramatic oligoclonal expansions in 3/3 individuals were confined to the CD56+ subset of CD8+ T cells. The CD56+ subset generally contained high amounts of intracellular perforin and granzyme B. Finally, direct cytolytic capacity was closely restricted to the CD56+(CD45RAhigh) cells, better than to CD27-CD45RAhigh cells in 5/5 individuals analyzed. Thus, the phenotype corresponding to the circulating effector CD8+ T cell pool may be simplified and more precisely defined by the use of just two surface markers: CD8 and CD56.
During the last decade, a large number of human tumor-associated antigens have been identified that are recognized by CTLs in a MHC-restricted fashion. The apoptosis inhibitor protein survivin is overexpressed in most human cancers, and inhibition of its function results in increased apoptosis. Therefore, this protein may serve as a target for therapeutic CTL responses. Here, using CTL epitopes deduced from survivin, we describe specific T-cell reactivity against this antigen in peripheral blood from chronic lymphatic leukemia patients and in tumor-infiltrated lymph nodes from melanoma patients by ELISPOT analysis. CTL responses against two survivin-deduced peptide epitopes were detected in three of six melanoma patients and three of four chronic lymphatic leukemia patients. No T-cell reactivity was detected in peripheral blood lymphocytes from six healthy controls. Thus, survivin may serve as an important and widely applicable target for anticancer immunotherapeutic strategies.
CD4(+) T cells control the effector function, memory, and maintenance of CD8(+) T cells. Paradoxically, we found that absence of CD4(+) T cells enhanced adoptive immunotherapy of cancer when using CD8(+) T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4(+)CD25(-) Th cells (Th cells) with tumor/self-reactive CD8(+) T cells and vaccination into CD4(+) T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4(+) T cells that contained a mixture of Th and CD4(+)CD25(+) T regulatory cells (T(reg) cells) or T(reg) cells alone prevented effective adoptive immunotherapy. Maintenance of CD8(+) T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2(-/-) mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring T(reg) cells to be effective.
T cell differentiation is a progressive process characterized by phenotypic and functional changes. By transferring tumor-specific CD8+ T cells into tumor-bearing mice at various stages of differentiation, we evaluated their efficacy for adoptive immunotherapy. We found that administration of naive and early effector T cells, in combination with active immunization and IL-2, resulted in the eradication of large, established tumors. Despite enhanced in vitro antitumor properties, more-differentiated effector T cells were less effective for in vivo tumor treatment. Several events may underlie this paradoxical phenomenon: (a) downregulation of lymphoid-homing and costimulatory molecules; (b) inability to produce IL-2 and access homeostatic cytokines; and (c) entry into a proapoptotic and replicative senescent state. While the progressive acquisition of terminal effector properties is characterized by pronounced in vitro tumor killing, in vivo T cell activation, proliferation, and survival are progressively impaired. These findings suggest that the current methodology for selecting T cells for transfer is inadequate and provide new criteria for the generation and the screening of optimal lymphocyte populations for adoptive immunotherapy.
Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8+ T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4+CD25+ regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8+ T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The gammaC cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of gammaC cytokine-responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8+ T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells.
Adoptive cell transfer after host preconditioning by lymphodepletion represents an important advance in cancer immunotherapy. Here, we describe how a lymphopaenic environment enables tumour-reactive T cells to destroy large burdens of metastatic tumour and how the state of differentiation of the adoptively transferred T cells can affect the outcome of treatment. We also discuss how the translation of these new findings might further improve the efficacy of adoptive cell transfer through the use of vaccines, haematopoietic-stem-cell transplantation, modified preconditioning regimens, and alternative methods for the generation and selection of the T cells to be transferred.
Multiple isoforms (TAG-1, TAG-2a, TAG-2b, and TAG-2c) of a novel cancer/testis antigen gene have been identified and are expressed in 84-88% of melanoma cell lines tested. The tumor antigen (TAG) genes are also expressed in K562, a myelogenous leukemia cell line, and they have homology to two chronic myelogenous leukemia-derived clones and a hepatocellular carcinoma clone in the human expressed sequence tags (EST) database, thus indicating that their expression is not restricted to melanomas. In contrast to the fact that many cancer/testis antigens are poorly immunogenic, the TAG-derived peptide, RLSNRLLLR, is recognized by HLA-A3-restricted, melanoma-specific CTLs that were obtained from a melanoma patient with spontaneous reactivity to the peptide. Unlike most cancer/testis antigen genes which are located on the X chromosome, the TAG genes are located on chromosome 5. The genes have the additional unusual features of being coded for in an open reading frame that is initiated by one of three nonstandard initiation codons, and the sequence coding the RLSNRLLLR peptide crosses an exon-exon boundary. The properties of the TAG antigens indicate that they are excellent vaccine candidates for the treatment of melanoma and perhaps other cancers.
8510 Background: Treatment of metastatic melanoma patients with adoptively transferred Tumor Infiltrating Lymphocytes (TIL) has developed into an effective therapy. The use of unselected, short term cultured (young) TIL simplifies the laboratory production process. Our aim was to explore the effectiveness and toxicity of adoptive young TIL transfer in metastatic melanoma patients (ClinicalTrial.gov NCT00287131). Methods: Patients were eligible if they had progressed on prior IL2 based treatment and had adequate organ function. Amended protocol since 5/10 allowed recruitment of patients with CNS metastasis without major neurologic deficit. Patients received lympho-depleting chemotherapy with CTX and fludarabin before TIL administration followed by high dose bolus IL2 infusion. Results: Sixty five patients were enrolled in the study and Young-TIL cultures were successfully generated for 60 (92%). Ten patients clinically deteriorated during TIL production, resulting in a total drop-out rate of 23% (15/65). 2...
Activation-induced cell death (AICD) of lymphocytes is an important mechamism of self-tolerance. In CD4+ T cells, AICD is mediated by the Fas pathway and is enhanced by IL-2. To define the mechanisms of this pro-apoptotic action of IL-2, we analyzed CD4+ T cells from wild-type and IL-2−/− mice expressing a transgenic T cell receptor. T cells become sensitive to AICD after activation by antigen and IL-2. IL-2 increases transcription and surface expression of Fas ligand (FasL) and suppresses transcription and expression of FLIP, the inhibitor of apoptosis. The ability of IL-2 to enhance expression of a pro-apoptotic molecule, FasL, and to suppress an inhibitor of Fas signaling, FLIP, likely accounts for the role of this cytokine in potentiating T cell apoptosis.
Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has proven effective in metastatic melanoma and should therefore be explored in other types of cancer. The aim of this study was to examine the feasibility of potentially expanding clinically relevant quantities of tumor-specific T-cell cultures from TIL from patients with head and neck squamous cell carcinoma (HNSCC) using a more rapid expansion procedure compared with previous HNSCC studies.
In a two-step expansion process, initially TIL bulk cultures were established from primary and recurrent HNSCC tumors in high-dose interleukin (IL)-2. Secondly, selected bulk cultures were rapidly expanded using anti-CD3 antibody, feeder cells and high-dose IL-2. T-cell subsets were phenotypically characterized using flow cytometry. T-cell receptor (TCR) clonotype mapping was applied to examine clonotype dynamics during culture. Interferon (INF)-γ detection by Elispot and Cr(51) release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion.
TIL bulk cultures were expanded in 80% of the patients included, showing tumor specificity in 60% of the patients. Rapid expansions generated up to 3500-fold expansion of selected TIL cultures within 17 days. The cultures mainly consisted of T-effector memory cells, with varying distributions of CD8(+) and CD4(+) subtypes both among cultures and patients. TCR clonotype mapping demonstrated oligoclonal expanded cultures, ranging from approximately 10 to 30 T-cell clonotypes. TIL from large-scale rapid expansions maintained functional capacity, and contained tumor-specific T cells.
The procedure is feasible for expansion of TIL from HNSCC, ensuring clinically relevant expansion folds within 7 weeks. The cell culture kinetics and phenotypes of the TIL resemble previously published results on TIL from melanoma, setting the stage for clinical testing of this promising treatment strategy for patients with HNSCC.
Treatment of metastatic melanoma patients with adoptively transferred tumor infiltrating lymphocytes (TIL) has developed into an effective therapy. Various studies reported objective responses of 50% and more. The use of unselected, minimally cultured, bulk TIL (Young-TIL) has simplified the TIL production process and may therefore, allow the accessibility of this approach to cancer centers worldwide. This article describes the precise process leading to the large-scale production of Young-TIL for therapy. We have enrolled 55 melanoma patients and optimized their Young-TIL generation process. Young-TIL cultures were successfully established for 51 of 55 (93%) patients in 16.7 ± 5.5 days. In a large-scale expansion procedure Young-TIL of 32 patients were further expanded to treatment levels, resulting in a final number of 4.5 x 10¹⁰ ± 2.0 x 10¹⁰ TIL. Fifteen of 31 (48%) patients, who were evaluated, achieved a clinical response, including 4 complete and 11 partial responses. We confirmed the significant correlation between short culture duration, high number of infused cells, and tumor regression. A high percentage of CD8 T cells in the infusion product was beneficial to achieve an objective response. All responding patients were treated with Young-TIL cultures established in < 20 days. In summary, we describe here an efficient and reliable method to generate Young-TIL for adoptive transfer therapy, which may easily be adopted by other cancer centers and can lead to objective responses in 50% of refractory melanoma patients. In the future this approach may be used also in other types of malignancies.
In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). We sought to characterize the immune cells responsible for this regression. Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56(+)) with few T cells (12% CD3(+)). A majority (88%) of the NK cells were CD56(bright)CD16(-). TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56(+)CD16(-)CD3(-) subset showed reactivity similar to TIL-2742 while the CD56(-)CD16(-)CD3(+) cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic and prostate cancer cell lines and could lyse the autologous tumor as well as pancreas and prostate cancer lines. Reactivity was partially abrogated by blockade of TRAIL. We thus identified a unique subset of NK cells (CD56(bright)CD16(dim)) isolated from a regressing metastatic pancreatic cancer in a patient responding to Ipilimumab. This represents the first report of CD56(+)CD16(-) NK cells with apparent specificity for pancreatic and prostate cancer cell lines and associated with tumor regression following the treatment with an immune modulating agent.
NK cells can be divided into two subsets, CD56(dim) and CD56(bright) NK cells, based on their expression of CD56 and CD16. In the present study, we analyzed NK cell dysfunction in patients with esophageal squamous cell carcinoma (ESCC), with a particular focus on the expression of CD16 and CD56 molecules. Expression of CD16 and CD56, and the distribution of CD56(dim) or CD56(bright) NK cells gated on CD56(+)CD3(-) NK cells were compared between ESCC patients (n= 40) and healthy donors (n= 38). Purified NK cells were evaluated for Cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against epidermal growth factor receptor (EGFR)-expressing ESCC cell lines. Although there were no significant differences in the distribution of CD56(dim) and CD56(bright) NK cells between ESCC patients and healthy donors, down-regulated CD16 and up-regulated CD56 were significantly observed on NK cells of ESCC patients, paralleling the impairment of Cetuximab-mediated ADCC, in comparison with healthy donors. After patients received curative resections of ESCC, the down-regulated CD16 and up-regulated CD56 were significantly restored to the levels of healthy donors. Moreover, TGF-beta1 partially contributed to down-regulation of CD16 on NK cells. Down-regulated CD16 and up-regulated CD56 molecules on NK cells were observed in ESCC patients, resulting in NK cell dysfunction.
Adoptive cell therapy (ACT) is the best available treatment for patients with metastatic melanoma. In a recent series of three consecutive clinical trials using increasing lymphodepletion before infusion of autologous tumor infiltrating lymphocytes (TIL), objective response rates between 49% and 72% were seen. Persistence of infused cells in the circulation at one month was highly correlated with anti-tumor response as was the mean telomere length of the cells infused and the number of CD8+ CD27+ cells infused. Responses occur at all sites and appear to be durable with many patients in ongoing response beyond three years. In the most recent trial of 25 patients receiving maximum lymphodepletion, seven of the 25 patients (28%) achieved a complete response. Of the 12 patients in the three trials who achieved a complete response all but one are ongoing between 18 and 75 months. We recently demonstrated that ACT using autologous lymphocytes genetically modified to express anti-tumor T cell receptors can mediate tumor regression and this approach is now being applied to patients with common epithelial cancers.
Human natural killer (NK) cells can be subdivided into different populations based on the relative expression of the surface markers CD16 and CD56. The two major subsets are CD56(bright) CD16(dim/) (-) and CD56(dim) CD16(+), respectively. In this review, we will focus on the CD56(bright) NK cell subset. These cells are numerically in the minority in peripheral blood but constitute the majority of NK cells in secondary lymphoid tissues. They are abundant cytokine producers but are only weakly cytotoxic before activation. Recent data suggest that under certain conditions, they have immunoregulatory properties, and that they are probably immediate precursors of CD56(dim) NK cells. CD56(bright) NK cell percentages are expanded or reduced in a certain number of diseases, but the significance of these variations is not yet clear.
Telomere length analysis has been greatly simplified by the quantitative flow cytometry technique FISH-flow. In this method, a fluorescein-labeled synthetic oligonucleotide complementary to the telomere terminal repeat sequence is hybridized to the telomere sequence and the resulting fluorescence measured by flow cytometry. This technique has supplanted the traditional laborious Southern blot telomere length measurement techniques in many laboratories, and allows single cell analysis of telomere length in high-throughput sample formats. Nevertheless, the harsh conditions required for telomere probe annealing (82 °C) has made it difficult to successfully combine this technique with simultaneous immunolabeling. Most traditional organic fluorescent probes (i.e. fluorescein, phycoerythrin, etc.) have limited thermal stability and do not survive the high temperature annealing process, despite efforts to covalently crosslink the antigen-antibody-fluorophore complex. This loss of probe fluorescence has made it difficult to measure FISH-flow in complex lymphocyte populations, and has generally forced investigators to use fluorescent-activated cell sorting to pre-separate their populations, a laborious technique that requires prohibitively large numbers of cells.
Adoptive cell therapy (ACT) with tumor-reactive lymphocytes in patients with refractory melanoma can result in tumor regression and prolonged survival. Generating tumor-reactive lymphocyte cultures is technically difficult and resource intensive; these limitations have restricted the widespread application of ACT. Tumor-infiltrating lymphocytes (TIL) from melanoma contain tumor antigen-reactive cells. The "standard" method for producing TIL cultures for clinical administration requires extended in vitro expansion in interleukin-2, then identification of tumor-reactive cells by immunologic assays. We show here that limitations in reagents and methods during screening underrepresent the actual reactivity of TIL cultures. Furthermore, the extended culture times necessitated by the screening assays resulted in telomere shortening and reduced expression of CD27 and CD28 in the TIL cultures, properties that our prior studies showed are correlated with in vivo persistence and clinical response. We have thus developed an alternative "young" TIL method that demonstrated superior in vitro attributes compared with standard TIL. This approach uses the entire resected tumor to rapidly expand TIL for administration without in vitro testing for tumor recognition. Our observations suggest that younger TIL can have an undetermined but high level of antigen reactivity, and other advantageous attributes such as long telomeres and high levels of CD27 and CD28. We suggest that minimally cultured, unselected lymphocytes represent an alternative strategy for generating TIL cultures suitable for use in ACT that, if effective in vivo, may facilitate the widespread application of this approach to a broader population of patients with melanoma.
Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G(0/1) cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length.
Telomere Flow FISH is a recently developed method which allows the measurement of telomere length in purified subsets of cells using flow cytometry. However, the harsh conditions required for flow FISH have precluded its use with conventional cell surface staining, thus limiting its utility for large scale clinical studies. We have now developed a method which permits simultaneous analysis of cell surface markers along with telomere length estimation by flow cytometry. This new assay employs the covalent crosslinking of monoclonal antibodies conjugated with a heat stable fluorochrome to the cell surface prior to flow FISH. Using this technique we have confirmed that human germinal center B cells (IgD(-)/CD38(+)) have dramatically longer telomeres than pre-germinal center founder B cells (IgD(+)/CD38(+)). This approach simplifies the analysis of complex cell populations and will facilitate widespread investigation of telomere length in health and disease states.
We have developed culture conditions for the efficient expansion of cytotoxic effector cells from peripheral blood mononuclear cells (PBMC) by the timed addition of cytokine-rich supernatants collected from allogeneic PBMC cultures stimulated with anti-CD3 monoclonal antibody (mAb) (allogeneic CD3 supernatants; ACD3S). These cytotoxic effectors belonged primarily to CD56(+) natural killer (NK) cells, and the cell subset with the greatest cytotoxic activity was an otherwise rare population of CD3(+)CD56(+) cells (NKT cells) that expand dramatically under these conditions. CD3(+)CD56(+) cytotoxic effectors were generated from the PBMC of 16 patients with several types of cancer. The CD3(+)CD56(+) cell subset expanded significantly and efficiently lysed NK- as well as lymphokine-activated killer (LAK)-sensitive targets. More importantly, ACD3S-activated CD3(+)CD56(+) cells were capable of efficiently lysing autologous tumor cells including metastatic colorectal, ovarian, breast, lung and pancreatic tumor cells as well as melanoma cells. ACD3S-expanded CD3(+)CD56(+) cells exhibited increased levels of cytoplasmic interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma) and perforin. CD3(+)CD56(+) cell-mediated cytotoxicity was not restricted by major histocompatibility complex (MHC) gene products, since it was not blocked by anti-MHC class I mAb but was highly inhibited in the presence of CD2- and CD18-specific mAb. These data suggest that CD3(+)CD56(+) cells expanded under the presence of ACD3S may be utilized in clinical protocols for cancer immunotherapy.
Background:
Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens.
Methods:
Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis.
Results:
The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques.
Conclusions:
The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.
Natural killer (NK) cells (CD56(+)/CD3(-)) in the circulation of cancer patients were reported to have low NK activity and undergo spontaneous apoptosis. A possible relationship between apoptosis and impaired NK activity was studied by Annexin V-binding and NK-cell assays performed with peripheral blood mononuclear cells of patients with head and neck cancer (HNC), breast cancer (BC) and normal controls (NC). Cells stained with Annexin V (Anx) and antibodies to CD56, CD3, CD95, CD25, CD122 or CD132 were examined by flow cytometry. NK activity was tested against K562 targets in 4-h (51)Cr-release assays. The ratio of CD56(dim)/CD56(bright) NK cells was significantly different in patients vs. controls (10 vs. 16; p<0.01). A significantly greater percentage of CD56(dim) NK cells bound Anx in HNC patients (27+/-17%, median +/- SD) or BC (46+/-18%) than in NC (15+/-18%, p<0.04 and p<0.0002, respectively). CD56(dim) NK cells were preferentially targeted for apoptosis. NK activity was significantly lower in patients with HNC and BC than in NC (p<0.009). An inverse correlation between NK activity and the percent of Anx(+)CD56(dim) NK cells was observed in cancer patients (p =0.002) but not in NC. In patients, circulating CD56(dim) NK cells were targeted for apoptosis, leading to low levels of NK activity.
The generation of T lymphocytes with specific reactivity against tumor antigens is a prerequisite for effective adoptive transfer therapies. Melanoma-specific lymphocyte cultures can be established from tumor infiltrating lymphocytes (TILs) by in vitro culture in high levels of IL-2. We have optimized methods for generating melanoma-reactive TIL cultures from small resected tumor specimens. We report a retrospective analysis of 860 attempted TIL cultures from 90 sequential melanoma biopsy specimens from 62 HLA-A2+ patients. Multiple independent TIL derived from a single tumor often exhibited substantial functional and phenotypic variation. Tumor specific activity was detected in TIL from 29 (81%) of 36 patients screened. TIL cultures selected for high activity were generally capable of large numerical expansion using a single round of a rapid expansion protocol. Limited clonal T-cell populations in an oligoclonal TIL culture could confer specific tumor recognition in these highly selected, highly expanded TIL cultures. These methods were efficient at generating TILs suitable for adoptive transfer therapy.
We isolated pure, viable populations of tumor-cytolytic T cells directly from patient blood samples using flow cytometric quantification of the surface mobilization of CD107a-an integral membrane protein in cytolytic granules-as a marker for degranulation after tumor stimulation. We show that tumor-cytolytic T cells are indeed elicited in patients after cancer vaccination, and that tumor reactivity is strongly correlated with efficient T-cell recognition of peptide-bearing targets. We combined CD107a mobilization with peptide-major histocompatibility complex (P-MHC) tetramer staining to directly correlate antigen specificity and cytolytic ability on a single-cell level. This showed that tumor-cytolytic T cells with high recognition efficiency represent only a minority of peptide-specific T cells elicited in patients after heteroclitic peptide vaccination. We were also able to expand these cells to high numbers ex vivo while maintaining their cytolytic potential. These techniques will be useful not only for immune monitoring of cancer vaccine trials, but also for adoptive cellular immunotherapy after ex vivo expansion. The ability to rapidly identify and isolate tumor-cytolytic T cells would be very useful in cancer immunotherapy.
In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen-specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA- CD62 ligand- (CD62L-) CC chemokine receptor 7- (CCR7-) interleukin-7 receptor alphaLo (IL-7RalphaLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7Ralpha in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen-specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+)CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L- CCR7- profile, which may explain in part their ability to mediate tumor destruction.
B-cell lymphoma 2 (Bcl-2) is a pivotal regulator of apoptotic cell death and it is overexpressed in many cancers. Consequently, the Bcl-2 protein is an attractive target for drug design, and Bcl-2-specific antisense oligonucleotides or small-molecule Bcl-2 inhibitors have shown broad anticancer activities in preclinical models and are currently in several clinical trials. The clinical application of immunotherapy against cancer is rapidly moving forward in multiple areas, including the adoptive transfer of anti-tumor-reactive T cells and the use of "therapeutic" vaccines. The overexpression of Bcl-2 in cancer and the fact that immune escape by down-regulation or loss of expression of this protein would impair sustained tumor growth makes Bcl-2 a very attractive target for anticancer immunotherapy. Herein, we describe spontaneous T-cell reactivity against Bcl-2 in peripheral blood from patients suffering from unrelated tumor types (ie, pancreatic cancer, breast cancer, acute myeloid leukemia [AML], and chronic lymphocytic leukemia [CLL]). Additionally, we show that these Bcl-2-reactive T cells are indeed peptide-specific, cytotoxic effector cells. Thus, Bcl-2 may serve as an important and widely applicable target for anticancer immunotherapeutic strategies (eg, in the combination with conventional radiotherapy and chemotherapy).
The lack of persistence of transferred autologous mature lymphocytes in humans has been a major limitation to the application of effective cell transfer therapies. The results of a pilot clinical trial in 13 patients with metastatic melanoma suggested that conditioning with nonmyeloablative chemotherapy before adoptive transfer of activated tumor-reactive T cells enhances tumor regression and increases the overall rates of objective clinical responses. The present report examines the relationship between T cell persistence and tumor regression through analysis of the TCR beta-chain V region gene products expressed in samples obtained from 25 patients treated with this protocol. Sequence analysis demonstrated that there was a significant correlation between tumor regression and the degree of persistence in peripheral blood of adoptively transferred T cell clones, suggesting that inadequate T cell persistence may represent a major factor limiting responses to adoptive immunotherapy.
Recent studies have indicated that adoptive immunotherapy with autologous antitumor tumor-infiltrating lymphocytes (TILs) following nonmyeloablative chemotherapy mediates tumor regression in approximately 50% of treated patients with metastatic melanoma, and that tumor regression is correlated with the degree of persistence of adoptively transferred T cells in peripheral blood. These findings, which suggested that the proliferative potential of transferred T cells may play a role in clinical responses, led to the current studies in which telomere length as well as phenotypic markers expressed on the administered TILs were examined. TILs that were associated with objective clinical responses following adoptive transfer possessed a mean telomere length of 6.3 kb, whereas TILs that were not associated with significant clinical responses were significantly shorter, averaging 4.9 kb (p < 0.01). Furthermore, individual TIL-derived T cell clonotypes that persisted in vivo following adoptive cell transfer possessed telomeres that were longer than telomeres of T cell clonotypes that failed to persist (6.2 vs 4.5 kb, respectively; p < 0.001). Expression of the costimulatory molecule CD28 also appeared to be associated with long telomeres and T cell persistence. These results, indicating that the telomere length of transferred lymphocytes correlated with in vivo T cell persistence following adoptive transfer, and coupled with the previous observation that T cell persistence was associated with clinical responses in this adoptive immunotherapy trial, suggest that telomere length and the proliferative potential of the transferred T cells may play a significant role in mediating response to adoptive immunotherapy.
Proper T cell function relies on the integration of signals delivered by Ag, cytokine, and costimulatory receptors. In this study, the interactions between IL-2, CD27, and its ligand CD70 and their effects on human T cell function were examined. Unstimulated CD8(+) T cells expressed relatively low levels of CD70 and high levels of CD27. Incubation in vitro with high doses of IL-2 (3,000 IU/ml) or administration of IL-2 in vivo resulted in substantial up-regulation of CD70 expression and the concomitant loss of cell surface CD27 expression on CD8(+) cells. Withdrawal of IL-2 from activated CD8(+) T cells that had been maintained in IL-2 resulted in a reversal of the expression of these two markers, whereas reciprocal changes were seen following treatment of PBMCs with IL-2. The proliferation observed in cells stimulated with IL-2 primarily occurred in a subset of the CD70(+)CD8(+) T cells that up-regulated IL-2 receptor expression but did not occur in CD70(-)CD8(+) T cells. Blocking CD70 resulted in a significant reduction of T cell proliferation induced by high-dose IL-2, indicating that the interaction of CD70 with CD27 played a direct role in T cell activation mediated by IL-2. Finally, studies conducted on tumor-infiltrating lymphocyte (TIL) samples that were administered to melanoma patients indicated that the size of the pool of CD27(+)CD8(+) T cells in bulk TILs was highly associated (p = 0.004) with the ability of these TILs to mediate tumor regression following adoptive transfer.
A subset of human T lymphocytes expresses the natural killer (NK) cell-associated receptor CD56 and is capable of major histocompatibility complex (MHC)-unrestricted cytotoxicity against a variety of autologous and allogeneic tumor cells. CD56+ T cells have shown potential for immunotherapy as antitumor cytotoxic effectors, but their capacity to control adaptive immune responses via cytokine secretion is unclear. We have examined the inducibility of CD56+ T cells from human blood in vitro and compared the kinetics of Th1, Th2, and regulatory cytokine secretion by CD56+ T cells with those of conventional CD56- T cells. CD56 was induced on CD8+ and CD4- CD8- T cells by CD3/T-cell receptor (TCR)-mediated activation, particularly when grown in the presence of interleukin (IL)-2. Activation-induced CD56+ T cells proliferated less vigorously but displayed enhanced natural cytotoxicity compared with CD56- T cells. CD56+ T cells released interferon-gamma (IFN-gamma) and interleukin-13 (IL-13), but not IL-10, upon TCR stimulation. Flow cytometric analysis demonstrated that, compared with CD56- T cells, elevated proportions of CD56+ T cells expressed IFN-gamma, IL-4, and IL-13 within hours of activation. These acquired cytolytic and cytokine secretion activities of CD56+ T cells make them potential targets for immunotherapy for infectious and immune-mediated disease.
Transfer of autologous tumor-specific tumor infiltrating lymphocytes (TILs) in adoptive immunotherapy can mediate the regression of tumor in patients with metastatic melanoma. In this procedure, TILs from resected tumors are expanded in vitro, then administered to patients and further stimulated to proliferate in vivo by the administration of high dose IL-2. After in vitro expansion, TILs are often dominated by a few specific clonotypes, and recently it was reported that the persistence in vivo of one or more of these clonotypes correlated with positive therapeutic response. We and others have previously shown that repeated in vitro stimulation and clonal expansion of normal human T lymphocytes results in progressive decrease in telomerase activity and shortening of telomeres, ultimately resulting in replicative senescence. In the studies reported here, we therefore compared telomerase activity and telomere length in persistent and nonpersistent TIL clonotypes before transfer in vivo, and found a correlation between telomere length and clonal persistence. We also observed that TILs proliferate extensively in vivo in the days after transfer, but fail to induce substantial telomerase activity, and undergo rapid decreases in telomere length within days after transfer. Thus, in vivo loss of telomeres by clonotypes that have the shortest telomeres at the time of administration may drive these clones to replicative senescence, whereas cells with longer telomeres are able to persist and mediate antitumor effects. These findings are relevant both to predicting effectiveness of adoptive immunotherapy and in deriving strategies for improving effectiveness by sustaining telomere length.
The ovarian tumour marker MUC16 (CA125) inhibits the cytotoxic responses of human natural killer (NK) cells and down-regulates CD16. Here we show that approximately 10% of the peripheral blood NK cells (PBNK) from the epithelial ovarian cancer (EOC) patients are CD16(-) CD56(br) whereas 40% of the peritoneal fluid NK (PFNK) carry this phenotype, which is usually associated with NK cells from the lymph nodes or human decidua. PBNK from healthy donors exposed to PF show a significant increase in the CD16(-) CD56(br) population. This shift in phenotype is not caused by increased apoptosis of the CD16(+) CD56(dim) cells or selective proliferation of the CD16(-) CD56(br) NK cells. Thus, the terminal differentiation of the CD16(-) CD56(br) NK cells to CD16(+) CD56(dim) subset that occurs during normal NK cell development may actually be a reversible step. A majority of the NK cell receptors (NKp46, NKp44, NKG2D, CD244, CD226, CD158a, CD158b, and CD158e) studied were down-regulated in the PFNK. MUC16 binds selectively to 30-40% of CD16(+) CD56(dim) NK cells in EOC patients indicating that phenotypic alterations in these cells are mediated by tumour-derived soluble factors. Similar to EOC, MUC16 in early pregnancy also binds to NK cells suggesting shared mechanisms of NK cell suppression in feto-maternal tolerance and immune evasion by ovarian cancers.
Despite natural killer (NK) cells being originally identified and named because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltrating malignant tumors, especially in humans.
NK cells infiltrating human nonsmall cell lung cancers (NSCLC) were analyzed with the aim of identifying their potential protective role in an antitumor immune response. Both relevant molecule expression and functions of NK cells infiltrating NSCLC were analyzed in comparison with autologous NK cells isolated from either peritumoral normal lung tissues or peripheral blood.
The CD56 bright CD16(-) NK cell subset was consistently observed as being highly enriched in tumor infiltrate and displayed activation markers, including NKp44, CD69, and HLA-DR. Remarkably, the cytolytic potential of NK cells isolated from cancer tissues was lower than that of NK cells from peripheral blood or normal lung tissue, whereas no difference was observed regarding their capability of producing cytokines. With regard to their localization within tumor, NK cells were found in tumor stroma, whereas they were not in direct contact with cancer cells.
For the first time NK cells infiltrating NSCLC have been characterized and it is shown that they are mainly capable of producing relevant cytokines rather than exerting direct cancer cell killing.