Activation of human tumor-infiltrating lymphocytes by monoclonal antibodies directed to the CD3 complex.

We have used high concentrations of recombinant-methionyl human interleukin 2 (rIL-2) for the initial growth and expansion of human tumor-infiltrating lymphocytes (TIL). Early in the life of the TIL bulk culture, cytotoxicity was non-major histocompatibility complex restricted. Under these culture conditions antitumor cytotoxicity was observed to decline with increasing age of the bulk culture. In addition, TIL became refractory to rIL-2-induced expansion. We have used solid-phase anti-CD3 antibodies for TIL activation followed by culture in reduced concentrations of rIL-2 to reactivate TIL previously grown in high concentrations of rIL-2. TIL refractory to rIL-2 in terms of growth and antitumor cytotoxicity proved sensitive to anti-CD3 activation. The use of solid-phase anti-CD3 was also more effective than high concentrations of rIL-2 in the expansion of TIL when used at the start of culture. Finally, TIL could be induced to secrete IL-2 following solid-phase activation with anti-CD3. These data suggest that human TIL are susceptible to activation by signals directed at the CD3 complex of the TIL cell surface.

Solid-phase anti-CD3 antibody activation of murine tumor-infiltrating lymphocytes.

Seventeen consecutive s.c. murine tumors, derived from a sarcoma and a colon adenocarcinoma, were cultured in the presence of recombinant interleukin 2 (rIL-2) for growth of tumor-infiltrating lymphocytes (TIL). Identical cultures were activated by solid-phase monoclonal antibody directed against the murine CD3 epsilon-chain, in conjunction with rIL-2. Forty-eight h later, cells were replaced in rIL-2 alone. Proliferation of anti-CD3-stimulated cultures was 1- to 17-fold greater than those cultured with rIL-2 alone (P less than 0.05). Both culture conditions yielded TIL which stained greater than 80% Thy-1.2+/Lyt-2+ (P greater than 0.05), less than 7% Thy-1.2+/L3T4+ (P greater than 0.05). Regardless of culture condition, longitudinal studies of in vitro cytotoxicity generated from 10 TIL preparations revealed no significant differences between the ability of TIL to lyse the murine natural killer-sensitive line YAC or heterologous or autologous tumor (P greater than 0.05). In vivo antitumor activity of TIL was tested by the adoptive transfer of suboptimal doses of TIL plus systemic rIL-2 to mice with pulmonary micrometastatic disease. No difference in tumor regression was noted between the TIL cultured with anti-CD3 plus rIL-2 or with rIL-2 alone (P greater than 0.05). Anti-CD3 stimulation of murine TIL cultures significantly increases lymphocyte cell yield without alteration of their phenotype, in vitro tumoricidal activity, or in vivo therapeutic effect.

Adoptive immunotherapy of human cancer using low-dose recombinant interleukin 2 and lymphokine-activated killer cells.

The adoptive transfer of recombinant-methionyl human interleukin 2 (rIL-2)-activated autologous peripheral blood mononuclear lymphokine-activated killer (LAK) cells to cancer patients is being evaluated as an alternative to conventional cancer therapy. We have independently developed an alternative regimen to previously reported adoptive immunotherapy protocols using rIL-2 and LAK cells which features the prolonged administration of low-dose rIL-2 (30,000 units/kg) and an automated, entirely enclosed system of peripheral blood cell procurement, culture, harvest, and reinfusion of activated cells. The cell culture system was tested with a murine tumor model in which LAK cells generated in plastic culture bags were reinfused into tumor-bearing mice. Tumor regression was as effective with cells activated in the bags as in conventional culture flasks. Twenty-eight cancer patients were treated for 5 consecutive days with low-dose rIL-2, followed by leukapheresis, infusion of LAK cells, and prolonged IL-2 administration. At least 50% tumor regression was observed in 46% of all patients treated. These data imply that human peripheral blood mononuclear cells retain fully their capacity for rIL-2-induced activation and effector cell function under this alternative approach, and further, that a low-dose rIL-2 regimen with markedly reduced toxicities can be as effective as high-dose rIL-2 regimens if low-dose rIL-2 is given for a prolonged period of time following LAK cell infusion.

Adoptive immunotherapy with tumor-infiltrating lymphocytes and interleukin-2 in patients with metastatic malignant melanoma and renal cell carcinoma: a pilot study.

PURPOSE: The objective of this study was to determine the tolerance and effect of moderate-dose recombinant human interleukin-2 (rHu IL-2) and tumor-infiltrating lymphocytes (TIL) in patients with metastatic melanoma (MM) or renal cell carcinoma (RCC) refractory to standard therapy. PATIENTS AND METHODS: Twenty-six patients (18 MM and eight RCC) were entered onto this pilot study. TIL were isolated from fresh biopsy material and activated with anti-CD3 antibody, OKT3, for 48 hours and expanded in 100 IU/mL r-methionyl Hu IL-2 alanine 125 (r-met Hu IL-2 [ala-125]). At least 10(10) TIL were reinfused intravenously in three divided injections on days 2, 4, and 6 of the protocol. A maximum dose of 30,000 U/kg of IL-2 per injection was administered every 8 hours from day 2 through day 11 for a total of 28 doses. RESULTS: Sixteen melanoma patients completed the study. Of these, three (19%) showed a durable complete response (CR), nine (56%) had no response (NR), and four (25%) had progressive disease (PD). One nonresponder demonstrated complete tumor regression within 1 year of treatment. Of four assessable RCC patients, two experienced a minor response (MR) and two showed NR. All TIL cultures showed comparably high cytotoxic activity as determined by antibody-redirected lysis (ARL). More importantly, melanoma TIL from responders possessed significantly higher cytotoxicity against autologous tumor cells than TIL from nonresponders (P < .05). Production of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-gamma), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and IL-4 was similar for TIL from melanoma responders and nonresponders, or TIL from RCC patients. CONCLUSIONS: Immunotherapy with polyclonally activated TIL and moderate-dose IL-2 could be successfully used for the treatment of immunogenic tumors with less toxicity and lower costs as compared with high-dose IL-2 protocols.

A new regimen of interleukin 2 and lymphokine-activated killer cells. Efficacy without significant toxicity.

Adoptive immunotherapy with high-dose interleukin 2 and lymphokine-activated killer (LAK) cells has proved to be successful in the treatment of some patients with metastatic cancer, but not without a significant degree of associated toxic effects. The primary goal of this study was to substantially reduce the toxicity of this complex and expensive treatment, while maintaining or improving efficacy. To this end, 29 patients were treated with LAK cells in conjunction with a low-dose regimen of interleukin 2 and a prolonged period of administration following LAK cell infusion. This protocol resulted in a considerable reduction in toxicity, as compared with that described in previous studies, without compromising the efficacy. This study offers further confirmation that adoptive immunotherapy of metastatic cancer can be clinically beneficial to patients for whom no other effective therapy is presently available.

Adaptation of the Müller method to allow quantitative characterization of the affinity and cross-reactivity of antibodies by competitive radioimmunoassay.

A quantitative expression is derived for the evaluation of antigen-antibody affinity constants from radioimmunoassays for the completely general situation in which antigen and antibody are both multivalent. The theoretical analysis is then extended to encompass quantitative characterization of the competitive inhibition observed in screening tests for cross-reactivity of antibody with structural analogs of the eliciting antigen. These procedures are illustrated with a radioimmunological study of the cross-reactivity of a desipramine-elicited monoclonal antibody with other tricyclic antidepressants. An unexpected finding to emerge from this immunochemical study is the demonstration that a single affinity constant suffices to describe the interaction of desipramine with a polyclonal antibody elicited by this univalent antigen.

Partial characterization of chicken spleen cell culture supernatants stimulated with Staphylococcus aureus.

The role of accessory cells in the proliferative response of chicken spleen cells to Staphylococcus aureus Cowan I (SAC) was examined. It was found that chicken spleen cells cultured with SAC produced a soluble molecule capable of causing proliferation when culture supernatants were added to spleen cells. The molecules responsible for this activity were stable in terms of exposure to extremes of heat and pH. Gel filtration of culture supernatants revealed biological activity, over a broad range of molecular weights, as measured by spleen cell proliferation. Similar findings were obtained when SAC was sonicated and evaluated following gel filtration. Exposure of culture supernatants to trypsin abrogated biological activity. The pivotal role of adherent cells in the generation of biologically active molecules is suggested by the ability of peritoneal exudate cells incubated with SAC to produce biologically active supernatants. In addition, the proliferative response of spleen cells to SAC was sensitive to chloroquine.

The role of soluble protein A in chicken spleen cell activation.

Soluble protein A (SpA) caused chicken spleen cell proliferation despite considerable evidence that SpA has no binding affinity for avian immunoglobulin (Ig). This biological activity was examined by several approaches. SpA-stimulated spleen cell cultures demonstrated no difference in proliferative responses regardless of the addition of human gamma globulin (HGG) or keyhole limpet hemocyanin. Adsorbents composed of HGG or hemoglobin were equally ineffective in abrogating the ability of SpA to induce spleen cell proliferation. In addition, ion exchange purified SpA was observed to induce comparable levels of spleen cell proliferation as ion exchange-affinity purified preparations. The lymphocyte subpopulation responsible for the observed proliferative response to SpA resides in the nylon wool adherent population. Nylon wool nonadherent lymphocytes failed to proliferate to SpA, but did proliferate in response to phytohemagglutinin as did nylon wool adherent cells. These data indicate that SpA is not responsible for the observed biological activity and suggest that components from Staphylococcus aureus other than SpA copurify during the preparation of SpA and are responsible for the activation of spleen cells.

B cell activation in chickens induced by Staphylococcus aureus.

Conventional mammalian polyclonal B cell activators were evaluated for activity in chicken spleen and peripheral blood lymphocyte (PBL) cultures. Although lipopolysaccharide was found to have a marginal influence on proliferation, two strains of the bacterium Staphylococcus aureus (Cowan I and Wood 46 strains) induced moderate proliferation in both spleen and PBL cultures. In spleen cell cultures the proliferating cell population was identified as the B cell. The mitogenic response required the presence of adherent cells since their removal eliminated the response. Evidence of in vitro polyclonal immunoglobulin synthesis could not be obtained. However, when administered intravenously, S. aureus induced polyclonal immunoglobulin synthesis.

Approaches to immunotherapy of cancer: characterization of lymphokines as second signals for cytotoxic T-cell generation.

Lymphokines, the soluble molecules produced by cells of the immune system, regulate cell-cell interactions and, consequently, the functional status of the immune system. Altering immunoregulatory pathways with lymphokines in vivo may provide a mechanism for controlling a variety of immunologic disorders. Although normally produced in vivo in very small quantities, the widespread availability of recombinant lymphokines has made it possible to study the molecular signals involved in production of lymphocyte effectors with activity against tumor. For example, interleukin-2-based cancer immunotherapy programs have, in certain clinical situations, suggested that immunologic intervention can influence the regression of metastatic cancer. Ultimately the successful application of these biologic agents requires an understanding of the interaction between the immune system and tumor on a molecular level. To induce a given biologic effect, it is necessary both to classify the required lymphokines and to identify the relevant effector cell populations. This review will examine the progress made in identifying the requirements for lymphokine-induced cytotoxic T-lymphocyte function.

T-cell recognition of ovarian cancer.

BACKGROUND: The existence of a tumor-specific T-cell immune response to human malignant melanoma has been well documented. In contrast, the existence of tumor-specific cytotoxic T lymphocyte to ovarian cancer remains controversial despite the abundant lymphocytic infiltrates in the malignant ascites and solid tumor of these patients. METHODS: Tumor-associated lymphocytes (TAL) from the malignant ascites and tumor-infiltrating lymphocytes (TIL) from the solid tumors were isolated from six untreated patients with ovarian cancer. TAL and TIL were grown with initial anti-cluster of differentiation of T cells (CD3), low-dose interleukin-2, and tumor stimulation. T-cell lines were analyzed in functional studies. RESULTS: At 5 weeks, TAL and TIL from five of six patients were > 50% CD8+, and one of six was > 70% CD4+. In all five pairs of CD8 positive cultures, both TAL and TIL exhibited high levels of tumor-specific cytotoxicity for ascite and solid tumor, respectively. T-cell recognition of tumor was mediated through the T-cell receptor-CD3 complex and was human leukocyte antigen class I restricted. TAL and TIL lysed autologous ascitic tumor equally well; however, TAL-mediated tumoricidal activity against autologous solid tumor was consistently and significantly poorer than TIL-mediated killing. CONCLUSIONS: Tumor-specific cytotoxic T lymphocytes can be expanded from both TAL and TIL. However, TAL do not kill solid tumor as efficiently as TIL. This suggests the requirement of TIL, or a combination of TIL and TAL, for effective immunotherapy.

Ibuprofen causes reduced toxic effects of interleukin 2 administration in patients with metastatic cancer.

Metastatic cancer was treated with interleukin 2 and lymphokine-activated killer cells with the addition of the cyclooxygenase inhibitor ibuprofen in an attempt to reduce side effects in 13 patients (eight male and five female). Twenty-six patients treated with only interleukin 2 and lymphokine-activated killer cells formed the control group. After interleukin 2 administration, a significantly increased number of lymphokine-activated killer cells were transfused in ibuprofen-treated patients. Cytotoxic effects were not significantly different in the treated and untreated groups. With regard to cell phenotype, both groups of patients manifested significant activation of the immune system as measured by T10 and OK1a. Symptom scores were dramatically reduced in patients treated with ibuprofen. Temperature above 37 degrees C were rare. Ibuprofen did not significantly alter rate of response in this immunotherapy trial (38% vs 42%). Ibuprofen is now routinely used in all of our current immunotherapy trials.

Survival characteristics of metastatic renal cell carcinoma patients treated with lymphokine-activated killer cells plus interleukin-2.

The immunologic manipulation of patients with metastatic renal cell carcinoma using lymphokine-activated killer (LAK) cells in conjunction with systemic interleukin-2 (IL-2) has been examined under conditions in which the life-threatening toxicities associated with IL-2 treatment have been virtually eliminated. We have examined tumor regression in vivo as well as the survival characteristics of 12 patients with metastatic renal cell carcinoma following immunotherapy. Five of 12 (42%) patients experienced tumor regression exceeding 50 percent following treatment. To determine if immunotherapy had influenced the length of survival, all patients were followed until the time of death. Previous studies have characterized the length of survival of metastatic renal cell cancer patients according to a combination of risk factors unique for each patient. In this model, patients were categorized into risk groups based on the number of risk factors. Survival was found to be dependent on risk factors such as performance status, time from initial diagnosis, number of metastatic sites, recent weight loss, and prior cytotoxic chemotherapy. On completion of the LAK cell immunotherapy protocol, patients were categorized as nonresponders or responders. In addition, they were assigned to risk groups based on their unique profile of risk factors at the time of entry into the protocol. Using this model, we found the median survival of nonresponders (23 months) to be no different from responders (24 months), p > 0.05. This was directly attributable to differences in risk factors which characterized members in these two response groups. However, the observed median survival of nonresponders following therapy was 1.9-fold longer than their projected survival based on the risk factors. Furthermore, the observed median survival of responders was 3.4-fold longer than projected from their risk factors. These results suggest that regardless of response status to therapy, cellular immunotherapy may play a role in mediating a significant palliative effect on the metabolic characteristics of these patients leading to extended survival.

The biological effects of immunosuppression on cellular immunotherapy.

Cytoreductive chemotherapy and immunosuppression have been postulated as possible adjuncts to cancer immunotherapy in studies using murine tumour-infiltrating lymphocytes (TIL). Treatment of animals with cyclophosphamide (Cy) therapy alone caused two distinct biological activities that altered the relationship between host and tumour. These two in vivo activities were distinguished by altering the timing and dose of Cy administration relative to tumour implantation. Cy administered 3 days following tumour injection caused a significant decline in the number of pulmonary micrometastases and greater survival compared to untreated controls in proportion to the dose of Cy administered. Further reduction in pulmonary disease was observed when Cy-treated mice were given TIL therapy. The possible role(s) of Cy-induced immunosuppression was studied by injecting Cy 24 h prior to tumour injection. This treatment failed to cause the cytoreductive effect observed when Cy was administered 3 days after tumour since Cy-administration prior to tumour resulted in a significantly higher number of pulmonary metastases and diminished survival compared to untreated controls. Despite the increased number of pulmonary metastases and decreased survival in mice treated with Cy before administration of tumour, therapy with TIL significantly diminished pulmonary disease compared to animals treated with Cy alone. Immunosuppression (without concomitant cytoreductive therapy) prior to TIL treatment significantly prolonged survival. Additional studies with TIL therapy indicate that the survival of animals immunosuppressed prior to tumour injection was significantly longer than controls which received immunotherapy alone. These results suggest that the combustion of immunosuppression plus cellular immunotherapy, which leads to significant survival advantage in these murine tumour models, may possibly augment the clinical response in human TIL trials.

Secondary cytokine production by lymphoid cells used in cellular immunotherapy.

Interleukin-2 (IL-2) has been used extensively in cellular immunotherapy trials as a systemic activator of the immune system as well as an ex vivo stimulant for lymphoid cell function. Despite the measurement of several in vitro and in vivo immunologic parameters related to cellular immunotherapy, determinants of successful cellular immunotherapy remain unknown. To further delineate the consequences of exposing peripheral blood lymphocytes to high concentrations of IL-2, we assessed the supernatants of IL-2-activated peripheral blood lymphocytes for production of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). Exposure of normal monocyte-depleted peripheral blood mononuclear cells (PBMC) to IL-2 caused a dose-dependent increase in secretion of TNF and IFN-gamma which increased linearly after 48 h in culture. Analysis of positively selected, highly purified PBMC subpopulations exposed to IL-2 revealed that TNF-alpha and TNF-beta were produced by both CD3+ and CD16+ subpopulations but not by CD22+ cells. These studies were extended to supernatants obtained from PBMC cultures used in the adoptive cellular immunotherapy of patients with advanced cancer. Patients treated with lymphokine-activated killer (LAK) cell immunotherapy were classified as responders (N = 14) or non-responders (N = 17) to therapy. We found no significant difference in the production of TNF between responders and nonresponders (22 +/- 9 U ml-1 vs. 20 +/- 6 U ml-1), P > 0.05. However, LAK cell supernatants harvested from non-responders contained a significantly higher level of IFN-gamma (232 +/- 94 U ml-1) compared with responders (42 +/- 14), P < 0.05. Furthermore, the linear association between IFN-gamma and TNF-alpha production was different between these two response groups (rs = -0.19 for non-responders and rs = 0.48 for responders). These results suggest that secondary cytokine production by adoptively transferred lymphocytesmay play an important role in the host response to cellular immunotherapy.

The role of interleukin-2 in cancer immunotherapy.

The arena of cellular immunotherapy is an emerging field of study. The field has strong foundations in numerous animal tumor models, which provide avenues of research for refinements in human immunotherapy. Experience from a number of research centers can be generalized: LAK cell trials in humans have demonstrated that PBMCs activated with IL-2 possess reproducible antitumor activity in vitro. Exogenous IL-2 administration to patients is required to maintain the biologic activity of the activated cells, although the requirement for LAK cells in tumors like melanoma is not clear-cut. Overall, the response rate to immunotherapy in humans is approximately 30%. The toxicity of immunotherapy, which is related to the dose of systemic IL-2, can be significantly reduced. The next generation of trials of cellular immunotherapy in humans will use TILs. Murine experiments indicate that this population of cells is more powerful and potentially more specific for tumor than LAK cells. Like LAK cells, TILs also require exogenous IL-2 for optimum performance in vivo. The difficulty in reproducible TIL growth can be overcome by the use of monoclonal antibody activation and IL-2. Improvements in TIL therapy are anticipated from the use of TILs combined with hybrid antibodies, the addition of other biologic response modifiers, and the implementation of genetically engineered cells and cell products.

Human tumor-infiltrating lymphocyte (TIL) cytotoxicity facilitated by anti-T-cell receptor antibody.

Long-term growth of tumor-infiltrating lymphocytes (TIL) in high concentrations of rIL-2 is required for generation of therapeutic numbers of cells for adoptive immunotherapy of human cancer. Under these conditions rIL-2 promotes both anti-tumor cytotoxicity and lymphocyte growth from tumors of several histological types. In a series of 16 consecutive tumors, studies of TIL-mediated cytotoxicity against different tumor targets were characterized by an initial strong tumor-non-specific cytotoxicity. With time, TIL bulk cultures became non-cytotoxic against all targets (median time = 38 days). Non-cytotoxic TIL bulk populations were capable of mediating strong cytotoxic responses if pre-treated with anti-T-cell antigen receptor antibody (TcR) before addition to targets. TIL populations were not, however, uniformly susceptible to anti-TcR-mediated cytotoxicity. Anti-TcR-mediated cytotoxicity was confined to CD8+ bulk populations (defined as populations with a CD4/CD8 ratio less than 1), with virtually no cytotoxicity observed in CD4+ populations (CD4/CD8 ratio greater than 2, (p less than 0.01). Both CD4 and CD8 populations expressed TcR antigen reactive with anti-TcR antibody. These results indicate that, despite poor in vitro anti-tumor cytotoxicity in short-term assays, CD8+ TIL are fully competent cytotoxic effector cells when subjected to strong activation signals via the TcR complex. In addition, these results imply that adoptively transferred CD4+ populations of TIL have in vivo biologic functions quite distinct from those of CD8+ populations and, further, that disparate clinical outcomes could reasonably be expected from the adoptive transfer of either population alone.

Isolation of genes overexpressed in freshly isolated breast cancer specimens.

A number of approaches have been used to identify genes important in breast cancer. In one approach the genes already shown to be involved in other tumors, such as p53 and Her2neu, were examined. A second approach examined genes detected through genetic screening of families with a high incidence of breast cancer, for example, BRCA-1 and BRCA-2. We used a third approach, subtractive hybridization, to identify and clone genes that were preferentially expressed in breast cancer cells compared to normal mammary epithelium. Instead of analyzing breast cancer cell lines, we examined fresh human breast cancer specimens. By subtracting normal mammary epithelial cDNA from breast cancer cDNA, we were able to clone several genes overexpressed in breast cancer. Two of these genes, L19 and MLN70, were previously reported to be overexpressed in breast cancer. Three of these genes, L19, L34, and MLN70, were localized to a region on chromosome 17 where Her2/neu and BRCA-1 are found. In addition, we isolated a gene we call breast cancer associated gene-1 that was expressed almost exclusively in fresh breast cancer tissue and not in normal mammary epithelium or breast cancer cell lines. We were unable to detect expression of breast cancer associated gene-1 in cell lines from melanoma, renal cell carcinoma, lymphoma, or leukemia. The full-length sequence from two separate breast cancer specimens revealed one amino acid difference compared to the sequence from normal breast epithelial tissue. Further studies are necessary to determine whether these genes contribute to breast cancer development or can be used as therapeutic targets.

The systemic complement activation caused by interleukin-2/lymphokine-activated killer-cell therapy of cancer causes minimal systemic neutrophil activation.

Twenty-three cancer patients undergoing therapy with interleukin-2 and lymphokine-activated killer cells were studied for evidence of complement activation and systemic neutrophil activation occurring during the course of therapy. Patient plasma samples demonstrated evidence of marked complement activation, with 3-fold elevations of C3a desArg concentrations by the 8th day of therapy. Concentrations of C4a desArg were also elevated by the end of therapy. In vitro chemotaxis of patients' neutrophils both to C5a and to the synthetic peptide chemotaxin, FMLP, was initially normal and then fell progressively to 60% of normal by the end of treatment. Mean neutrophil cell-surface expression of complement receptor Type 1 and complement receptor Type 3 increased in inverse temporal relationship to the deficit in chemotaxis, but showed no consistent pattern for individuals and was only doubled at maximum. Thus, despite a degree of complement activation which should have produced pronounced neutrophil activation, the response of the circulating neutrophils was diminished. In view of this discrepancy, the toxicity of this therapy may not be mediated by activation of circulating neutrophils.