Analysis of in vitro immunization: generation of cytotoxic T-lymphocytes against allogeneic melanoma cells with tumor lysate-loaded or tumor RNA-transfected antigen-presenting cells

In this study we compared several protocols for in vitro induction of cytotoxic T lymphocytes (CTL) from na?ve HLA-A*0201(+) peripheral blood mononuclear cells (PBMC) against allogeneic melanoma cells. As immunization material we compared: (1) the lysate of apoptotic or live melanoma tumor cells [MSM-M14 (M14) and MSM-M3 (M3)]; and (2) total RNA extracted from the same melanoma cell lines, either unconjugated or conjugated with a charged carrier (DMRIE-C). Overall killing activity was very similar in CTL induced by tumor lysate or RNA. CTL induced by both methods preferentially killed an HLA class I-matched M14 melanoma cell line rather than HLA class I-unmatched M3 cells. Cytotoxicity could be partially blocked by anti-HLA class I antibodies. There were no significant differences in cytotoxicity and in other clonal characteristics in CTL lines induced by a lysate of apoptotic bodies as compared to lines induced by lysate of viable cells. However, CTL induced by DMRIE-C-bound total RNA demonstrated superior cytotoxicity when compared with CTL induced by unconjugated total RNA. Polyclonal CTL induced by tumor lysate contained a substantial percentage of tyrosinase(368-376 370N) tetramer-positive cells and demonstrated specific killing activity against tyrosinase(368-376 370N) peptide-labeled T2 cells, comparable to cytotoxicity of the CTL developed against this peptide alone. In contrast, there were no detectable tyrosinase(368-376 370N)-tetramer positive cells and no specific anti-tyrosinase peptide(368-376 370N) response in polyclonal CTL induced by immunization with tumor RNA. These data demonstrate that both total tumor RNA and tumor lysate are effective for inducing of cytotoxic anti-melanoma CTL, but tyrosinase(368-376 370N) specific cells were detected only in lysate-induced CTL cultures. This suggests that nature of the antigens present in tumor lysate might be different from those in tumor RNA.     

Chemoimmunotherapy of stage III breast carcinoma with BCG and a live allogeneic tumor cell vaccine

Summary Ten women with metastatic breast carcinoma were treated with CMF, BCG, and a live TCV in a phase I study to determine whether viable allogeneic tumor cells can safely be given to women receiving adjuvant chemotherapy. Side effects of fatigue, nausea, vomiting, alopecia, and myelosuppression were attributable to chemotherapy. Side effects of BCG therapy were malaise, fever, and pruritis at the injection site, as previously described. Four patients developed hepatitis B due to viral contamination of the human agamma serum in which the tumor cells were grown. This complication has now been eliminated by omitting human sera, and hence the possibility of hepatitis B virus, from tumor cell growth media. No local tumor growth was observed in any patient. Results of this study show that live TCV is safe for patients receiving chemotherapy, and could be tested in combination with BCG and chemotherapy for control of micrometastases from breast cancer following mastectomy. Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; CMF, cyclophosphamide, methotrexate, and 5-fluorouracil; DNCB, dinitrochlorobenzene; PPD, purified protein derivative; TCV, tumor cell vaccine(s).     

Immunotherapy with allogeneic immune peritoneal cells activated by BCG or pyran copolymer

Summary Both pyran copolymer and BCG augmented specific macrophage-mediated cytotoxicity against allogeneic MBL-2 leukemia cells in a synergistic fashion. Similarly, the local passive transfer of peritoneal exudates (PE) from MBL-2 alloimmune mice treated with pyran or BCG protected against MBL-2 tumor development in the syngeneic host, whereas exudates from immunized or adjuvant-treated animals alone were ineffective. Tumor neutralization by alloimmune-activated PE was specific for the immunizing cell type and required intimate cell contact with target cells. Both adherent and nonadherent PE cells were required for optimal therapeutic effect. Long-term survivors demonstrated resistance to subsequent tumor challenge.     

Inhibition of pulmonary metastases of B16 melanoma with irradiated tumor cells and BCG

Summary When the tumor-bearing leg of C57BL/6J mice was amputated 16 days after SC inoculation of 10⁶B16 melanoma cells, all the amputated mice died of pulmonary metastases. Transfer of lungs from the amputated to normal syngeneic mice revealed tumor cells in the lungs just after amputation. Repeated weekly injections of BCG and irradiated tumor cells, beginning 24 h after amputation of the tumor-bearing limb, prolonged the survival only of mice presensitized to BCG. Injections of BCG or irradiated melanoma cells alone, of neuraminidase- and mitomycin C-treated tumor cells or of Levamisole had no effect, but injections of ConA-coated tumor cells slightly prolonged the survival of the amputated mice. Both BCG and B16 cells induced humoral and cell-mediated immunity but there was no cross-reactivity between BCG and B16 cells. Abbreviations used: ConA, concanavalin A; SC, subcutaneous; IP, intraperitoneal; IV, intravenous; ID, intradermal; IT, intratumoral; PBS, phosphate-buffered saline (0.01 M sodium phosphate, pH 7.1); VCN, Vibrio cholerae neuraminidase; HBSS, Hank's balanced salt solution; RPMIM, Roswell Park Memorial Institute medium     

Long-term outcomes in patients with metastatic melanoma vaccinated with melanoma peptide-pulsed CD34+ progenitor-derived dendritic cells

Between March 1999 and May 2000, 18 HLA-A*0201⁺ patients with metastatic melanoma were enrolled in a phase I trial using a dendritic cell (DC) vaccine generated by culturing CD34⁺ hematopoietic progenitors. This vaccine includes Langerhans cells. The DC vaccine was loaded with four melanoma peptides (MART-1/MelanA, tyrosinase, MAGE-3, and gp100), Influenza matrix peptide (Flu-MP), and keyhole limpet hemocyanin (KLH). Ten patients received eight vaccinations, one patient received six vaccinations, one patient received five vaccinations, and six patients received four vaccinations. Peptide-specific immunity was measured by IFN-γ production and tetramer staining in blood mononuclear cells. The estimated median overall survival was 20 months (range: 2–83), and the median event-free survival was 7 months (range: 2–83). As of August 2005, four patients are alive (three patients had M1a disease and one patient had M1c disease). Three of them have had no additional therapy since trial completion; two of them had solitary lymph node metastasis, and one patient had liver metastasis. Patients who survived longer were those who mounted melanoma peptide-specific immunity to at least two melanoma peptides. The present results therefore justify the design of larger follow-up studies to assess the immunological and clinical outcomes in patients with metastatic melanoma vaccinated with peptide-pulsed CD34-derived DCs.Joseph W. Fay and A. Karolina Palucka have equally contributed to this work     

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     

A phase II trial of sorafenib in metastatic melanoma with tissue correlates

Background

Sorafenib monotherapy in patients with metastatic melanoma was explored in this multi-institutional phase II study. In correlative studies the impact of sorafenib on cyclin D1 and Ki67 was assessed.

Methodology/Principal Findings

Thirty-six patients treatment-naïve advanced melanoma patients received sorafenib 400 mg p.o. twice daily continuously. Tumor BRAF^V600E mutational status was determined by routine DNA sequencing and mutation-specific PCR (MSPCR). Immunohistochemistry (IHC) staining for cyclin D1 and Ki67 was performed on available pre- and post treatment tumor samples. The main toxicities included diarrhea, alopecia, rash, mucositis, nausea, hand-foot syndrome, and intestinal perforation. One patient had a RECIST partial response (PR) lasting 175 days. Three patients experienced stable disease (SD) with a mean duration of 37 weeks. Routine BRAF^V600E sequencing yielded 27 wild-type (wt) and 6 mutant tumors, whereas MSPCR identified 12 wt and 18 mutant tumors. No correlation was seen between BRAF^V600E mutational status and clinical activity. No significant changes in expression of cyclin D1 or Ki67 with sorafenib treatment were demonstrable in the 15 patients with pre-and post-treatment tumor samples.

Conclusions/Significance

Sorafenib monotherapy has limited activity in advanced melanoma patients. BRAF^V600E mutational status of the tumor was not associated with clinical activity and no significant effect of sorafenib on cyclin D1 or Ki67 was seen, suggesting that sorafenib is not an effective BRAF inhibitor or that additional signaling pathways are equally important in the patients who benefit from sorafenib.

Trial registration

Clinical Trials.gov {"type":"clinical-trial","attrs":{"text":"NCT00119249","term_id":"NCT00119249"}}NCT00119249     

Vaccination of melanoma patients using dendritic cells loaded with an allogeneic tumor cell lysate

The aim of the present phase I/II study was to evaluate the safety, immune responses and clinical activity of a vaccine based on autologous dendritic cells (DC) loaded with an allogeneic tumor cell lysate in advanced melanoma patients. DC derived from monocytes were generated in serum-free medium containing GM-CSF and IL-13 according to Good Manufacturing Practices. Fifteen patients with metastatic melanoma (stage III or IV) received four subcutaneous, intradermal, and intranodal vaccinations of both DC loaded with tumor cell lysate and DC loaded with hepatitis B surface protein (HBs) and/or tetanus toxoid (TT). No grade 3 or 4 adverse events related to the vaccination were observed. Enhanced immunity to the allogeneic tumor cell lysate and to TAA-derived peptides were documented, as well as immune responses to HBs/TT antigens. Four out of nine patients who received the full treatment survived for more than 20 months. Two patients showed signs of clinical response and received 3 additional doses of vaccine: one patient showed regression of in-transit metastases leading to complete remission. Eighteen months later, the patient was still free of disease. The second patient experienced stabilization of lung metastases for approximately 10 months. Overall, our results show that vaccination with DC loaded with an allogeneic melanoma cell lysate was feasible in large-scale and well-tolerated in this group of advanced melanoma patients. Immune responses to tumor-related antigens documented in some treated patients support further investigations to optimize the vaccine formulation. Margarita Salcedo and Nadège Bercovici both contributed equally to this work     

Murine macrophage heparanase: inhibition and comparison with metastatic tumor cells

Circulating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16-BL6 melanoma; ESb T-lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monlayer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = -labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intracellularly but it is instead found pericellularly and requires a continuous cell-matrix contact at the optimal pH for maintaining cell growth. The degradation of [35S]O4 = -labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 micrograms/ml), arteparon (10 micrograms/ml), and heparin at a concentration of 3 micrograms/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo-beta-glucuronidase D-saccharic acid 1,4-lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two-step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases--alpha 2-macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)--failed to inhibit this degradation process, and only alpha 1-antitrypsin inhibited the heparanase activity. B16-BL6 metastatic melanoma cell heparanase, which is also a cell-associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 micrograms/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood-borne tumor metastasis.     

Analysis of T-cell responses in metastatic melanoma patients vaccinated with dendritic cells pulsed with tumor lysates

In melanoma patients, CD8⁺ cytotoxic T cells have been found recognizing self-proteins of which the expression is restricted to the melanocytic lineage. These melanocyte differentiation antigens are expressed in normal melanocytes as well as in 80–100% of primary and metastatic melanoma. In this report, six HLA-A*0201–subtyped metastatic melanoma patients vaccinated with dendritic cells (DCs) pulsed with autologous tumor lysates and keyhole limpet hemocyanin (KLH) were screened for the presence of CD8⁺ T cells specific for three HLA-A*0201–binding peptides derived from the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase. For this purpose, nonstimulated as well as in vitro peptide-stimulated peripheral blood mononuclear cells (PBMCs) were tested for peptide-specific IFN- release by enzyme-linked immunosorbent spot (ELISpot) assays. Furthermore, expression of the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase in tumor lesions was analyzed by immunohistochemistry before and after vaccination. We also used the ELISpot technique to investigate whether KLH-specific T cells were induced and whether these cells released type 1 (IFN-) and/or type 2 (IL-13) cytokines. Our data show induction of CD8⁺ T cells specific for the melanosomal peptides MART-1/Melan-A_27–35 or tyrosinase_1–9, as well as IFN-–releasing KLH-specific T cells, in two of six vaccinated melanoma patients, but do not support an association between the induction of these T cells and clinical responses.     

Interaction of monocytes with tumor cells coated with complement with or without antibody

Raji, a human B lymphoblastoid cell line has the ability to activate the complement cascade by alternate pathway mechanisms with subsequent fixation of C3 to receptors on the Raji cell membrane. Using this property, we examined the role that complement plays in mediating a cytolytic event between human peripheral blood monocytes and Raji cells coated with C3b, antibody, or both. Presence of C3 was confirmed by immune adherence. IgG bound to the Raji membrane was quantitated using I¹²⁵ Staphylococcal protein A assay. The presence of alternate pathway-activated C3 on Raji cells failed to produce monocyte-mediated cytotoxicity. These same target cells subsequently coated with antibody concentration ranging from 200 to >600,000 SPA molecules per Raji cell produced neither enhancement nor inhibition of antibody-dependent, cell-mediated cytotoxicity (ADCC). ADCC was enhanced by complement when complement activation and binding of C3 to the cell surface occurred by classical pathway mechanisms. ADCC of 32% ± 3.2 occurred with undiluted antiserum (625,000 SPA molecules bound/Raji cell) with enhancement to 52% ± 1.1 in the presence of C3. IgG inhibition of ADCC was unaffected by the presence of membrane-bound C3.     

T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

Activation of specific suppressor cells with heat-treated allogeneic tumor cells

The ability of heat-treated allogeneic cells to induce suppressor cells was examined. The tumor cell lines EL-4 (H-2^b) and P815-X2 (H-2^d), were heated to 56 °C for 10 min and injected intravenously into mice of the DBA/2J (H-2^d) and C57BL/6J (H-2^b) strains, respectively. After 4 days, the splenocytes of the treated mice were mixed with normal spleen cells and cultured for 5 days with allogeneic tumor cells. The cytotoxic T-cell response was reduced in cultures of these cell mixtures. An allogeneic difference was required to induce suppression because the syngeneic combination did not induce suppressor cell activity. Furthermore, the induction of cytotoxic T cells to the C118 cell line (H-2^k) was not suppressed by this procedure, which suggests that the suppression was haplotype specific. These suppressor cells were sensitive to anti-Thy 1.2 and complement, cortisone, and cyclophosphamide, but insensitive to irradiation. These are characteristics similar to suppressor cells activated by intact cells. Heat treatment abrogated the tumor cell's ability to induce a proliferative and a primary, but not a secondary, cytotoxic T-cell response. The heat-treated cells also lost their ability to function as cold target inhibitor cells, but retained the same quantity of serologically detected antigens as the intact cells. These results suggest that the serologically detected antigens are responsible for the activation of the suppressor cells of the cytotoxic T-cell response.     

Immunoprophylaxis with attenuated ehrlich ascites tumor cells in admixture with BCG

Summary During propagation in tissue culture, the Ehrlich ascites carcinoma was found to lose some of its tumor-producing capacity (oncogenicity) when implanted IP or SC into CF-1 mice. On the other hand, attenuated cells retained their immunoprotective capacity; immunization of mice with a single dose (1×10⁴) of these cells induced a high degree of resistance against a challenge 1 month later with virulent Ehrlich cells maintained by IP transplantation. The admixture of BCG (1×10⁶ viable units) with attenuated cells further improved their immunogenicity. The immunogenicity of attenuated cells was almost completely abolished by gamma-irradiation (2,500 rads), but this property was significantly restored by the addition of BCG. Some evidence is presented that suggests that attenuated cells have a higher immunoprotective capacity than the corresponding virulent cells.     

Vaccination of Stage IV patients with allogeneic IL-4- or IL-2-gene-transduced melanoma cells generates functional antibodies against vaccinating and autologous melanoma cells

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.     

A mathematical model of tumor growth. III. comparison with experiment

In this paper a model of nonuniform inhibitor production is used to discuss some experimental results obtained by Folkman and Hochberg for multicellular spheroids (of V-79 Chinese hamster lung tissue). Built into the mathematical model is a parameter b which is a measure of the degree of nonuniformity of the inhibitor production rate. The “inverse problem” is solved by finding the value of b necessary to account for results with which the uniform model is incompatible [6]. The resulting value of b enables a good estimate to be made for the observed width of peripheral mitotic zones in the V-79 spheroids, and the resulting stability parameters lie in appropriate ranges for the model to be a significant improvement over uniform models. Further improvements are discussed, including a heuristic model for estimating the destabilizing effect of vascularization on tissue growth.