Bulk Tumor-infiltrating Lymphocytes Research Articles

Abstract 4052: Direct selection of PD1+ CD39+ tumor infiltrating lymphocytes (TIL) from tumor dissociates enrich for functional tumor-reactive cells

Abstract Background: The autologous cell therapy field has investigated numerous novel strategies and processes to improve response rates and expand the use of adoptive cell therapies (ACT) to patient suffering from a broad range of cancers. Tumor infiltrating lymphocytes (TIL) can be harvested from tumors, expanded in vitro, and infused to patients leading to substantial clinical benefits in some patient populations. Tumor reactivity of TIL can vary greatly, representing a key limiting factor of bulk TIL therapies. The ability to generate an autologous cell product enriched in tumor reactive cells while limiting the presence of potentially detrimental and/or competitive non-reactive cells is highly desirable for next generation TIL products. Here we demonstrate that direct selection of antigen experienced TIL from tumor dissociates is feasible and allows to obtain functional cells enriched for tumor reactivity from various tumor indications. Methods: Patient-derived tumor material from various indications, including ovarian, kidney, colorectal cancer and lung tumors were first processed into single cell suspensions, maximizing TIL recovery and viability. Antigen experienced TIL were immediately selected based on the co-expression of PD-1 and CD39, using fluorescence-activated single cell sorting (FACS). The sorted cells or the unselected counterparts were expanded in a rapid expansion protocol (REP), then analyzed for phenotypic and functional characteristics and reactivity against autologous tumor cells. Results: The PD-1+ CD39+ selection strategy consistently allowed sorting of a population of viable TIL that was amenable to in vitro culture from tumors of various cancer indications. The selected TIL successfully expanded in a rapid expansion protocol. TCR sequencing analysis revealed that the PD-1+ CD39+ sorted populations were enriched in unique clones compared to the unselected populations. Expanded PD-1+ CD39+ selected cells demonstrated the ability to produce key effector cytokines upon restimulation in polyclonal assays. Importantly, the PD-1+ CD39+ expanded TIL were enriched for tumor reactive T cells and showed improved cytotoxic activity against autologous tumors. Conclusions: PD-1+ CD39+ selected TIL can be successfully isolated and expanded in vitro, generating a TIL product of superior reactivity in multiple cancer indications. PD-1+ CD39+ selected TIL showed increased cytokine secretion and cytotoxic activity against autologous material, indicating that this selection strategy enriches for functional tumor-reactive lymphocytes, which is likely to be a key feature of successful ACT. Citation Format: Christophe Pedros, Larissa Pikor, Anna Fritzsche, Zachary K. Jilesen, Bethany Macleod, Niloufar Khojandi, Bryant Thompson, Matthew Thayer, Nikolas Bryan, Emily Carron, Sowbarnika S Ratliff, April Fraley, Jake Nikota, Robert Fisher, Sebastien Delpeut, Anna Kluew, Madysson Scott, Christian Laing, Leo He, Antoine Bernard, Nathalie Brassard, Simon Turcotte, Timothy J Langer, David Stojdl, Stewart Abbot, Barbara Sennino. Direct selection of PD1+ CD39+ tumor infiltrating lymphocytes (TIL) from tumor dissociates enrich for functional tumor-reactive cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4052.

CD103 and CD39 coexpression identifies neoantigen-specific cytotoxic T cells in colorectal cancers with low mutation burden

BackgroundExpression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+ T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific...

Enriching for tumor-reactive CD8 TIL (AGX148) leads to effective tumor clearance in a patient-derived xenograph model

Abstract Adoptive Cell Therapy (ACT) with Tumor Infiltrating Lymphocytes (TIL) can induce durable clinical responses in a subset of patients with melanoma; however, the efficacy of standard bulk TIL therapy can be limited. We hypothesize that the therapeutic efficacy of TIL ACT is dependent on the frequency and functionality of tumor-reactive T cells when infused back into patients. Our approach enhances the activity of the ACT product by enriching for the tumor-reactive CD8 TIL prior to ex vivo expansion by sorting on cells that co-express CD103 and CD39. The CD103/CD39 double positive (DP) CD8 TIL can be found in most human solid tumors but at varying frequencies in any given patient. Surgically resected human tumor samples were provided through a collaboration with the EACRI. Tumor samples were flow sorted and expanded ex vivo and patient-matched autologous tumor lines were generated. Tumors were then cocultured with the expanded autologous ACT product (AGX148) in vitro or used in xenograph mouse experiments. The AGX148 TIL products are effective at recognizing and killing autologous tumor cells, in vitro. Upon adoptive transfer into patient-derived tumor-bearing huIL-2 NOG mice, the transferred human T cells increased in frequency, migrated to the tumor, became activated in the tumor and recognized/destroyed established tumors. Interestingly there was little to no in vivo activity of CD8 TIL that did not express these two markers. We have generated a potent therapeutic ACT TIL product (AGX148) through the selective isolation and expansion of tumor-reactive T cells. This TIL product effectively induces durable curative responses in hosts harboring autologous tumors providing rationale for clinical investigation.

Abstract LB-069: Persistence of cryopreserved tumor-infiltrating lymphocyte product lifileucel (LN-144) in C-144-01 study of advanced metastatic melanoma

Abstract Adoptive cell transfer utilizing tumor-infiltrating lymphocytes (TIL) is recognized as an effective treatment in metastatic melanoma and other solid tumors eliciting durable and complete responses in heavily pretreated patients, presumably by targeting somatic mutations specific to each tumor1. We have treated patients with anti-PD-1-refractory advanced melanoma with lifileucel and reported an overall response rate of 38%2. Here, we analyzed the composition of the initial TIL products and the T cells circulating 42 days post-infusion (D42) to uncover a potential link between clonal diversity, TIL in vivo persistence, and anti-tumor activity. Since TIL products are preparations of polyclonal autologous T cells, each T cell clone expresses a unique T cell receptor (TCR) that can be identified by its complementary determining region 3 (CDR3). CDR3 of TIL products and corresponding post-infusion peripheral blood samples were subjected to RNA-seq, using iRepertoire technology (Huntsville, AL). The average number of unique TCR CDR3 sequences (uCDR3) was 17511 [3574-110797] across TIL products, with Shannon diversity indexes varying from 2.7 to 10.8. Correlation analyses revealed no association between either parameter and clinical response, suggesting that tumor-reactive T cells may be present in low and high diversity bulk TIL products. At D42, TIL clones could be detected in the circulation of all treated patients. Numbers of Shared uCDR3 ranged from 28 to 6964 unique clonotypes and represented highly variable fractions of both the TIL product and the D42 circulating T cells. A link between Shared T cell clones and clinical response was previously hypothesized3. In this study, comparable percentages of Shared uCDR3 were detected in responders and non-responders. Importantly, the majority of Shared uCDR3 were not detected in the patients’ peripheral blood at the time of enrollment, indicating that they represented intratumoral clonotypes that persisted after TIL administration. In addition, Shared uCDR3 clones represented at either high or low frequencies in the TIL product could persist for at least 6 weeks post-infusion. Finally, more than 97% of persisting clones were uniquely present in individual responding and non-responding patients, indicating a unique repertoire in each TIL preparation. Overall, the data demonstrate that a fraction of TIL clones persisted in all patients. The uniqueness of the clonal profiles associated with response highlights the challenge of identifying a single TCR as mediator of activity and supports using a polyclonal product such as bulk TILs to treat solid tumors with their associated unique, patient-specific, mutational and neoantigen spectra. 1 Rosenberg et al. CCR 2011; 2 SITC Nov2018; 3 Robbins et al. J Immunol 2004 Citation Format: Viktoria Gontcharova, Sam Suzuki, Michelle R. Simpson-Abelson, Michelle Blaskovich, Cécile Chartier. Persistence of cryopreserved tumor-infiltrating lymphocyte product lifileucel (LN-144) in C-144-01 study of advanced metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-069.

Abstract B039: Peptide-MHC-directed expansion of multifunctional antigen-responsive T-cells

Abstract Functional properties and antigen specificity of expanded T-cells are crucial for the efficacy of adoptive cell transfer-based therapies in cancer. Most current strategies involve nonspecific expansion of bulk tumor-infiltrating lymphocytes, often providing growth preference to co-infiltrated virus specific T-cells and driving an exhausted phenotype of the expanded T-cell product.A potential way to resolve this challenge, is the use of artificial antigen-presenting scaffolds providing both an antigen specific stimulation through peptide-MHC interaction and additional the required co-stimulatory and growth signals through associated stimulatory molecules and cytokines. We have designed such antigen-presenting scaffolds; build on a dextran-polysaccharide, carrying both peptide-MHC and relevant stimulatory molecules. The artificial antigen-presenting scaffolds interacts specifically with T-cells based on recognition of the peptide-MHC molecule and effectively expand and functionally stimulate specific T-cells in a peptide-MHC-directed fashion, while leaving all other T-cell specificities untouched. Results from in vitro experiments have showed that antigen specific CD8 T-cells stimulated with these artificial antigen-presenting scaffolds express a less differentiated phenotype and low PD-1 expression, associated with high proliferation potential and enhanced antitumor effect in vivo. Furthermore, this expansion strategy provides a high frequency of multifunctional antigen specific CD8 T-cells expressing IFN-γ, TNF-α, and CD107a upon target recognition and provide improved cancer cell killing over IL2-driven expansion of tumor-infiltrating lymphocytes. Furthermore, the current strategy allows for simultaneous expansion of numerous different T-cell populations, required to generate T-cell products with broad recognition profiles based on the personal cancer-antigen and mutational profile. All of these characteristics are of significant importance for in vivo tumor cell killing following adoptive transfer of expanded T-cell products. Thus, the present strategy represents an optimized method for expansion of cancer-restricted T-cells for adoptive cell therapy. Citation Format: Vibeke Mindahl Rafa, Mona Bodenhöfer, Amalie Kai Bentzen, Tripti Tamhane, Marco Donia, Inge Marie Stentoft Svane, Søren Nyboe Jakobsen, Christian Schmess, Sine Reker Hadrup. Peptide-MHC-directed expansion of multifunctional antigen-responsive T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B039.

Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens.

The antigenicity of cells is demarcated by the peptides bound by their Human Leucocyte Antigen (HLA) molecules. Through this antigen presentation, T cell specificity response is controlled. As a fraction of the expressed mutated peptides is presented on the HLA, these neo-epitopes could be immunogenic. Such neoantigens have recently been identified through screening for predicted mutated peptides, using synthetic peptides or ones expressed from minigenes, combined with screening of patient tumor-infiltrating lymphocytes (TILs). Here we present a time and cost-effective method that combines whole-exome sequencing analysis with HLA peptidome mass spectrometry, to identify neo-antigens in a melanoma patient. Of the 1,019 amino acid changes identified through exome sequencing, two were confirmed by mass spectrometry to be presented by the cells. We then synthesized peptides and evaluated the two mutated neo-antigens for reactivity with autologous bulk TILs, and found that one yielded mutant-specific T-cell response. Our results demonstrate that this method can be used for immune response prediction and promise to provide an alternative approach for identifying immunogenic neo-epitopes in cancer.

Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

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

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

Phenotype and Function of T Cells Infiltrating Visceral Metastases from Gastrointestinal Cancers and Melanoma: Implications for Adoptive Cell Transfer Therapy

Adoptive cell transfer of tumor-infiltrating lymphocytes (TILs) can mediate cancer regression in patients with metastatic melanoma, but whether this approach can be applied to common epithelial malignancies remains unclear. In this study, we compared the phenotype and function of TILs derived from liver and lung metastases from patients with gastrointestinal (GI) cancers (n = 14) or melanoma (n = 42). Fewer CD3(+) T cells were found to infiltrate GI compared with melanoma metastases, but the proportions of CD8(+) cells, T cell differentiation stage, and expression of costimulatory molecules were similar for both tumor types. Clinical-scale expansion up to ~50 × 10(9) T cells on average was obtained for all patients with GI cancer and melanoma. From GI tumors, however, TIL outgrowth in high-dose IL-2 yielded 22 ± 1.4% CD3(+)CD8(+) cells compared with 63 ± 2.4% from melanoma (p < 0.001). IFN-γ ELISA demonstrated MHC class I-mediated reactivity of TIL against autologous tumor in 5 of 7 GI cancer patients tested (9% of 188 distinct TIL cultures) and in 9 of 10 melanoma patients (43% of 246 distinct TIL cultures). In these assays, MHC class I-mediated up-regulation of CD137 (4-1BB) expression on CD8(+) cells suggested that 0-3% of TILs expanded from GI cancer metastases were tumor-reactive. This study implies that the main challenge to the development of TIL adoptive cell transfer for metastatic GI cancers may not be the in vitro expansion of bulk TILs, but the ability to select and enrich for tumor-reactive T cells.

Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells

Significant tumor regressions have been observed in up to 70% of patients receiving adoptively transferred autologous melanoma-reactive tumor infiltrating lymphocytes (TIL) 1,2, and in pilot trials, 40% of treated patients experienced complete regressions of all measurable lesions for at least five years following treatment 3. To evaluate the potential association between the ability of TIL to mediate durable regressions and their ability to recognize potent antigens that presumably include mutated gene products, a novel screening approach was developed that involved mining whole exome sequence data to identify the mutated proteins that were expressed in patient tumors. Candidate mutated T cell epitopes that were identified using an MHC binding algorithm 4 were then synthesized and evaluated for recognition by TIL. Using this approach, mutated antigens expressed on autologous tumor cells were identified as targets of three TIL that were associated with objective tumor regressions following adoptive transfer. This simplified approach, which avoids the need to generate and laboriously screen cDNA libraries from tumors, may represent a generally applicable method for identifying mutated T cell antigens expressed in melanoma as well as other tumor types.

Abstract 1807: Tumor-infilitrating lymphocytes derived from pancreatic adenocarcinoma exhibit an activated, pro-inflammatory phenotype

Abstract Contrary to current dogma, which maintains that pancreatic adenocarcinoma (PA) is infiltrated by predominantly inhibitory, regulatory CD4+ T cells (Treg), we demonstrate expansion of activated, pro-inflammatory tumor-infiltrating lymphocytes (TILs) from PA that could be used for adoptive cell therapy. TILs were grown in vitro in high-dose IL-2 from fragments of primary or metastatic PA. Pancreatic TILs were successfully expanded in 9 of 10 tumors and were phenotyped by flow cytometry. CD3+ TILs were predominantly CD4+ (mean frequency 76%, range 37-97%). CD4+ TILs were assessed for Treg frequency by CD25 and FoxP3 co-expression as a percent of total CD3+ TILs. Tregs were found to comprise the minority of CD3+ TILs (mean=3.1%, range=0.36-6.6%). We assessed CD4+ TIL expression of CD69, a known marker of T cell activation, and found a relatively high percentage of CD4+ TILs expressed CD69 (mean=25%, range=9-46%), which was significantly higher than the mean percent of Treg (p=0.0006). To confirm pro-inflammatory function, bulk TILs were stimulated in vitro by PMA/ionomycin, and expression of the pro-inflammatory cytokine IFNγ and the cytolytic marker CD107a was assessed. CD4+ TILs demonstrated a relatively high level of expression of IFNγ (mean=35%, range=20-47%) and CD107a (mean=19%, range=10-26%). Despite a low relative frequency, CD8+ TILs (mean=17%, range=1.8-41%) also expressed CD69 (mean=46%, range=17-83%) and exhibited a pro-inflammatory function upon stimulation as demonstrated by IFNγ (mean=43%, range=31-61%) and CD107a (mean=42%, range=22-69%) expression. After CD4 depletion, CD8-enriched TILs were effectively expanded with α-CD3 and maintained an activated, pro-inflammatory phenotype. This is the first report demonstrating that CD4+ TILs derived from PA have a predominantly pro-inflammatory phenotype. CD8+ TILs, though in the minority, also displayed an activated phenotype and preserved this phenotype throughout selective expansion. Further work is ongoing to support a phase I clinical trial involving adoptive TIL therapy in PA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1807. doi:10.1158/1538-7445.AM2011-1807

Expansion and Characterization of Human Melanoma Tumor-Infiltrating Lymphocytes (TILs)

BackgroundVarious 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.Principal FindingsTILs 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 ≥3×107 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.ConclusionsTILs 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.

Enrichment of CD8+ Cells From Melanoma Tumor-infiltrating Lymphocyte Cultures Reveals Tumor Reactivity for Use in Adoptive Cell Therapy

Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for metastatic melanoma has shown objective response rates as high as 72%. The successful application of this therapy requires the selection of unique tumor-reactive lymphocyte cultures for each patient. This is a technically and logistically difficult undertaking, and patients who do not have tumor-reactive TIL are not considered eligible for treatment. To simplify the methods of TIL generation and extend TIL-based immunotherapy to additional patients, methods were developed to use unselected, minimally cultured ("young") TIL. Young TIL cultures contain a variable number of CD8(+), CD4(+), and CD3(-)CD56(+) natural killer cells. In this study we retrospectively investigated a role for these subsets in the clinical outcome of patients treated with TIL derived from selected microcultures. This analysis demonstrated a suggestive but nonsignificant association between the number of CD8(+) cells administered and tumor regression. We therefore investigated the feasibility of selecting CD8(+) cells from young TIL cultures for ACT therapy. The available methods for clinical scale CD8(+) enrichment proved inadequate for TIL, so an optimized CD8(+) enrichment method was developed and is reported here. We observed that CD8 (+)enrichment of some TIL cultures revealed in vitro tumor recognition that was not evident in bulk culture, and an improved in vitro recognition of tumor in other TIL cultures. In addition, the enriched CD8(+) young TIL expanded more reliably and predictably in rapid expansions than the bulk TIL. Thus, optimized CD8(+) selection combines the benefits of antigen-selected TIL and young TIL for generating lymphocyte cultures for ACT, and should be evaluated in cell transfer therapy protocols.

TGF-β1 Induces Preferential Rapid Expansion and Persistence of Tumor Antigen-specific CD8+ T Cells for Adoptive Immunotherapy

Adoptive cell transfer of expanded, autologous tumor-infiltrating lymphocytes (TIL) into lymphodepleted melanoma patients can induce the regression of bulky, metastatic disease. To generate the large numbers of T cells needed for infusion, TIL undergo a rapid expansion protocol (REP) in vitro using anti-CD3 antibody, interleukin-2, and irradiated peripheral blood feeder cells that typically results in an approximately 1000-fold expansion over 14 days. However, we have found that the conventional REP (C-REP) often favors the expansion of CD4+ T cells at the expense of tumor antigen-specific CD8+ T cells, which are the most potent cytolytic effector cells. In this study, we demonstrate that addition of transforming growth factor (TGF)-beta1 to the TIL culture at the onset of rapid expansion (T-REP ) maintained the percentage of CD8+ T cells while not inhibiting overall T-cell expansion. Of T cells expanded from different melanoma patient tumors, 13 of 15 TIL demonstrated improved yields and percentages of both CD8+ and MART-1 melanoma antigen-specific T cells after 14 days of expansion in TGF-beta1 compared with the C-REP. This was associated with a marked improvement in the antitumor activity of the resulting bulk TIL culture in terms of interferon-gamma production and melanoma tumor-specific cytotoxic T-lymphocyte activity. In addition, T-REP T cells demonstrated a higher potential for continued expansion in vitro for up to 3 weeks after the expansion compared with C-REP T cells, suggesting that they may also be capable of increased persistence after adoptive cell transfer. Our results suggest that TGF-beta1-expanded TIL have attributes that might predict efficacy superior to that of conventional TIL.

Clinical Responses in a Phase II Study Using Adoptive Transfer of Short-term Cultured Tumor Infiltration Lymphocytes in Metastatic Melanoma 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.

Tumor‐infiltrating lymphocytes derived from human renal cell carcinoma: Clonal analysis of its characteristics

To assess the characteristics of activated tumor-infiltrating lymphocytes (TIL), we report the isolation, growth response, and functional analysis of a CD4(-) CD8(+) TIL-clone derived from human renal cell carcinoma (RCC). Bulk TILs were expanded from a human RCC and the lymphocytes were separated into a CD8(+) enriched population. Subsequently, using the limiting dilution technique, a TIL clone was established and its growth response, phenotype and cytotoxic activity were analyzed. A clone, T16-13, by day 94 numbering 1 x 10(7) cells, was harvested and characterized as a CD4(-) CD8(+) clone. On day 144, the cytotoxic activity of this clone against the autologous tumor was relatively high (2.3 +/- 0.7 LU(30)/10(6) cells). Meanwhile, against allogeneic renal tumors, there was no cytotoxic activity (-0.1 LU(30)/10(6) cells). A TIL clone possessing modest autologous tumor-specific cytotoxicity can be isolated from human RCC. The characteristics analysis of various TIL clones may provide a better understanding of an RCC tumor microenvironment and may help to establish new modalities for the treatment of patients with metastatic kidney cancer.

T cells associated with tumor regression recognize frameshifted products of the CDKN2A tumor suppressor gene locus and a mutated HLA class I gene product.

The dramatic tumor regression observed following adoptive T cell transfer in some patients has led to attempts to identify novel Ags to understand the nature of these responses. Nearly complete regression of multiple metastatic melanoma lesions was observed in patient 1913 following adoptive transfer of autologous tumor-infiltrating lymphocytes. The autologous 1913 melanoma cell line expressed a mutated HLA-A11 class I gene product that was recognized by the bulk tumor-infiltrating lymphocytes as well as a dominant T cell clone derived from this line. A second dominant T cell clone, T1D1, did not recognize the mutated HLA-A11 product, but recognized an allogeneic melanoma cell line that shared expression of HLA-A11 with the parental tumor cell line. Screening of an autologous melanoma cDNA library with clone T1D1 T cells in a cell line expressing the mutated HLA-A11 gene product resulted in the isolation of a p14ARF transcript containing a 2-bp deletion in exon 2. The T cell epitope recognized by T1D1, which was encoded within the frameshifted region of the deleted p14ARF transcript, was also generated from frameshifted p14ARF or p16INK4a transcripts that were isolated from two additional melanoma cell lines. The results of monitoring studies indicated that T cell clones reactive with the mutated HLA-A11 gene product and the mutated p14ARF product were highly represented in the peripheral blood of patient 1913 1 wk following adoptive transfer, indicating that they may have played a role in the nearly complete tumor regression that was observed following this treatment.

Analysis of Cytokine Secretion by Melanoma-Specific CD4+T Lymphocytes

Although specific antitumor immune reactivity has been documented extensively in CD8+ T cells derived from melanoma patients, relatively little is known about CD4+ T cell responses against melanoma. Tumor-infiltrating lymphocytes (TIL) cultured from metastatic lesions in five patients yielded cytolytic CD8+ T cells with specific activity against autologous and MHC class I-compatible allogeneic melanoma targets. In four of the five cases studied, CD4+ T cells purified from bulk TIL cultures also reacted specifically with autologous melanoma cells, as manifested by the secretion of various cytokines (GM-CSF, TNF-alpha, and IFN-gamma) after a 24 h cocultivation. Cytokine secretion by CD4+ T cells was MHC class II restricted, and proved to be a more reliable indicator of the immunologic reactivity of CD4+ T cells than cytolysis. Three of the four reactive CD4+ TIL failed to recognize allogeneic melanomas, suggesting recognition of Ag with limited expression in the patient population. Cloning such Ags may provide clues to optimizing current antitumor immunization strategies.

CD4+ T lymphocytes infiltrating human breast cancer recognise autologous tumor in an MHC-class-II restricted fashion.

Tumor-infiltrating lymphocytes (TIL) were derived from primary breast tumors, metastatic lymph nodes and malignant pleural effusions from 34 patients with breast cancer. TIL were cultured for approximately 30 days and studied for phenotype, cytotoxicity, and the ability to secrete cytokines in response to autologous tumor stimulation. Tumor specimens were obtained from two different sites in 7 patients, resulting in 41 samples from which 38 TIL cultures were established. In addition to screening 38 bulk TIL cultures, TIL from 21 patients were separated into CD4+ and CD8+ subsets and extensively studied. Three CD4+ TIL were found specifically to secrete granulocyte macrophage-colony-stimulating factor and tumor necrosis factor alpha when stimulated by autologous tumor and not by a large panel of stimulators (24-34) consisting of autologous normal cells, allogeneic breast or melanoma tumors and EBV-B cells. This cytokine release was found to be MHC-class-II-restricted, as it was inhibited by the anti-HLA-DR antibody L243. These 3 patients' EBV-B cells, when pulsed with tumor lysates, were unable to act as antigen-presenting cells and induce cytokine secretion by their respective CD4+ TIL. These findings demonstrate that MHC-class-II-restricted CD4+ T cells recognising tumor-associated antigens can be detected in some breast cancer patients.

Isolation and characterization of tumor-infiltrating lymphocytes from cervical carcinoma.

The evidence that virus-induced tumors generally elicit T-cell responses prompts the notion that HPV-related cervical carcinoma would be amenable to treatment by T-cell-mediated adoptive therapy. Therefore, we cultured and cloned tumor-infiltrating lymphocytes (TIL) from a patient with cervical carcinoma and studied the in vitro characteristics of these TIL by using the established autologous tumor-cell line. After stimulation of bulk TIL cultures with 1,000 Units/ml recombinant interleukin 2 (rIL-2), followed by limiting dilution, T-cell clones were generated in the presence of 20 U/ml rIL-2 and irradiated autologous tumor cells, PBLs and EBV-transformed B-cell lines. Phenotypically, all clones were CD3/CD8-positive with a heterogeneous CD56 expression. All expressed preferential cytolytic activity against autologous tumor cells, did not lyse autologous lymphoblasts, and were cytotoxic against the NK-sensitive cell line K562. A minor lytic capacity was detectable on allogeneic cervical tumor-cell lines or tumor-cell lines of other histologic types. Cytotoxicity against the autologous tumor could be inhibited by anti-CD3, anti-CD8 and anti-ICAM1 but not by anti-HLA class-1 (W6/32, B9.12.1), anti-allele-specific HLA determinants and anti-LFA-3 antibodies. We demonstrate a highly specific autologous lytic activity of cervical carcinoma TIL, in which a CD3-associated surface antigen recognition is involved. These results may prove useful in further studies on adoptive immunotherapy of cervical cancer patients.