Active specific immunotherapy with Newcastle-diseasevirus-modified autologous tumor cells following resection of liver metastases in colorectal cancer

Summary A group of 23 colorectal cancer patients were treated by a new type of active specific immunotherapy (ASI) following complete surgical resection of liver metastases (R0 resection). For ASI treatment we used a vaccine consisting of 1 × 10⁷ autologous, irradiated (200 Gy) metastases-derived tumor cells incubated with 32 hemagglutination units (HU) of Newcastle disease virus (NDV). The adjuvant vaccine therapy was started 2 weeks after surgery and was repeated five times at 14-days intervals followed by one boost 3 months later. The delayed-type hypersensitivity (DTH) skin reactions to the vaccine were measured as well as the DTH reactions to a challenge test of 1 × 10⁷ non-virus-modified autologous tumor cells from liver metastases or 1 × 10⁷ autologous normal liver cells. In addition 32 HU NDV alone and a standard antigen test (Merieux test) were applied pre- and post-vaccination. The vaccination was well tolerated. In 13 of 23 patients an increasing reactivity against the vaccine was observed during the vaccination procedure. Nine patients (40%) experienced an increased DTH reactivity against autologous tumor cells following vaccination, while 17% or fewer showed an increased reactivity to Merieux test antigens, NDV, or normal liver cells. The increased antitumor response was not correlated to responsiveness to NDV alone, autologous liver cells, enzymes and culture medium used for vaccine preparation or standard antigens (Merieux test). After a follow-up of at least 18 months 61% of the vaccinated patients developed tumor recurrence in comparison to 87% of a matched control groups from the same institution that had been only surgically treated. The results of this phase II trial are encouraging and should stimulate further prospective randomized studies.     

Active specific immunotherapy of Dukes B2 and C colorectal carcinoma: Comparison of two doses of the vaccine

Summary The ability of active specific immunotherapy to enhance immune responses to autologous tumor-associated antigens (TAA) and to prolong the disease-free interval was evaluated in patients with Dukes B₂ and C colorectal carcinoma who had undergone potentially curative resections. Patients were sensitized in the early postoperative period with irradiated autologous adenocarcinoma cells mixed with bacillus Calmette-Guérin (BCG) to yield either a low-dose vaccine (3×10⁶ tumor cells) or a high-dose vaccine (1×10⁷ tumor cells). Six of seven patients who received the low-dose vaccine developed delayed-type hypersensitivity (DTH) responses to autologous tumor cells upon completion of the vaccination, whereas all four patients receiving high-dose vaccine displayed a positive DTH response. However, DTH responses to autologous TAA waned within 3 months in all patients receiving the low-dose vaccine; DTH responses persisted for 3 months in three of the four high-dose vaccine patients. In vitro lymphoproliferative responses to TAA correlated with DTH responses to autologous tumor cells. Active specific immunotherapy appeared to induce specific immune responses either in vitro or in vivo to autologous TAA because it did not induce responses to autologous mucosa cells. There were no complications caused by BCG or tumor cells. This series demonstrates that active specific immunotherapy is a nontoxic treatment that augments immunity to autologous TAA.This project was supported by grants from Cutter Laboratories, Inc., the Annual Campaign of the University of Texas System Cancer Center, and the National Cancer Cytology Center     

Effect of the dose and composition of an autologous hapten-modified melanoma vaccine on the development of delayed-type hypersensitivity responses

We have reported that treatment of melanoma patients with a vaccine consisting of autologous tumor cells modified with the hapten, dinitrophenyl (DNP) and preceded by low-dose cyclophosphamide induces delayed-type hypersensitivity (DTH) to autologous, unmodified tumor cells and that this response is a significant predictor of survival. We analyzed the vaccines prepared for 284 patients who were treated following resection of regional or distant metastases to find out whether the dose and composition determined the immunological response. A positive DTH response (> or =5 mm induration) to unmodified autologous tumor cells was induced in 57% of the patients (median: 5 mm; range: 0-22 mm). Regression analysis showed no significant association between the magnitude of DTH and the number of live (trypan blue exclusion) melanoma cells per dose over a dosage range of 0.5-25.0 x 10(6). Surprisingly, there was a small but significant positive relationship between the mean number of dead cells in the vaccines of a given patient and that patient's maximum DTH to unmodified melanoma cells. Only 37% of patients whose vaccines contained >50% live cells developed DTH, as compared with 69% and 65% of patients whose vaccines contained 26% to 50% or < or =25% live cells, respectively. Thus, it appears that dead tumor cells contribute to the immunogenicity of the DNP vaccine, but other factors such as the administration schedule may be more important determinants of immunological and clinical outcome.     

Immunotherapy of spontaneous mammary tumors in mongrel dogs with autologous tumor cells and neuraminidase

Summary The effect produced on tumor progression by the injection of either VCN-treated tumor cells or tumor cells mixed with VCN to dogs with spontaneous mammary tumors was investigated. Dogs of different breeds and ages with at least two palpable spontaneous mammary tumors were selected. One tumor was left in the animal for further clinical examination, whereas the other tumor(s) was (were) excised for histologic diagnosis and for preparation of a single-cell suspension. Autologous M-cells were treated with VCN, subsequently extensively washed and injected SC into the neck of the dog on the day of operation and on the next day or different numbers of autologous M tumor cells (10⁵, 10⁶, 10⁷, 5×10⁷, 10⁸) were mixed with different amounts of VCN (0, 0.65, 6.5, 65 mU), and these various mixtures were injected ID at different sites to each dog on the day of operation. This procedure has been called chessboard vaccination (Seiler and Sedlacek, 1978). Altogether 79 dogs were blindly distributed into six groups in three consecutive studies. The results show that the therapeutic effect of the injection of VCN-treated autologous tumor cells depends on the number of tumor cells injected: injection of 2×10⁷ tumor cells repeatedly induced regression of the residual tumor mass (Studies I, II, and III) in most dogs and prevention of metastasis (Study I), while the application of 1×10⁸ tumor cells caused enhanced tumor proliferation in all and early metastasis in most of the dogs (Study I). The injection of 2×10⁶ tumor cells induced only a transient regression, with subsequent progression of the residual mammary tumor (Study II). Repetition of the injection of 2×10⁶ tumor cells three times every 4 weeks did not improve this effect (Study II). The chessboard vaccination proved to be at least as effective as the injection of 2×10⁷ VCN-treated tumor cells (Study III), although 1×10⁸ or more tumor cells had been injected; this number of cells caused tumor enhancement when the cells were treated with VCN only and injected SC (Study I). Moreover, the DTH reaction after ID injection of autologous tumor cells could be increased by the addition of VCN: low numbers of tumor cells and high amounts of VCN or high numbers of tumor cells and low amounts of VCN caused the most pronounced skin response. The relevance of these data to overcoming the risk of tumor enhancement after injection of an inadequate number of VCN-treated tumor cells and the possible diagnostic and therapeutic relevance of the DTH response after chessboard vaccination will be discussed.The abbreviations used in this work are: VCN, vibrio cholerae neuraminidase; M, mitomycin-treated; M-VCN, treated with mitomycin and VCN; DTH, delayed-type hypersensitivity; PBS, phosphate-buffered saline     

Human papillomavirus type 16 and 18 E7-pulsed dendritic cell vaccination of stage IB or IIA cervical cancer patients: a phase I escalating-dose trial

The safety and immunogenicity of the human papillomavirus type 16 (HPV16) or HPV18 (HPV16/18) E7 antigen-pulsed mature dendritic cell (DC) vaccination were evaluated for patients with stage IB or IIA cervical cancer. Escalating doses of autologous DC (5, 10, and 15 × 10⁶ cells for injection) were pulsed with recombinant HPV16/18 E7 antigens and keyhole limpet hemocyanin (KLH; an immunological tracer molecule) and delivered in five subcutaneous injections at 21-day intervals to 10 cervical cancer patients with no evidence of disease after they underwent radical surgery. Safety, toxicity, delayed-type hypersensitivity (DTH) reaction, and induction of serological and cellular immunity against HPV16/18 E7 and KLH were monitored. DC vaccination was well tolerated, and no significant toxicities were recorded. All patients developed CD4⁺ T-cell and antibody responses to DC vaccination, as detected by enzyme-linked immunosorbent spot (ELISpot) and enzyme-linked immunosorbent assays (ELISA), respectively, and 8 out of 10 patients demonstrated levels of E7-specific CD8⁺ T-cell counts, detected by ELISpot during or immediately after immunization, that were increased compared to prevaccination baseline levels. The vaccine dose did not predict the magnitude of the antibody or T-cell response or the time to detection of HPV16/18 E7-specific immunity. DTH responses to intradermal injections of HPV E7 antigen and KLH were detected for all patients after vaccination. We conclude that HPV E7-loaded DC vaccination is safe and immunogenic for stage IB or IIA cervical cancer patients. Phase II E7-pulsed DC-based vaccination trials with cervical cancer patients harboring a limited tumor burden, or who are at significant risk of tumor recurrence, are warranted.     

Monocyte chemoattractant protein-1 (MCP-1) gene transduction: an effective tumor vaccine strategy for non-intracranial tumors

Recently, there has been renewed interest in the concept of tumor vaccines using genetically engineered tumor cells expressing a variety of cytokines to increase their immunogenicity. Human MCP-1 (JE) is a potent chemoattractant and activator of monocytes and T lymphocytes and thus a good candidate gene for a tumor vaccine. We therefore evaluated the efficacy of vaccines consisting of irradiated tumor cells transduced with the murine MCP-1 gene in the syngeneic 9L gliosarcoma brain tumor model. 9L cell lines stably expressing murine MCP-1 (9L-JE) and control cell lines expressing neomycin 3 phosphotransferase (9L-Neo) were generated by infection with a Moloney murine leukemia retroviral vector. Fisher 344 rats were immunized with intradermal injections of 5×10⁵ or 2×10⁶ irradiated (5000 cGy) 9L-JE, 9L-Neo, and wild-type 9L (9L-WT) cells. Two weeks later immunized an non-immunized animals were challenged with varyious doses of intradermal (5×10⁶–5×10⁷) or intracerebral (2×10⁴–5×10⁵) 9L-WT cells. Intradermal tumors grew in all non-immunized animals. No tumors grew in animals immunized with irradiated 9L-JE or 9L-Neo cells and challenged with inocula of fewer than 5×10⁵ 9L-WT cells. With higher inocula up to 10⁷ cells, tumors appeared in all the animals. Tumors in animals immunized with 9L-JE were always smaller than tumors in the other groups. In addition, only the 9L-JE vaccine protected against tumor inocula of 5×10⁷ cells. Thus vaccination with MCP-1-expressing cells was able to protect animals against at least a 100-fold larger number of challenge tumor cells than vaccination with control cells. In contrast to studies with intradermal tumors, immunization with 9L-JE and 9L-Neo produced only minimal protection against intracerebral tumors. There was no significant difference between the 9L-JE and 9L-Neo vaccines in intracerebral challenge. This study suggests that tumor vaccines expressing cytokine genes such as MCP-1 can increase the antitumor response. However, the protective effect of these vaccines appears to be largely limited to intradermal tumors rather than intracerebral tumors.     

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.     

Induction of specific T cell immunity in patients with prostate cancer by vaccination with PSA146–154 peptide

T cell immunotherapy of prostate cancer (CaP) offers the potential for less toxic, more effective outcomes. A clinical trial was conducted in 28 patients with locally advanced or metastatic CaP to determine whether an HLA-A2 binding epitope of prostate-specific antigen, PSA146–154 (PSA-peptide), can induce specific T cell immunity. Patients were vaccinated either by intradermal injection of PSA-peptide and GM-CSF or by intravenous administration of autologous dendritic cells pulsed with PSA-peptide at weeks 1, 4 and 10. Delayed-type hypersensitivity (DTH) skin testing was performed at weeks 4, 14, 26 and 52. Fifty percent of the patients developed positive DTH responses to PSA-peptide. The size of the DTH induration progressively increased over time in the majority of responding patients. Skin biopsies from seven DTH-positive patients were available and T cells that developed in situ were also characterized. The phenotype of recovered T cells demonstrated variable proportions of CD4⁺CD8⁻, CD4⁻CD8⁺ and CD4⁺CD8⁺ T cell populations. Cytokine analysis of PSA-peptide stimulated T cells per bead array assay exhibited specific IFN-γ and TNF-α response in six of seven patients. Specific IL-4 response was observed in five patients, while IL-10 response was detected in one patient. Purified CD4⁻CD8⁺ T cells isolated from four patients demonstrated specific cytolytic activity per chromium release assay. In conclusion, immunization with PSA-peptide induced specific T cell immunity in one-half of the patients with locally advanced and hormone-sensitive, metastatic CaP. DTH-derived T cells exhibited PSA-peptide-specific cytolytic activity and predominantly expressed a type-1 cytokine profile.     

Selective induction and inhibition of direct and lectin-dependent cell-mediated cytotoxic reactivity

The immune reactivity of mice (C57BL/6, H-2^b) which had been challenged with various numbers (10²–10⁸) of allogeneic tumor cells (P815, H-2^d) was assessed at various times after challenge. Challenge with a high dose (10⁸) of tumor cells resulted in the development of direct cytotoxicity (DCMC), lectin-dependent cytotoxicity (LDCC), delayed-type hypersensitivity (DTH), and antibody production, whereas challenge with lower doses (< 10⁶) of tumor cells favored development of DTH and LDCC with marginal or no DCMC or antibody production. Spleen cells from low-dose alloimmune animals failed to produce DCMC when cultured with P815 cells in vitro and were capable of nonspecifically suppressing the DCMC response of normal spleen cells in MLC. Treatment with cyclophosphamide (100 mg/kg) prior to alloimmunization did not alter the pattern of DTH and cytotoxic reactivity, although treatment after alloimmunization was immunosuppressive for all forms of reactivity. When low-dose challenge was followed by cyclophosphamide treatment and a subsequent high-dose challenge, selective inhibition of DTH, LDCC, and suppressor activity, but not DCMC, was observed. The data suggest that (a) the initial challenge dose plays a significant role in determining which effector and regulatory populations will be activated and what direction the expression of immune reactivity will take; (b) the activated responding populations of DTH, DCMC, and LDCC effector cells are sensitive to cyclophosphamide treatment, whereas the precursors of each are resistant to the effects of the drug; (c) low-dose alloimmunization may be used in combination with cyclophosphamide treatment to modulate DTH, DCMC, and LDCC reactivity in a selective manner; (d) the cytotoxic effector cells responding to highdose challenge and mediating DCMC and those responding to low-dose challenge and mediating LDCC appear to arise from distinct precursor populations.     

Colon cancer cell vaccine prepared with replication-deficient vaccinia viruses encoding B7.1 and interleukin-2 induce antitumor response in syngeneic mice

 A replication-deficient recombinant vaccinia virus, NYVAC, was developed by deleting 18 open reading frames in the vaccinia virus genome. Recombinant NYVAC, encoding the murine T cell co-stimulatory gene B7.1 (CD 80) (NYVAC-B7.1) and the murine interleukin-2 gene (NYVAC-IL-2), were prepared and the expression of B7.1 and the secretion of IL-2 were respectively confirmed in vitro. The use of these viruses to prepare a potent tumor cell vaccine was studied in a syngeneic murine CC-36 colon adenocarcinoma model. Mice were immunized on days 1 and 8 with 10⁶ irradiated CC-36 cells that were infected with 10⁷ plaque-forming units of either NYVAC-B7.1, NYVAC-IL-2 or a control virus, NYVAC-HR, which encodes a vaccinia virus host-range gene. These mice were then challenged with 10⁸ viable CC-36 tumor cells on day 15. All mice (10/10) in a group that had received no vaccination and all mice (20/20) in a group that had received a control vaccine of CC-36/NYVAC-HR developed tumor 4-weeks after tumor cell challenge. Interestingly, only 16/20 mice in a group that had received CC-36/NYVAC-B7.1 showed the development of tumor after the same interval. The protection against tumor development and the reduction in tumor burden (as mean tumor diameter, 4 weeks after tumor challenge) were significant in this group when compared to groups that were either unvaccinated or vaccinated with CC-36/NYVAC-HR (mean tumor diameter = 6.51±3.2 mm compared to 26.5±0.9 mm or 26.2±1.8 mm respectively) (P = < 0.05). The protection against tumor in a group of mice that received CC-36/NYVAC-IL-2 vaccination was similar to that in the unvaccinated group or the group receiving a CC-36/NYVAC-HR control vaccination. However, in a survival experiment, mice that received either CC36/NYVAC-B7.1 or CC-36/NYVAC-IL-2 vaccination on the day of tumor transplantation survived significantly longer than mice that had not been vaccinated (median survival 60+ days, 60+ days or 23.5 days respectively) (P = <0.05). Interestingly, when a therapeutic tumor vaccination was performed on day 4 after tumor transplantation, mice that had been vaccinated with either CC36/NYVAC-B7.1 or CC-36/NYVAC-IL-2 did not show an improved survival when compared to mice in the control that had not been vaccinated (median survival 28 days compared to 26 days or 25 days respectively). However, mice that had received a therapeutic vaccination with CC-36 cells infected with both NYVAC-B7.1 and NYVAC-IL-2, 4 days after tumor transplantation, survived significantly longer than control mice that had not received any vaccination (median survival 29.5 days compared to 25 days respectively) (P<0.05). These results suggest that a replication-deficient recombinant NYVAC encoding the B7.1 gene and NYVAC encoding the IL-2 gene can be used to produce an effective vaccinia-virus-augmented tumor cell vaccine. Received: 2 March 1998 / Accepted 23 March 1998     

Genetic Control of Immune Responses to HIV-1 env DNA Vaccine

We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4⁺8^? and Ia— T cells. However, CD8⁺ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.     

Inhibition of H-thymidine incorporation by hydroxyurea: Atypical response of mycoplasma-infected cells in culture

Contamination with Mycoplasma hyorhinis was demonstrated in long-term cultures of HeLa, BICR/M1R_K rat mammary tumor, and NV1C rat neurinoma cells, by microbiological, equilibrium sedimentation, and autoradiographic techniques. In non-infected DNA-synthesizing cells, hydroxyurea (HU) in concentrations 10⁻⁴ M typically inhibits ³H-thymidine (³H-TdR) incorporation into acid-insoluble material. This effect was lacking in the contaminated cell lines, although HU did block nuclear DNA replication, as shown by pulse-cytophotometric analyses. The response to HU could be restored to normal by supplementing the culture medium either with the anti-mycoplasma agent Tylosin or with fresh rat serum. The total ³H-activity in non-infected (or anti-mycoplasma treated) versus infected cells, in the absence of HU, was up to four times higher in the former. The data indicate that (i) incorporation of ³H-TdR into the nuclear DNA of contaminated cells was strongly reduced, probably due to a ‘scavenger effect’ (i.e. utilisation and rapid cleavage) by the mycoplasma; (ii) mycoplasmal ³H-TdR incorporation, contrary to nuclear DNA replication, was insensitive to HU in concentrations 10⁻² M. If equally valid for other species of mycoplasma, the observed phenomenon provides a criterion (together with the possibility of a rapid test) for the presence of mycoplasmal contamination in cell cultures.     

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.     

Pre-immunotherapy serum CA27.29 (MUC-1) mucin level and CD69+ lymphocytes correlate with effects of Theratope® sialyl-Tn-KLH cancer vaccine in active specific immunotherapy

Patients with metastatic breast, colorectal or ovarian cancers received active specific immunotherapy (ASI) with Theratope® sialyl-Tn-KLH (keyhole limpet hemocyanin) cancer vaccine emulsified in Detox? adjuvant. The median log₂ anti-STn IgG titer generated by ASI, estimated by enzyme-linked immunosorbent assay with solid-phase ovine submaxillary mucin, was 5.322 (range = 0?–?9.322). Following ASI, 51 patients who generated titers higher than the median value for anti-STn⁺ mucin IgG survived longer than 46 patients who generated lower titers below the median. 38 of the patients were phenotyped for CD69 prior to ASI. The patients with lower numbers of CD69⁺ peripheral blood lymphocytes prior to immunotherapy (pre-ASI) also had low serum CA27.29 cancer antigen (MUC-1) levels, and had longer times to disease progression and improved survival following ASI. Elevated pre-ASI serum CA27.29 tumor antigen levels were associated with higher numbers of CD69⁺ PBL, with decreased anti-STn antibody production and decreased survival following ASI. The data are compatible with the hypothesis that elevated serum MUC-1 mucin is specifically immunosuppressive.     

Generation of dendritic cell–tumor cell hybrids by electrofusion for clinical vaccine application

Vaccination with hybrids comprising fused dendritic cells (DCs) and tumor cells is a novel cancer immunotherapy approach designed to combine tumor antigenicity with the antigen-presenting and immune-stimulatory capacities of DCs. For clinical purposes, we have incorporated a large-scale process for the generation of clinical-grade DCs together with novel electrofusion technology. The electrofusion system provides for ease and standardization of method, efficient DC–tumor cell hybrid formation, and large-quantity production of hybrids in a high-volume (6-ml) electrofusion chamber. In addition, we have evaluated DC electrofusion with a variety of allogeneic human tumor cell lines with the rationale that these tumor cell partners would prove a ready, suitable source for the generation of DC–tumor cell hybrid vaccines. The DC production process can generate 6×10⁸ to 2×10⁹ DCs from a single leukapheresis product (~180 ml). As determined by FACS analysis, electrofusion of 6×10⁷ total cells (1:1 ratio of DC and tumor cells) resulted in a consistent average of 8–10% DC–tumor cell hybrids, irrespective of the tumor type used. Hybrids were retained in the population for 48 h postfusion and following freezing and thawing. Upon pre-irradiation of the tumor cell partner for vaccine purposes, the overall fusion efficiency was not altered at doses up to 200 Gy. Evaluation of DC–tumor cell hybrid populations for their ability to stimulate T-cell responses demonstrated that electrofused populations are superior to mixed populations of DCs and tumor cells in generating a primary T-cell response, as indicated by IFN- release. Moreover, hybrids comprising HLA-A^*0201 DCs and allogeneic melanoma tumor cells (Colo 829 cell line) stimulated IFN- secretion by antigen-specific CD8⁺ T cells, which are restricted for recognition of a melanoma gp100 peptide antigen (gp100_209–217) within the context of the DC HLA haplotype. Maturation of the DC-Colo 829 cell hybrid population served to further improve this T-cell gp100-specific response. Overall, our results are promising for the large-scale generation of electrofused hybrids comprising DCs and allogeneic tumor cells, that may prove useful in human vaccine trials.     

Immunization with a tumor-cell-lysate-loaded autologous-antigen-presenting-cell-based vaccine in melanoma

The discoveries of human melanoma-associated antigens in molecular terms have renewed interest in peptide- or peptide- and antigen-presenting-cell (APC)-based cancer vaccines. Considering the limited scope of immunization using defined peptides, we have studied an alternative approach of specific immunization with tumor-lysate-loaded autologous APC (adherent peripheral mononuclear cells cultured in 1000 U granulocyte/macrophage-colony-stimulating factor for 14 days) as a surrogate vaccine. Seventeen patients (11 with active metastatic disease) were intradermally immunized with the vaccine in a phased dose escalation (10⁵–10⁷ cells/injection) monthly for 4 months. Thirteen patients completed all four immunizations showing no toxicity (3 patients had to be taken off study because of progressive disease and 1 patient went off study as a result of myocardial infarction due to multi-vessel coronary artery disease). None has shown any immediate or delayed toxicity attributable to the immunization and none has shown any evidence of autoimmunity. One patient showed a partial regression of a subcutaneous nodule. Thirteen patients are alive after 4+ months to 30+ months (17-month median survival for the group). Nine patients showed evidence of delayed-type hypersensitivity at the vaccine sites. Monitoring of biological response in conventional natural killer or cytolytic T lymphocyte assays with pre- and post-immune peripheral blood lymphocytes revealed no consistent differences. The vaccine-infiltrating lymphocytes (VIL) from nine specimens were adequately expanded following in vitro stimulation with the respective autologous-lysate-loaded APC for phenotypic and functional analyses. Five of the nine ex vivo expanded VIL were predominantly CD8⁺. Evidence of an antigen-specific CD8⁺ T cell response (cytotoxicity and/or tumor necrosis factor production) was detected in three of the five CD8⁺ VIL. These observations suggest that this type of vaccine is feasible, that it has biological activity, and that the approach may be improved through additional strategic manipulations. Received: 27 March 1998 / Accepted: 14 May 1998     

Antitumor activity of two BCG vaccine preparations against the lewis lung carcinoma in mice

Summary Two BCG vaccine preparations were prepared following different production methods. Immuno-BCG Pasteur F was produced by surface culture on Sauton medium; BCG-RIV was a homogenous stirred deep culture.The antitumor effects of the two BCG vaccines were investigated on the Lewis lung carcinoma (3LL) in C57Bl/6 mice. A direct relationship exists in this tumor model between the log₁₀ dose of single-cell suspension inoculated subcutaneously in the hind footpad of mice and the onset and the degree of local tumor growth and the time of death, which is directly related to the lung metastases. No significant difference from control mice was observed in the two groups of BCG-immunized mice when 3LL tumor cells were injected 2 weeks after BCG immunization. When varying numbers of viable units of the two BCG vaccines were injected together with 10⁵ tumor cells in separate groups of normal mice, a dose-dependent local reaction was observed with Immuno-BCG Pasteur F, which was associated with a delay in the onset and development of tumor growth and an increase in the mean survival time. The local inflammatory reaction produced with BCG-RIV was of lower magnitude, and only the highest concentration (1.8×10⁶ viable units) led to some delay in tumor occurrence and mortality. The antitumor effect of a specific local delayed-type hypersensitivity (DTH) elicited by varying amounts of the two BCG preparations injected together with 10⁵ tumor cells in separate groups of normal or BCG-immunized mice showed that the challenge injection of Immuno-BCG Pasteur F was in all cases more effective than the BCG-RIV, but these two vaccines were more effective in BCG-RIV-immunized mice than in Immuno-BCG F Pasteur-immunized mice.When the same number of viable units within each BCG vaccine was used as a criterion of comparison, Immuno-BCG Pasteur F produced a higher specific and nonspecific local inflammatory reaction (which was associated with a local antitumor effect) than BCG-RIV. But within 2 weeks, the latter was much better able to sensitize the mice to mycobacterial antigens. This was confirmed by the evaluation of local granuloma formation and tuberculin hypersensitivity. BCG vaccines prepared as surface-grown pellets and mechanically dispersed always sensitized mice to a lesser degree and after a much longer period of time than did the well-dispersed deep-cultured vaccine.     

Curative potential of GM-CSF-secreting tumor cell vaccines on established orthotopic liver tumors: Mechanisms for the superior antitumor activity of live tumor cell vaccines

In preclinical studies, tumor cells genetically engineered to secrete cytokines, hereafter referred to as tumor cell vaccines, can often generate systemic antitumor immunity. This study investigated the therapeutic effects of live or irradiated tumor cell vaccines that secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) on established orthotopic liver tumors. Experimental results indicated that two doses (3 × 10⁷ cells per dose) of irradiated tumor cell vaccines were therapeutically ineffective, whereas one dose (3 × 10⁶ cells) of live tumor cell vaccines caused complete tumor regression. In vivo depletion of CD8+ T cells, but not natural killer cells, restored tumor formation in the live vaccine-treated animals. Additionally, the treatment of cells with live vaccine induced markedly higher levels of cytotoxic T lymphocyte activity than the irradiated vaccines in the draining lymph nodes. The higher levels of cytokine and antigen loads could partly explain the superior antitumor activity of live tumor cell vaccines, but other unidentified mechanisms could also play a role in the early T cell activation in the lymph nodes. A protocol using multiple and higher dosages of irradiated tumor cell vaccines also caused significant regression of liver tumors. These results suggest that the GM-CSF-secreting tumor cell vaccines are highly promising for orthotopic liver tumors if higher levels of immune responses are elicited during early tumor development.     

Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats

Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 10^9.8 50% egg infective doses (EID₅₀)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 10^8.3 EID₅₀ completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.     

Effects of natural interferon α, natural tumor necrosis factor α and their combination on human mesothelioma xenografts in nude mice

Effects of human natural interferon (nIFN) alone, human natural tumor necrosis factor (nTNF) alone and their combination (OH-1) were tested on three human mesothelioma lines implanted in nude mice. Tumors were transplanted subcutaneously by trocar on treatment day –12. nIFN was given intraperitoneally (i.p.) at a dose of 2 × 10⁷ or 2 × 10⁸ IU kg^–1 day^–1, 5 days a week for 3 weeks. nTNF was given i.p. at a dose of 2 × 10⁷ or 2 × 10⁸ U kg^–1 day^–1 in the same schedule as that of nIFN. Tumor diameters were serially measured and tumor volumes were calculated. Antitumor effects were assessed by two methods: comparison of final tumor volumes in treated and control groups (T/C), and changes in median average total tumor volume. The treatment produced no clinically discernible toxicities. nIFN had strong inhibitory activity against all three human mesothelioma lines. nTNF alone had modest activity only at the high dose used. The combination of the two produced activity essentially similar to that produced by nIFN alone. High-dose nIFN may have a role as an active agent in the treatment of patients with mesothelioma.