Adjuvant adoptive immunotherapy with tumour-infiltrating lymphocytes and modulated doses of interleukin-2 in 22 patients with melanoma, colorectal and renal cancer, after radical metastasectomy, and in 12 advanced patients

 Adoptive tumour infiltrating lymphocytes (TIL) in combination with a modulated dosage of interleukin-2 (IL-2) can be used with acceptable toxicity in the treatment of immunogenic tumours. Following an experience of reinfusion in advanced melanoma, colorectal and renal cancer patients, treatment was given to disease-free patients after metastasectomy. The high risk of relapse and favourable ratio between reinfused TIL and possible microscopic residual disease determined this choice of adjuvant treatment. A group of 12 patients with advanced disease (7 melanoma, 4 colorectal carcinoma, 1 kidney carcinoma) were treated with TIL (median 5.8×10¹⁰ cells) and IL-2 (West’s schedule) modulated towards a lower dosage (from 12 to 6 MIU/day) in order to maintain an acceptable level of toxicity. As treatment was well tolerated, it was offered to another 22 patients in an adjuvant setting after metastasectomy (11 melanoma, 10 colorectal carcinoma, 1 renal cancer), the median dose of TIL reinfused being 4.95×10¹⁰ cells. No objective response was observed in advanced patients: all patients progressed after a median of 1.5 months (0–8 months) and median survival was 8 months (3–22+ months). Thirteen patients from the second group are still disease-free after a median of 23+ months (9+–47+ months). The remaining 9 patients relapsed after a median of 5 months (3–18 months). Toxicity was moderate as clinical and hepatic/renal function parameters were used to assess the need for dose reductions. Consequently, there was great diversity in IL-2 dosages administered. In particular, there seemed to be a difference in IL-2 doses administered between disease-free cases and those who progressed (17.5 MIU/day versus 7 MIU/day in melanoma patients; 11.2 MIU/day versus 7.1 MIU/day in colorectal cancer patients). By contrast, no differences were observed between number of TIL reinfused and clinical response. Phenotypical characteristics of reinfused TIL were similar to those reported in the literature: 97% were CD3 and 92% were CD8. Aspecific cytolytic activity was evaluated on 12 cases whereas, in 2 melanoma cases, autologous tumour tissue was available for the specific cytotoxicity test. Perforin levels in TIL measured at the end of culture were generally high or very high. Cytokine levels were measured on the supernatant at the end of culture, with an estreme variability in results. Finally, ζ chain and p56^lck were histologically assessed on the resected tissue from which TIL were cultivated. There were virtually none of the former and a complete absence of the latter, which concurs with data reported in the literature. The same immunocytochemical analysis was carried out on TIL at the end of culture. This time an almost complete restoration of both functions was seen, especially in melanoma patients, who are still free from disease. The study is on-going and it has been decided to focus on disease-free patients after metastasectomy in order to increase the number and possibility of clinical and histological correlations.     

Evaluation of natural killer cell expansion and activation in vivo with daily subcutaneous low-dose interleukin-2 plus periodic intermediate-dose pulsing

 Natural killer (NK) cells may be expanded in vivo with a prolonged course of daily subcutaneous interleukin-2 (IL-2). However, cellular activation requires higher concentrations of IL-2 than are achieved with low-dose therapy. The objective of the current trial was to determine the toxicity and immunological effects of periodic subcutaneous intermediate-dose IL-2 pulses in patients receiving daily low-dose therapy. A group of 19 patients were treated with daily subcutaneous low-dose IL-2 at 1.25×10⁶ International Units (1.25 MIU) m^–2 day^–1. After 4–6 weeks, patients received escalating 3-day intermediate-dose IL-2 pulses administered as single daily subcutaneous injections, repeated at 2-week intervals. The maximum tolerated pulse dose was 15 MIU m^–2 day^–1, with transient hypotension, fatigue, and nausea/vomiting dose-limiting. Subcutaneous IL-2 resulted in in vivo expansion of CD56⁺ NK cells (796±210%) and CD56^bright natural killer (NK) cells (3247±1382%). Expanded NK cells coexpressed CD16, and showed lymphokine-activated killer activity and antibody-dependent cellular cytotoxicity in vitro. Intermediate-dose pulsing resulted in serum IL-2 concentrations above 100 pM. Cellular activation was suggested by rapid margination of NK cells following pulsing, coincident with peak IL-2 levels, with return to baseline by 24 h. In addition, interferon γ production in response to lipopolysaccharide was augmented. Subcutaneous daily low-dose IL-2 with intermediate-dose pulsing is a well-tolerated outpatient regimen that results in in vivo expansion and potential activation of NK cells, with possible application in the treatment of malignancy and immunodeficiency. Received: 31 December 1997 / Accepted: 20 April 1998     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion

 The immunological properties of tumor-infiltrating (TIL) and peripheral blood lymphocytes (PBL) from 29 patients with renal cell carcinomas were characterized with respect to their phenotypic expression and cytokine production. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation. To eliminate all non-lymphoid cells, CD3-positive cells were specifically separated from these cell fractions with anti-CD3 magnetic beads. These pure CD3-positive PBL (CD3⁺PBL) and TIL (CD3⁺TIL) were cultured with pokeweed mitogen and the levels of the cytokines interleukin-1α (IL-1α), IL-1β, IL-2, interferon γ (IFNγ), and tumor necrosis factor α (TNFα) measured in the 4-day post-inductional cell culture supernatants. In all cell cultures a wide range of cytokine values was found, indicating a large variation in the immunological activity of the lymphocytes of each individual. When the cell cultures of the CD3⁺TIL and CD3⁺PBL were compared in each patient similar values for IL-1α, IL-1β, IFNγ and TNFα were found. However CD3⁺TIL produced significantly lower levels of IL-2 than CD3⁺PBL upon mitogenic stimulation. This may be due to a lower CD4/CD8 ratio in the CD3⁺TIL as compared to the CD3⁺PBL. These results suggest that there are no fundamental qualitative and quantitative differences in the lymphokine-producing capacity of CD3⁺TIL and CD3⁺PBL derived from patients with renal cell carcinomas. Received: 8 August 1995 / Accepted: 23 January 1996     

Adoptive T-cell therapy for malignant melanoma patients with TILs obtained by ultrasound-guided needle biopsy

Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) can mediate objective responses in up to 50% of malignant melanoma patients with a good performance status refractory to standard treatments. Current protocols for generation of TILs rely on open surgery for access to tumor tissue. We obtained tumor material by ultrasound-guided core needle biopsy or surgery from melanoma patients with progressive disease and were able to isolate >5 × 10⁶ TILs from 23 of 24 patients who were subsequently treated with these cells. One-third of the individual TIL-positive cultures displayed interferon gamma activity after stimulation with relevant melanoma cell lines. When expanded TILs were used for treatment in combination with daily low dose s.c. IL-2 after prior lymphodepleting chemotherapy, we observed objective clinical responses in one patient treated with TILs obtained from surgery and 4 patients treated with TILs from core biopsies. The results of this study demonstrate for the first time the potential of core biopsies for generation of relevant numbers of TILs that can mediate objective responses in patients with metastatic malignant melanoma. Ultrasound-guided core needle biopsy is a robust, safe and inexpensive approach to obtain tumor tissue for TIL generation, and is especially valuable in instances where surgery is contraindicated.     

Bimodal ex vivo expansion of T cells from patients with head and neck squamous cell carcinoma: a prerequisite for adoptive cell transfer

Background aimsAdoptive 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.MethodsIn 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 Cr⁵¹ release assay determined the specificity and functional capacity of selected TIL pre- and post-rapid expansion.ResultsTIL 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.ConclusionThe 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.     

Adoptive immunotherapy of advanced melanoma patients with interleukin-2 (IL-2) and tumor-infiltrating lymphocytes selected in vitro with low doses of IL-2

Freshly isolated tumor-infiltrating lymphocytes (TIL) from stage IV melanoma patients were cultured for 2 weeks with low doses of interleukin-2 (IL-2; 120 IU/ml), to select potentially for tumor-specific lymphocytes present in the neoplastic lesion, followed by high doses (6000 IU/ml) to achieve lymphocyte expansion. TIL were serially analyzed for their expansion, phenotype and cytotoxic activity against autologous and allogeneic tumor cells. A preferential lysis of autologous melanoma cells was obtained in long-term cultures of 7/13 cases (54%), while the remaining ones showed a major-histocompatibility-complex-unrestricted, lymphokine-activated-killer(LAK)-like activity at the time of in vivo injection. Sixteen patients with metastatic melanoma were infused with TIL (mean number: 6.8×10⁹, range: 0.35 × 10⁹–20 × 10⁹) and IL-2 (mean dose: 130 × 10⁶ IU, range: 28.8 × 10⁶–231 × 10⁶ IU); 1 complete and 3 partial responses were observed in 12 evaluable patients (response rate 33%). In all responding patients, injected TIL showed an in vitro preferential lysis of autologous tumor cells, while in no cases were TIL with LAK-like activity associated with a clinical response. The mean autologous tumor cytotoxic activity of TIL at the time of in vivo injection was significantly higher in responding patients in comparison to nonresponding ones, suggesting that a marked and preferential cytolysis of autologous tumor cells is associated with the therapeutic efficacy of TIL.     

Characterization of interleukin-2-initiated versus OKT3-initiated human tumor-infiltrating lymphocytes from glioblastoma multiforme: Growth characteristics,cytolytic activity,and cell phenotype

Summary Outgrowth of tumor-infiltrating lymphocytes (TIL) from the human primary brain tumor glioblastoma multiforme was achieved by OKT3 initiation (10 ng/ml), followed by sustained expansion by interleukin-2 (IL-2; 200 U/ml). Tumor-infiltrating lymphocyte (TIL) initiation by this process was performed in parallel with the standard IL-2-only method. Of ten tumors, seven yielded TIL in response to OKT3/IL-2, whereas only three of these seven grew after initiation with IL-2 alone. On the basis of cell doubling times, at least 60 doublings, resulting in (hypothetically) up to 10²³ TIL from as few as 2 × 10⁵ cells in tumor suspensions, could be achieved using OKT3/IL-2. OKT3-initiated TIL proliferated in culture for as long as 288 days, although senescence of some cultures occurred at as early as 73 days. Significant heterogeneity of lymphocytes infiltrating the fresh tumors and heterogeneity of resultant TIL phenotype and function were apparent, yet several common trends were noted. In all cases after OKT3 initiation, significant net growth was not apparent until approximately 14 days. In contrast, in the three samples that grew in response to IL-2 alone, log-phase growth was always observed earlier. During the early phase of the cultures, all TIL expressed some killing activity toward a broad spectrum of tumors, including the autologous tumor. No consistent preference of TIL for lysis of autologous tumor was observed. Glioblastoma multiforme TIL cultures contained a mixture of CD8⁺ and CD4⁺ cells, with few CD16⁺ or NKH-1⁺. Of the six TIL examined in detail for population phenotype in relationship to time in culture, four eventually became exclusively CD4⁺. Further analysis of these CD4⁺ TIL indicated that all were of the helper-inducer class, 4B4⁺ and 2H4^–. Concurrent with the decline in CD8⁺ cells, a decline in the cytolytic activity of these TIL cultures occurred. Furthermore, in two TIL that remained CD8⁺, a decline in the cytolytic activity also occurred. Therefore, loss of killing activity was not merely a reflection of the major cell phenotype changes. These results indicate that the OKT3/IL-2 process provides an alternative to IL-2 alone for TIL initiation and growth, as well as providing a novel system for further analysis of tumorderived lymphocyte and accessory cell functional potential.This work was supported by a grant from the Dunn Foundation, Houston, Texas     

Pilot study of treatment of biochemotherapy-refractory stage IV melanoma patients with autologous dendritic cells pulsed with a heterologous melanoma cell line lysate

Eleven AJCC stage IV melanoma patients with progressive disease after treatment with biochemotherapy were treated with autologous dendritic cells pulsed with heterologous tumor cell lysates. The vaccine used mature DCs (CD1a⁺⁺⁺, CD40⁺⁺, CD80⁺⁺, CD83⁺, and CD86⁺⁺⁺) generated from peripheral blood monocytes in the presence of GM-CSF and IL-4. After 7 days, DCs were matured with a defined cocktail of cytokines (IL-1+IL-6+TNF-+PGE₂) and simultaneously pulsed with lysates of heterologous melanoma cell lines, for 2 days. A total of 4×10⁶ DCs was injected monthly under ultrasound control in an inguinal lymph node of normal appearance. The study was closed when all patients died as a consequence of tumor progression. No sign of toxicity was observed during the study. One patient experienced a partial response lasting 5 months, and two patients showed a mixed response which lasted 3 months. The median survival of the whole group was 7.3 months (range 3–14 months). This vaccination program had specific antitumoral activity in highly pretreated and large tumor burden stage IV melanoma patients and was well tolerated. The clinical responses and the median survival of the group of patients, together with the low toxicity of our DC vaccine, suggest that this approach could be applied to earlier AJCC stage IV melanoma patients.     

Adoptive immunotherapy with peripheral blood lymphocytes cocultured in vitro with autologous tumor cells and interleukin-2

A clinical trial of adoptive immunotherapy was carried out with peripheral blood lymphocytes (PBL), cocultured in vitro with autologous tumor cells and interieukin-2 (IL-2), in 14 patients with advanced melanoma. PBL from these patients were cocultured with irradiated autologous tumor cells for 7 days, which was followed by expansion in IL-2-containing medium. These lymphocytes were returned to the patient along with intravenous IL-2 at doses up to 2×10⁶ IU m^–2 day^–1. A dose of 300 mg/m² cyclophosphamide was administered to each patient intravenously 4 days prior to each treatment. Following coculture, the lymphocytes were primarily CD3⁺ T cells and they expressed varied degrees of cytotoxicity against autologous melanoma cells. In 9 patients the activated cells were al least 80% CD4⁺ and in 2 cases they were mostly CD8+. Some of the activated cells exhibited suppressor or helper activity in a functional regulatory coculture assay. No major therapeutic response was observed in this study. Minor responses were observed in 2 patients. Toxicities were those expected from the IL-2 dose administered.This work has been supported by an American Cancer Society Institutional Research Grant (ACS-IRG 91-230), by the University of Connecticut Clinical Research Center (grant 0021), and by the Hartford Hospital Research Fund (grant 1017-20-018). Dr. Sporn is a recipient of American Cancer Society Clinical Oncology Career Development Award 90-230     

Phase I trial of combined treatment with ch14.18 and R24 monoclonal antibodies and interleukin-2 for patients with melanoma or sarcoma

Purpose: We conducted a phase I trial of interleukin 2 (IL-2) in combination with chimeric 14.18 (ch14.18) and murine R24 antibodies to determine the maximal tolerated dose (MTD), immunological effects, and toxicity of this treatment combination. Experimental Design: Twenty-seven patients with either melanoma (23 patients) or sarcoma (4 patients) were enrolled to receive a combination therapy with ch14.18 and R24 antibodies together with continuous infusion of Roche IL-2 (1.5×10⁶ U/m²/day, 26 patients) or Chiron IL-2 (4.5×10⁶ U/m²/day, 1 patient) given 4 days/week for 3 weeks. The antibodies ch14.18 (2–7.5 mg/m²/day) and R24 (1–10 mg/m²/day) were scheduled to be administered for 5 days during the second week of IL-2 therapy. Results: When given in combination in this study, the MTD for ch14.18 was 5 mg/m²/day and the MTD for R24 was 5 mg/m²/day. Dose-limiting toxicities were severe allergic reactions to both ch14.18 and R24 as well as pain related to ch14.18. This ch14.18 MTD was lower than the 7.5 mg/m²/day MTD previously determined for ch14.18 given alone with the same dose and schedule of IL-2. Immunological effects included the induction of lymphokine-activated killer (LAK) activity and antibody-dependent cell-mediated cytoxicity (ADCC). Anti-idiotype response to ch14.18 was seen in six patients, including two melanoma patients who had a partial response to treatment. In addition to two partial responses, four patients had a stable disease and one patient remained without any evidence of disease. Conclusions: Immunotherapy with IL-2 in combination with ch14.18 and R24 antibodies augments LAK function and ADCC measured in vitro in all patients. While there exist theoretical advantages of combining these two antibodies, the MTD of ch14.18 and of R24 were lower than the MTD of each antibody in prior studies evaluating single antibody therapy with IL-2. As such, the combination of these two antibodies together with IL-2 therapy appeared to influence the MTD and toxicity of each of the administered antibodies. This work is supported by NIH grants M01-RR03186, R01-CA32685, and P30-CA14520     

Cytokine biosynthesis by tumor-infiltrating T lymphocytes from human non-small-cell lung carcinoma

The purpose of this work was to assess the capacity of tumor-infiltrating leukocytes (TIL) from human non-small-cell lung carcinoma (NSCLC) specimens to synthesize type-1 and type-2 cytokines. Methods: TIL were isolated from tumors following digestion with collagenase/DNase and further enriched by ficoll-hypaque gradient centrifugation. Membrane phenotypes and intracellular cytokine protein expression of TIL were assessed by flow cytometry. Results: The majority of TIL expressed the CD3 antigen with a CD4:CD8 ratio of approximately 2:1. Other leukocytes such as macrophages (CD14), B lymphocytes (CD20), and natural killer (NK) cells (CD56) were also found to infiltrate the tumors, but in significantly lower numbers. Owing to the limited recovery of non-CD3⁺ leukocytes, our analysis of cytokine biosynthesis has focused on T lymphocytes. In the absence of activation, a small percentage of CD3⁺ TIL synthesized cytokines ( <4%). Following activation with anti-CD3+interleukin-2 (IL-2), CD3⁺ TIL synthesized predominantly a type-1 cytokine profile; however, the type-2 cytokines, IL-6 and IL-10, were also detected in a small percentage of infiltrating cells. Following activation with phorbol 12-myristate 13-acetate + ionomycin, CD3⁺ TIL also expressed more type-1 than type-2 cytokines and in significantly greater numbers of cells. The CD3⁺CD8⁺ component of the TIL synthesized only type-1 cytokines, whereas the CD3⁺CD4⁺ component synthesized both type-1 and type-2 cytokines. Conclusion: These results show that the majority of the TIL isolated from NSCLC specimens are T lymphocytes with the capacity to synthesize type-1 cytokines. Received: 24 March 1999 / Accepted: 9 September 1999     

Clinical effects of monoclonal antibody 17-1A combined with granulocyte/macrophage-colony-stimulating factor and interleukin-2 for treatment of patients with advanced colorectal carcinoma

Granulocyte/macrophage-colony-stimulating factor (GM-CSF) has previously been indicated to enhance the therapeutic effect of the anti-colorectal carcinoma mAb17-1A as well as to augment in vivo immune effector functions. In vitro interleukin-2 (IL-2) augmented GM-CSF-induced antibody-dependent cellular cytotoxicity, a mechanism considered to be of significance for the therapeutic effect of mAb. A treatment regimen was elaborated that combined mAb17-1A (400 mg at day 3 of a 10-day treatment cycle) with the simultaneous administration of GM-CSF (250 μmg/m² once daily) and IL-2 (2.4 × 10⁶ U/m² twice daily) for 10 days. The treatment cycle was repeated once a month. Twenty patients with advanced colorectal carcinoma were included in the study. One patient obtained a partial remission and 2 patients stable disease for 7 and 4 months respectively. The median survival time from the start of mAb therapy was 8 months. Owing to allergic reactions, the planned mAb17-1A dose had to be reduced by repeated infusions. At the fourth treatment cycle only 25% received the planned mAb dose. In 3 patients the GM-CSF and IL-2 dose was reduced because of side-effects. The subjective tolerability of the treatment was considered good or acceptable in more than 80% of the patients. The increment in white blood cell subsets induced by the cytokines decreased by increasing number of courses. This particular regimen did not augment the therapeutic effect of mAb17-1A anticipated from in vitro data but rather hampered the clinical effect of the antibody. The reason for this is not clear but a possibility might be the induction of immune suppression in vivo resulting from an impaired human anti-(mouse Ab) and anti-idiotypic antibody response as well as antibody-dependent cellular cytotoxicity, on the basis of a comparison of mAb17-1A/GM-CSF/IL-2- and mAb17-1A/GM-CSF-treated patients. Received: 25 February 1999 / Accepted: 15 July 1999     

Treatment of patients with advanced cancer with the natural killer cell line NK-92

Background aimsNatural killer (NK) cells, either naive or genetically engineered, are increasingly considered for cellular therapy of patients with malignancies. When using NK cells from peripheral blood, the number of expanded NK cells can be highly variable and the need for NK cell enrichment can make the process expensive. The NK-92 cell line (CD56+/CD3?) that was isolated from a patient with lymphoma has predictable high cytotoxic activity and can be expanded under good manufacturing practice conditions in recombinant interleukin-2.MethodsFifteen patients (age, 9–71 years) with advanced, treatment-resistant malignancies, either solid tumors/sarcomas (n = 13) or leukemia/lymphoma (n = 2), received two infusions of NK-92 cells, given 48 h apart. Three cohorts of patients were treated with escalating doses of NK-92 cells (n = 7 at 1 × 10⁹, n = 6 at 3 × 10⁹ and n = 2 at 1 × 10¹⁰ cells/m²).ResultsNo infusion-related or long-term side effects were observed. The dose of 10¹⁰ cells/m² was considered the maximum expandable cell dose with the use of an established culture bag system. Three fourths of patients with lung cancer had some anti-tumor response. Only one patient of seven had development of human leukocyte antigen antibodies. The persistence of NK-92 cells (male origin) in the circulation was confirmed by Y chromosome–specific polymerase chain reaction in two female patients.ConclusionsInfusions of NK-92 cells up to 10¹⁰ cells/m² were well tolerated. Despite the allogeneic nature of NK-92, development of human leukocyte antigen antibodies in these patients with cancer appears to be rare. The cells can persist in the recipient's circulation for at least 48 h. Some encouraging responses were seen in patients with advanced lung cancer.     

Treatment of refractory Hodgkin’s disease with an anti-CD16/CD30 bispecific antibody

 A group of 15 patients with refractory Hodgkin′s disease were treated in a phase I/II trial with the natural-killer (NK)-cell-activating bispecific monoclonal antibody HRS-3/A9, which is directed against the Fcγ receptor III (CD16 antigen) and the Hodgkin’s associated CD30 antigen. The antibody was given four times every 3 – 4 days, starting with 1 mg/m². The treatment was well tolerated and the maximum tolerated dose was not reached at 64 mg/m². Side-effects were rare and consisted of fever, pain in involved lymph nodes and a maculopapulous rash. Nine patients developed human anti-(mouse immunoglobulin) antibodies. One complete and one partial remission (lasting 5 and 3 months, respectively), three minor responses (1 to 11+ months), and one mixed response were achieved. There was no clear-cut dose side-effect or dose/response correlation. NK cell activity increased in most of the patients treated with 4 mg/m² or higher doses but lasted no longer than 6 weeks after therapy. Our results encourage further clinical trials with this novel immunotherapeutic approach and emphasize the necessity to reduce the immunogenicity of the antibody to allow retreatment of responding patients. Accepted: 14 October 1997     

Active immunization of metastatic melanoma patients with interleukin-2-transduced allogeneic melanoma cells: evaluation of efficacy and tolerability

 From January 1994 to July 1996 we immunized metastatic melanoma patients with HLA-A2-compatible, interleukin-2 (IL-2)-secreting, immunogenic melanoma lines in an attempt to induce a systemic reaction that might also affect distant melanoma lesions. Twelve patients (6 male and 6 female) aged from 28 to 72 years, affected with visceral and/or subcutaneous (s.c.) melanoma metastases, were treated. Two different HLA-A2⁺ melanoma lines were transduced with the human IL-2 gene (14932/IL-2 and 1B6/IL-2) and used as vaccine. Two groups of 4 patients each were injected s.c. with 5×10⁷ and 15×10⁷ irradiated 14932/IL-2 melanoma cells respectively, whereas a third group received 5×10⁷ cells of the second line (1B6/IL-2). All patients received the vaccine on days 1, 13, 26; if no progression was evident, further immunizations were administered at monthly intervals. All patients were assessable for clinical response after at least three injections of the vaccine. In 4 cases a stabilization of disease lasting from 2 to 6 months was observed; in 2 of them a mixed type of response to treatment was noted with simultaneous evidence of regressing and non-responding lesions in the same patients. No signs of clinical response were found in the remaining patients. Nine patients died of disease between 3 and 24 months after the onset of therapy, whereas 3 were alive 3 months after the end of therapy. The local and systemic side-effects of treatment were mild. These results indicate that vaccination with cells bearing the appropriate antigens and releasing IL-2 locally can produce weak clinical responses, but also indicate that better results may be achieved through modifications of the vaccine, the schedule of immunization and/or a more appropriate selection of patients. Received: 20 December 1996 / Accepted: 27 February 1997     

Differences in TCR-Vβ Repertoire and Effector Phenotype between Tumor Infiltrating Lymphocytes and Peripheral Blood Lymphocytes Increase with Age

Tumor infiltrating lymphocytes (TIL) reflect the host''s anti-tumor immune response, and can be a valuable predictor of prognosis. However, many properties of TIL are not fully understood. In the present study, TCR-Vβ repertoires of cancer patients were primarily analyzed by flow cytometry. Abnormally expressed TCR-Vβ subfamilies were generally found in both TIL and peripheral blood lymphocytes (PBL) of each patient. Of note, increased patient age was associated with increasingly biased TCR-Vβ repertoire in TIL but not in PBL, and the dispersion degree of the differences of TCR-Vβ subfamilies between TIL and PBL correlated positively with age (P = 0.007). Utilizing immunoscope analysis, we identified the age-related reduction in TCR-Vβ diversity, but polyclonal pattern was predominant in significantly expanded TCR-Vβ subfamilies. In addition, we found that older patients possessed a decreased ratio of CD8⁺CD62L⁺ non-effector cells in TIL compared to PBL, implying age-related increase of CD8⁺CD62L⁻ effector cells in TIL. The colocalization analysis of CD8 and CD3, however, suggested the suppressed activity of these effector cells in tumor microenvironment. These findings further elucidate the properties of TIL, showing an increasing difference between TIL and PBL with age, which may provide insight for the development of effective immunotherapies for cancer patients of different ages.     

Phenotypic and functional analysis of lymphocytes infiltrating paediatric tumours,with a characterization of the tumour phenotype

Summary Tumour-infiltrating lymphocytes (TIL) of paediatric tumours obtained from 37 lesions of different histo-type (12 osteosarcomas, 5 Wilms' tumours, 7 soft-tissue sarcomas, 5 neuroblastomas and 8 miscellaneous) were studied to establish their potential for therapy. Fresh isolated TIL were cultured for the first 2 weeks with low doses of interleukin-2 (IL-2) (20 Cetus U/ml) to select for tumour-specific lymphocytes potentially present in the neoplastic lesion, followed by culture with high doses of IL-2 (1000 Cetus U/ml) to achieve TIL expansion. TIL were grown with more than 10-fold expansion in only 9 cases (mean expansion: 58-fold, range 13.5–346). In 17 cases no viable cells were obtained. After 30 days of culture with IL-2 the proliferative ability of TIL declined sharply in the majority of cases and TIL became refractory to any further stimulus, including addition of IL-4, tumour necrosis factor (TNF) or interferon , and activation with OKT3 in solid phase. In 20 out of 37 cases TIL were available for phenotypic and functional analysis. TIL after long-term culture were predominantly CD3⁺ but 2 cases of osteosarcoma showed a predominance of CD3⁺TcR / cells. The CD4/CD8 ratio was more than 1 in 10 cases, without correlation with tumour histology, site of lesion or TIL growth. The number of CD16⁺ and CD25⁺ lymphocytes decreased progressively during culture, the latter concomitantly with a reduction of TIL growth rate. The lytic pattern of TIL against allogeneic and autologous tumour (Auto-Tu) cells was variable, but specific lysis of Auto-Tu was seen in only one case (Wilms' tumour) after culture with TNF and irradiated Auto-Tu cells. The immunohistochemical analysis of tumour lesions revealed a limited lymphocyte infiltrate, a low expression of histocompatibility leukocyte antigens (HLA) class I and of the adhesion molecules ICAM1, LFA3, and a significant production of transforming growth factor (TGF). These data indicate that TIL obtained from paediatric patients are difficult to expand at levels required for immunotherapy and lack a significant number of tumour-specific T lymphocytes. A low expression of immunomodulatory molecules on tumour cells or the production of suppressive factors may prevent activation and expansion of TIL in paediatric tumours.     

Histopathological analysis of metastatic melanoma deposits in patients receiving adoptive immunotherapy with tumor-infiltrating lymphocytes

Tumor-infiltrating lymphocytes (TIL) from a wide range of human and murine tumors can be expanded in vitro using interleukin-2 (IL-2). These TIL are cytolytic T lymphocytes with in vivo and in vitro antitumor activity in mice and in humans. TIL from human melanoma can recognize autologous tumor in an MHC-restricted fashion, localize in vivo after¹¹¹In labeling, and mediate regression of large metastatic deposits. Although studied extensively in vitro, less is known in vivo about TIL activity associated with tumor regression. This study was undertaken, in association with a study of TIL localization, to investigate mechanisms of TIL action by evaluating histopathological changes that occur at the tumor site during TIL administration. A total of 106 pre- and post-treatment pathological specimens from 25 patients enrolled in phase II TIL treatment and¹¹¹In-TIL imaging protocols were examined blindly by a single pathologist. Histological subtype, lymphocytic infiltration, melanin content, vascularity, and necrosis were documented for each tumor specimen. Average baseline and post-treatment parameters were compared. Any significant changes were evaluated for correlation with clinical response and¹¹¹In-TIL localization to tumor. Melanin content and vascularity of the tumor did not change as a result of therapy or correlate with either response or TIL localization. However, both increased lymphocytic infiltration and tumor necrosis were present after TIL administration (P=0.044 and 0.032 respectively). Furthermore, increases in lymphocytic infiltration correlated with tumor imaging using¹¹¹In-TIL, and with the percentage of¹¹¹In-labeled injectate present per gram of tumor specimen (P=0.036 and 0.0041 respectively). This suggests that TIL either account for the increased lymphocytes directly, or localize to tumor and recruit endogenous lymphocytes. We were unable to demonstrate any pretreatment histopathological predictors of response or variables that significantly correlated with subsequent clinical response, although peak and average values of necrosis were higher in responding patients compared to non-responding patients.