Reconnaissance of tumor immune microenvironment spatial heterogeneity in metastatic renal cell carcinoma and correlation with immunotherapy response.

A clearer understanding of the tumor immune microenvironment (TIME) in metastatic clear cell renal cell carcinoma (ccRCC) may help to inform precision treatment strategies. We sought to identify clinically meaningful TIME signatures in ccRCC. We studied tumors from 39 patients with metastatic ccRCC using quantitative multiplexed immunofluorescence and relevant immune marker panels. Cell densities were analyzed in three regions of interest (ROIs): tumor core, tumor-stroma interface and stroma. Patients were stratified into low- and high-marker density groups using median values as thresholds. Log-rank and Cox regression analyses while controlling for clinical variables were used to compare survival outcomes to patterns of immune cell distributions. There were significant associations with increased macrophage (CD68+ CD163+ CD206+ ) density and poor outcomes across multiple ROIs in primary and metastatic tumors. In primary tumors, T-bet+ T helper type 1 (Th1) cell density was highest at the tumor-stromal interface (P = 0·0021), and increased co-expression of CD3 and T-bet was associated with improved overall survival (P = 0·015) and survival after immunotherapy (P = 0·014). In metastatic tumor samples, decreased forkhead box protein 3 (FoxP3)+ T regulatory cell density correlated with improved survival after immunotherapy (P = 0·016). Increased macrophage markers and decreased Th1 T cell markers within the TIME correlated with poor overall survival and treatment outcomes. Immune markers such as FoxP3 showed consistent levels across the TIME, whereas others, such as T-bet, demonstrated significant variance across the distinct ROIs. These findings suggest that TIME profiling outside the tumor core may identify clinically relevant associations for patients with metastatic ccRCC.

Interleukin 4 (B cell stimulatory factor 1) can mediate the induction of lymphokine-activated killer cell activity directed against fresh tumor cells.

Interleukin 4 (IL-4) expresses multiple biologic activities, including B cell, mast cell, and T cell stimulation. We showed that the incubation of resting splenocytes from C57BL/6 mice solely in purified native or recombinant mouse IL-4 results in the generation of lymphokine-activated killer (LAK) activity directed against fresh, syngeneic sarcoma cells. The precursor activated by IL-4 expresses surface asialo-GM1. In addition, IL-4 is capable of amplifying the splenic LAK activity induced by recombinant IL-2. The generation, by IL-4, of killer cells with broad antitumor reactivity raises the possibility of using IL-4 alone or in combination with IL-2 in the immunotherapy of cancer in animal models.

Interleukin 7 generates antitumor cytotoxic T lymphocytes against murine sarcomas with efficacy in cellular adoptive immunotherapy.

Interleukin 7 (IL-7) is a 25-kD cytokine that was initially described as a pre-B cell growth factor. This cytokine has also been shown to have T cell proliferative and differentiation effects. In this report, we demonstrate that antitumor cytotoxic T lymphocytes (CTL) generated by secondary in vitro sensitization of draining lymph node cells in IL-7 are effective in treating 3-day syngeneic methylcholanthrene (MCA) sarcoma pulmonary metastases in mice. In vivo titrations comparing IL-7 to IL-2 antitumor CTL show that they have equivalent potency in adoptive immunotherapy. IL-7 antitumor CTL generated against MCA sarcomas of weak immunogeneity are also tumor specific in their in vivo efficacy. This study represents the first successful use of a cytokine other than IL-2 for the generation of cells with in vivo efficacy in cellular adoptive transfer.

Interferon gamma and tumor necrosis factor have a role in tumor regressions mediated by murine CD8+ tumor-infiltrating lymphocytes.

We have investigated the mechanisms whereby adoptively transferred murine CD8+ lymphocytes mediate tumor regressions. Noncytolytic, CD8+ tumor-infiltrating lymphocytes (TIL) eradicated established lung tumors in irradiated mice. Many cytolytic and noncytolytic CD8+ TIL cultures specifically secreted interferon gamma (IFN-gamma) and tumor necrosis factor when stimulated with tumor cells in vitro. The effectiveness of TIL when adoptively transferred to mice bearing micrometastases correlated better with their ability to specifically secrete lymphokines than with their cytotoxicity in vitro. In 14 of 15 tests, therapeutically effective TIL specifically secreted IFN-gamma in vitro, whereas only 1 of 11 ineffective TIL specifically secreted IFN-gamma. In contrast, only 8 of 15 therapeutically effective TIL were cytolytic. Antibodies to TNF inhibited the effectiveness of two adoptively transferred TIL cultures. In five experiments, antibodies to IFN-gamma abrogated the ability of four different CD8+ TIL cultures to mediate tumor regressions, indicating that secretion of IFN-gamma is an essential part of the mechanism of action of TIL.

A nonimmunogenic sarcoma transduced with the cDNA for interferon gamma elicits CD8+ T cells against the wild-type tumor: correlation with antigen presentation capability.

To be recognized by CD8+ T lymphocytes, target cells must process and present peptide antigens in the context of major histocompatibility complex (MHC) class I molecules. The nonimmunogenic, low class I-expressing, methylcholanthrene (MCA)-induced murine sarcoma cell line, MCA 101, is a poor presenter of endogenously generated viral antigens to specific CD8+ T lymphocytes and cannot be used to generate tumor infiltrating lymphocytes (TIL). Since interferon gamma (IFN-gamma) has been shown to upregulate three sets of molecules important for antigen processing and presentation, we retrovirally transduced wild-type MCA 101 (101.WT) tumor with the mIFN-gamma cDNA to create the 101.NAT cell line. Unlike 101.WT, some clones of retrovirally transduced 101.NAT tumor expressed high levels of class I, and could be used to generate CD8+ TIL. More importantly, these TIL were therapeutic in vivo against established pulmonary metastases from the wild-type tumor. Although not uniformly cytotoxic amongst several separate cultures, these TIL did specifically release cytokines (IFN-gamma and tumor necrosis factor-alpha) in response to 101.WT targets. 101.WT's antigen presentation deficit was also reversed by gene modification with mIFN-gamma cDNA. 101.NAT had a greatly improved capacity to present viral antigens to CD8+ cytotoxic T lymphocytes. These findings show that a nonimmunogenic tumor, incapable of generating a CD8+ T cell immune response, could be gene-modified to generate a therapeutically useful immune response against the wild-type tumor. This strategy may be useful in developing treatments for tumor histologies not thought to be susceptible to T cell-based immunotherapy.

Antitumor activity of recombinant interleukin 6 in mice.

IL-6 possesses multiple biologic activities that affect a broad range of cells including those directly involved in immune responses as well as cells important in the systemic response to infection or trauma. We now show that purified human rIL-6, when administered alone at relatively high doses that are comparable to therapeutic levels of IL-2, mediated substantial reductions in the number of pulmonary and hepatic micrometastases from four distinct syngeneic tumors. Unlike IL-2, IL-6 injections resulted in neither observable toxicity nor death of the treated mice at the dose regimens used. Host immunosuppression by sublethal total-body irradiation before the initiation of therapy prevented the IL-6 antitumor effect, thus suggesting that IL-6 acted through a radiosensitive host component rather than directly on the tumor itself. Moreover, the systemic administration of relatively low doses of IL-6 in combination with subtherapeutic doses of TNF to mice bearing an established weakly immunogenic, syngeneic tumor at a subcutaneous site resulted in marked tumor regression and cure rates. These studies represent the first demonstration of tumor regression mediated by recombinant IL-6 in vivo.

Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2.

Incubation of resting lymphoid cells with recombinant interleukin 2 (IL-2) in vitro leads to the generation of lymphokine activated killer (LAK) cells capable of lysing fresh tumor cell suspensions in short-term chromium-release assays. Our previous studies (7) have demonstrated that the injection of LAK cells plus low doses of recombinant IL-2 were capable of inhibiting the growth of pulmonary metastases. We have now explored the ability of high doses of recombinant IL-2, administered systemically, to generate LAK cells in vivo, and to mediate antitumor effects directly. Administration of increasing doses of recombinant IL-2 intraperitoneally resulted in the generation of LAK cells in the spleens of recipient mice. Doses of 100,000 U recombinant IL-2 administered intraperitoneally approximately every 8 h for 5 d were capable of dramatically inhibiting established 3-d pulmonary metastases from the MCA-105 and MCA-106 syngeneic sarcomas and the syngeneic B16 melanoma in C57BL/6 mice. Grossly visible metastases present at 10 d after tumor injection also underwent regression following IL-2 therapy. Surprisingly, established 10 d pulmonary metastases were more susceptible to the effects of IL-2 than were the smaller 3 d pulmonary metastases. All antitumor effects of the systemic administration of recombinant IL-2 were eliminated if mice received prior treatment with 500 rad total body irradiation. The administration of high doses of recombinant IL-2 was also capable of inhibiting the growth of 3-d established subcutaneous tumors from the MCA-105 sarcoma, and of mediating the inhibition of growth and regression of established palpable subcutaneous MCA-105 sarcomas. Lymphocytes, which appeared morphologically to be activated, were present at the site of regressing tumor, and it appears that the mechanism of the antitumor effect of recombinant IL-2 administered systemically is via the generation of LAK cells in vivo, although this hypothesis remains to be proven. The ready availability of high doses of recombinant human IL-2, and the demonstration of antitumor effects seen in animal models have led us to the initiation of the clinical trials of recombinant IL-2 in humans.

Identification of human cancers deficient in antigen processing.

Intracellular antigens must be processed before presentation to CD8+ T cells by major histocompatibility complex (MHC) class I molecules. Using a recombinant vaccinia virus (Vac) to transiently express the Kd molecule, we studied the antigen processing efficiency of 26 different human tumor lines. Three cell lines, all human small cell lung carcinoma, consistently failed to process endogenously synthesized proteins for presentation to Kd-restricted, Vac-specific T cells. Pulse-chase experiments showed that MHC class I molecules were not transported by these cell lines from the endoplasmic reticulum to the cell surface. This finding suggested that peptides were not available for binding to nascent MHC molecules in the endoplasmic reticulum. Northern blot analysis of these cells revealed low to nondetectable levels of mRNAs for MHC-encoded proteasome components LMP-7 and LMP-2, as well as the putative peptide transporters TAP-1 and TAP-2. Treatment of cells with interferon gamma enhanced expression of these mRNAs and reversed the observed functional and biochemical deficits. Our findings suggest that downregulation of antigen processing may be one of the strategies used by tumors to escape immune surveillance. Potential therapeutic applications of these findings include enhancing antigen processing at the level of the transcription of MHC-encoded proteasome and transporter genes.

Adoptive immunotherapy of established pulmonary metastases with LAK cells and recombinant interleukin-2.

The activation of human peripheral blood leukocytes or murine splenocytes with interleukin-2 (IL-2) generated cells that were lytic in vitro for a variety of fresh tumor cells. The adoptive transfer of such lymphokine-activated killer (LAK) cells to mice with established pulmonary sarcoma metastases was highly effective in reducing the number (and size) of these tumor nodules when combined with repeated injections of recombinant IL-2. These findings provide a rationale for clinical trials of the infusion of human LAK cells generated with recombinant IL-2 as well as Phase I trials of the infusion of recombinant IL-2 systemically into humans.

Dendritic cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis.

Dendritic cells (DCs) are specialized antigen-presenting cells that migrate from the periphery to lymphoid tissues, where they activate and regulate T cells. Genetic modification of DCs to express immunoregulatory molecules would provide a new immunotherapeutic strategy for autoimmune and other diseases. We have engineered bone marrow-derived DCs that express IL-4 and tested the ability of these cells to control murine collagen-induced arthritis (CIA), a model for rheumatoid arthritis in which Th1 cells play a critical role. IL-4-transduced DCs inhibited Th1 responses to collagen type II in vitro. A single injection of IL-4-transduced DCs reduced the incidence and severity of CIA and suppressed established Th1 responses and associated humoral responses, despite only transient persistence of injected DCs in the spleen. In contrast, control DCs and IL-4-transduced T cells or fibroblastic cells failed to alter the course of the disease. The functional effects correlated well with the differential efficiency of DC migration from various sites of injection to lymphoid organs, especially the spleen. The ability of splenic T cells to produce IL-4 in response to anti-CD3 was enhanced after the administration of IL-4-transduced DCS: These results support the feasibility of using genetically modified DCs for the treatment of autoimmune disease.

Disseminated human malignant melanoma in congenitally immune-deficient (bg/nu/xid) mice.

Congenitally immune-deficient bg/nu/xid (BNX) mice are severely compromised in their ability to mount T-cell, B-cell, and lymphokine-activated killer (LAK) cell responses. Successful engraftment of BNX mice with human hematopoietic stem cells has been demonstrated recently. We have investigated the potential use of BNX mice for studies relating to the biology and immunotherapy of human malignant melanoma. The intravenous injection of fresh single-cell suspensions of human malignant melanomas into mice resulted in widely disseminated disease. Metastatic spread of human melanoma in BNX mice mimicked that observed in patients: eg, there were numerous tumor nodules identified in the subcutaneous tissues as well as in a variety of visceral organs, including spleen, kidneys, thyroid, adrenals, lungs, heart, and brain. BNX mouse lymph nodes were replaced consistently by human malignant melanoma cells. The presence of human tumor cells in these mice was confirmed by histologic analysis and microcytofluorometry analyses using human melanoma-specific monoclonal antibodies (MAbs). Moreover, human melanoma cells passaged in BNX mice remained lysable in vitro by specifically cytolytic, autologous human tumor-infiltrating lymphocytes (TILs). The capacity of fresh human malignant melanoma to disseminate widely in BNX mice may prove valuable not only for study of the biology of metastatic spread but also for studies of the immunotherapy of human melanoma using melanoma-specific MAbs and chemotherapeutic agents, as well as human TILs and LAK cells with or without retrovirus-mediated gene transfer modification.

Induction of tumor antigen-specific immunity in vivo by a novel vaccinia vector encoding safety-modified simian virus 40 T antigen.

BACKGROUND: Evidence that simian virus 40 (SV40) is associated with human mesotheliomas, osteosarcomas, and brain tumors suggests that a recombinant vaccine directed against lethal cancers expressing SV40 T antigen (Tag) could have clinical utility. To address this potential need, we designed a novel vaccinia virus construct that encodes an SV40 Tag in which oncogenic domains were excluded and immunogenic domains were preserved. We named this recombinant construct vaccinia-encoding safety-modified SV40 Tag (vac-mTag). METHODS: Purified vac-mTag was characterized by DNA sequencing, reverse transcription-coupled polymerase chain reaction, western blot analysis, and immunocytochemical techniques. Induction of Tag-specific immunity was examined by cytolytic T-cell assays, and the efficacy of vac-mTag in protecting animals against Tag-expressing tumors and in treating pre-established microscopic tumors was evaluated in vac-mTag-immunized BALB/c mice. RESULTS: The immune response elicited by vac-mTag in C57BL/6 and BALB/c mice included an SV40 Tag-specific cytolytic T-lymphocyte activity against syngeneic (identical genetic background) SV40 Tag-expressing tumor targets. Immunization of mice with a single dose of vac-mTag resulted in potent protection against subsequent challenge with a lethal mouse cancer expressing SV40 Tag. In addition, single-dose vac-mTag immunization coadministered with interleukin 2 produced a possible therapeutic effect against a preadministered microscopic (but lethal) burden of Tag-expressing tumor cells in vivo. CONCLUSION: vac-mTag induces an effective immune response in mice that is specific for a tumor-associated antigen. This response protects against a lethal tumor challenge and results in a possible therapeutic effect against Tag-expressing tumors in vivo. Thus, vac-mTag provides a new avenue for the development of therapies for human cancers thought to be associated with SV40.

Antitumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo: successful immunotherapy of established pulmonary metastases from weakly immunogenic and nonimmunogenic murine tumors of three district histological types.

We have recently shown that the systemic administration of lymphokine activated killer cells (LAK cells) plus relatively low doses of recombinant interleukin 2 (RIL-2) or the administration of high doses of RIL-2 alone can reduce the number of established pulmonary metastases from the weakly immunogenic MCA-105 sarcoma in mice. We have now analyzed the therapeutic efficacy of these treatments on both weakly and nonimmunogenic tumors of three distinct histological types in two different mouse strains. In all experiments, LAK cells were administered i.v. on days 3 and 6 and RIL-2 was injected i.p. from days 3 through 8 after tumor induction. The MCA-101 sarcoma was completely nonimmunogenic as defined by its inability to successfully immunize C57BL/6 mice. Nevertheless, administration of LAK cells plus 7,500-10,000 units RIL-2 was highly effective in reducing the number of established 3-day pulmonary metastases from this sarcoma [at 7,500 units RIL-2, mean number of metastases 37 +/- 11 (SE); P less than 0.05; at 100,000 units, 2 +/- 1; P less than 0.05] when compared to Hanks' balanced salt solution treated control animals (116 +/- 9). Likewise, RIL-2 alone at doses of 20,000 units/injection or greater had significant antimetastatic effects (77 +/- 12; P less than 0.05). Established 3-day pulmonary metastases from the MCA-38 adenocarcinoma in C57BL/6 mice and the M-3 melanoma in C3H mice were also susceptible to adoptive immunotherapy with LAK cells plus RIL-2 and with high dose RIL-2 alone. Treatment of mice with LAK cells alone or with low doses of RIL-2 alone (less than or equal to 20,000 units/injection) had little if any antitumor effects. LAK cells were tested for cytolytic activity in vitro against tumor target cells of a variety of histological types; there was no discernible relationship between susceptibility to lysis by LAK cells in vitro and therapeutic efficacy in vivo. These findings have thus demonstrated that the successful immunotherapy of established pulmonary metastases with LAK cells plus RIL-2 or with high dose RIL-2 alone includes: tumors that are immunogenic and nonimmunogenic; tumors of distinct histological types such as sarcoma, adenocarcinoma, and melanoma; and tumors in at least two different mouse strains, C57BL/6 and C3H, and that there is little correlation between the in vitro lysability of tumor cells by LAK effectors and the susceptibility of these same tumors to successful immunotherapy in vivo.

Comparative analysis of necrotic and apoptotic tumor cells as a source of antigen(s) in dendritic cell-based immunization.

There is considerable controversy as to whether necrotic (lysate) or apoptotic tumor cells serve as the superior source of multiple tumor-associated antigens (TAAs) to pulse dendritic cells (DCs) for immunotherapeutic applications. Here, we show that standard procedures to induce apoptosis by UVB irradiation unequivocally result in a mixed population of viable, apoptotic, and necrotic tumor cells, necessitating additional purification. We used highly enriched apoptotic versus lysate of B16 melanoma cells to examine whether or not there are important distinctions between these two sources of TAAs for loading of DCs. Our results demonstrate that although some differences exist between the two forms of TAAs in expression of heat shock proteins, as well as production of interleukin-12 by pulsed DCs, their respective capacities to mature DCs phenotypically, as well as to elicit both effective immune priming and antitumor therapeutic efficacy in vivo when presented by DCs, are equivalent.

Synergistic antitumor effects of immunotherapy with recombinant interleukin-2 and recombinant tumor necrosis factor-alpha.

The antitumor activity of combination therapy with recombinant tumor necrosis-alpha (rhTNF-alpha) and recombinant interleukin-2 (rhIL-2) was assessed against established immunogenic (MCA-106) and nonimmunogenic (MCA-102) sarcomas at both s.c. and visceral (hepatic) sites. C57BL/6 (B6) mice were treated with a single i.v. dose of rhTNF-alpha (2, 4, 6, or 8 micrograms) followed by rhIL-2 (25,000 U) i.p. thrice daily for 5 consecutive days. Synergistic effects as measured by regression of tumor, prolongation of survival, and improved cure rates were found using the combination of rhTNF-alpha plus rhIL-2 compared to rhTNF-alpha alone or rhIL-2 alone in the treatment of the immunogenic sarcoma MCA-106. No significant antitumor effects were observed against the nonimmunogenic MCA-102 sarcoma. These findings were similar for both s.c. and large single hepatic tumor models. The effect of the timing of rhIL-2 injections in relation to rhTNF-alpha administration (concurrent, 2, 4, or 6 days post single rhTNF-alpha dose) was also evaluated. Substantial tumor regression and increased survival times were seen in mice with s.c. tumors when rhIL-2 therapy was delayed as much as 48 h after rhTNF-alpha administration. No antitumor response was noted with the combination compared to rhTNF-alpha alone when rhIL-2 was delayed for greater than 4 days. No increase in lethal toxicity during treatment course of the combination of rhTNF-alpha and rhIL-2 was noted at any schedule compared to single agent rhTNF-alpha therapy. A possible role of rhTNF-alpha in regulation of IL-2-dependent antitumor activity in vivo is discussed.

CD4+ T-cells from mice immunized to syngeneic sarcomas recognize distinct, non-shared tumor antigens.

We have utilized a newly developed culture system to study the properties of antitumor CD4+ T-cells relevant to the rejection of syngeneic methylcholanthrene sarcomas. Fresh syngeneic dendritic cells prepared from spleen, then pulsed with crude lysates of methylcholanthrene sarcomas, evoke antigen-specific proliferation by CD4+ but not by CD8+ T-cells from tumor-immune mice. Unfractionated splenocytes display similar antigen presenting capacity if they are not irradiated before the pulse with tumor lysate. CD4+ T-cells from mice immunized to individual methylcholanthrene sarcomas proliferate cross-reactively to dendritic cells pulsed with fresh tumor digests, but not to dendritic cells pulsed with cultured tumor cells. This apparent shared recognition of sarcoma lysates was demonstrated to be a result of sensitization to bacterial collagenase during the immunization procedure. Therefore, the murine CD4+ T-cell response to tumor immunization is similar to the CD8+ response in that sensitization occurs predominantly to tumor specific transplantation antigens rather than to shared tumor antigens. Strategies to avoid artefactual tumor cross-recognition by CD4+ T-cells are discussed.

Studies of effects of recombinant human tumor necrosis factor on autochthonous tumor and transplanted normal tissue in mice.

The acute hemorrhagic necrosis of tumor nodules caused by the systemic administration of recombinant human tumor necrosis factor alpha (rhTNF-alpha) has been partially attributed to changes in tumor neovascularity. In this study, the effects of rhTNF-alpha were tested on primary autochthonous sarcomas induced in C57BL/6 mice by 3-methylcholanthrene, on spontaneous mammary tumors in C3H/HEN mammary tumor virus positive mice, and on the rejection of normal tissue transplants at different stages of maturity in C57BL/6 mice. Primary i.m. tumors induced by injection of 3-methylcholanthrene grew slowly over a 3-month period and became acutely necrotic after i.v. injection of rhTNF-alpha (2-6 micrograms). In addition, rhTNF-alpha caused a reduction in tumor area of 24% over 10 days compared to a 43% increase in tumor area in control mice receiving excipient (P2 less than 0.01). Histopathologically, tumors underwent central necrosis with a neutrophilic infiltration as was observed previously for serially transplanted tumors following rhTNF-alpha administration. Spontaneous, virally induced mammary tumors underwent a 11% regression on administration of rhTNF-alpha (4-6 micrograms) compared to a 24% growth in mice receiving excipient (P2 less than 0.05). Normal mice were grafted with syngeneic (C57BL/6) or partially allogeneic (C57BL/10 to C57BL/6) skin and were treated with a single dose of rhTNF-alpha (5-20 micrograms) i.v. at either 5, 10, or 15 days posttransplantation. rhTNF-alpha administration had no effect on the integrity of the skin grafts at any maturation point tested (syngeneic graft survival at 60 days: excipient, 35 of 36 versus 20 micrograms rhTNF-alpha, 35 of 36; allogeneic graft survival: excipient, 46 +/- 8 days versus 20 micrograms rhTNF-alpha, 48 +/- 10 days). In addition, rhTNF-alpha had no effect on the integrity of a syngeneic neonatal s.c. heart graft (graft survival at 60 days, excipient, 35 of 36 versus rhTNF-alpha, 30 of 33). Thus, although rhTNF-alpha administration led to marked necrosis and growth inhibition of vascularized tumor, no effect was observed on vascularized normal tissue transplants. To evaluate possible systemic effects of the tumor bearing state on the maturing neovascularity of normal tissue grafts, the three transplant models were studied in mice bearing a 9-day established MCA-106 s.c. sarcoma. After treatment with rhTNF-alpha (2-6 micrograms), acute necrosis and tumor size reduction was apparent in the s.c. tumors; however, no effect was seen in any of the normal tissue transplants.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of tumor lysate- and peptide-pulsed dendritic cell-based vaccines by the addition of foreign helper protein.

We have evaluated whether the addition of a foreign helper protein, keyhole limpet hemocyanin (KLH), can augment the efficacy of tumor lysate-pulsed dendritic cells and peptide-pulsed DC immunizations in vivo. Besides being used as a "surrogate antigen" in approaches to measure immunological response in cancer patients, KLH is also an immunogenic carrier protein to elicit T-cell help. Using the D5 subline of B16 melanoma, we demonstrate that DCs pulsed with both KLH and tumor lysate mediate enhanced immune priming and rejection of established metastases in vivo, which is dependent on host-derived T cells. Interleukin 2 augments the enhancement afforded by KLH, as measured by cure rates and overall survival, in the absence of autoimmune depigmentation. KLH added to DC immunizations markedly enhances tumor-specific T cell production of IFN-gamma. D5 melanoma exposed to similar levels of IFN-gamma results in substantial expression of MHC class I molecules. DCs pulsed with KLH and mouse tyrosinase-related protein-2 peptide results in enhanced reduction of B16 melanoma metastases; the effect is most pronounced in a setting where tyrosinase-related protein-2 peptide-pulsed DCs alone are completely ineffective. Collectively, these findings demonstrate that KLH addition to tumor antigen-pulsed DC immunizations can augment IFN-gamma production and enhance in vivo antitumor activity.

Combination cytokine immunotherapy with tumor necrosis factor alpha, interleukin 2, and alpha-interferon and its synergistic antitumor effects in mice.

The antitumor activity of combination therapy with recombinant human tumor necrosis factor alpha (rhTNF-alpha), recombinant human interleukin 2 (rhIL-2), and recombinant hybrid alpha-interferon A/D (rhIFN-alpha A/D) was assessed against established weakly immunogenic (MCA-106) and nonimmunogenic (MCA-102) sarcomas at both s.c. and visceral (hepatic) sites. C57BL/6 mice were treated with a single i.v. dose of rhTNF-alpha followed by rhIL-2 (75,000 units) and rhIFN-alpha A/D (75,000 units) i.p. twice daily for 5 consecutive days. Substantial improvements were observed when the combination of rhTNF-alpha, rhIL-2, and rhIFN-alpha A/D was administered, as measured by regression of tumor, prolongation of survival, and improved cure rates, compared with any combination of two cytokines or any cytokine alone against the MCA-106 sarcoma. These findings were consistent in both the s.c. and single hepatic tumor models. For example, treatment of the MCA-106 s.c. tumor bearers with the triple cytokine combination resulted in cures of 16 of 18, 17 of 18, and 12 of 18 mice receiving rhTNF-alpha dosages of 2, 4, and 6 micrograms, respectively, compared with 2 of 18, 7 of 18, and 9 of 18 at 2, 4, and 6 micrograms of rhTNF-alpha plus rhIL-2 without rhIFN-alpha A/D. Established 10-day single liver tumor weights when treated with the triple combination therapy were 54 and 25 mg in treatment groups receiving 2 and 4 micrograms of rhTNF-alpha, compared with 376 and 302 mg with the same amounts of rhTNF-alpha alone (P less than 0.004). Mice bearing hepatic sarcomas treated with triple cytokine combination therapy had cure rates of 50% and 67% at rhTNF-alpha doses of 2 and 4 micrograms, compared with no survivors with rhTNF-alpha alone. No improved antitumor effects resulted from therapy with any cytokine alone or in combination against the nonimmunogenic MCA-102 sarcoma. Possible in vivo mechanisms by which these three cytokines synergize are discussed.

Augmentation of antitumor efficacy by the combination of recombinant tumor necrosis factor and chemotherapeutic agents in vivo.

We evaluated the in vivo antitumor effects of the combination of recombinant human tumor necrosis factor (rhTNF) and three chemotherapeutic agents in an established murine tumor model. C57BL/6 mice bearing a subdermal weakly immunogenic 3-methylcholanthrene-induced sarcoma (MCA-106) received one i.v. dose of cyclophosphamide (Cy) (100 mg/kg), doxorubicin (5 mg/kg), or 5-fluorouracil (75 mg/kg) on either Day 8, 10, or 12. All animals received one i.v. dose of rhTNF (4 or 6 micrograms/mouse) on Day 10. The most effective time for administration of the chemotherapeutic agent was determined to be 48 h following rhTNF administration of all agents tested. The combined results of four separate experiments evaluating tumor size on Day 28 following tumor inoculation revealed that the groups treated with 4 or 6 micrograms of rhTNF and Cy (on Day 12) had tumor size reductions of 70 and 94%, respectively, compared to untreated controls (P2 less than 0.005). Mice treated with Cy alone, or with 4 or 6 micrograms of rhTNF alone had tumor size reductions of 30, 35, and 41%, respectively, compared to untreated controls (P2 less than 0.02). Analysis of cure rates demonstrates that the combination of Cy with 4 or 6 micrograms tumor necrosis factor cured 35 and 48% of the animals, respectively (P2 less than 0.01), compared to 10, 0, and 14% of mice treated with single agent Cy, 4 micrograms rhTNF, or 6 micrograms rhTNF, respectively. The timing of Cy and TNF administration was critical since administration of Cy prior to or concurrent with rhTNF was not effective in reducing tumor area or increasing cure rates over those achieved with either agent alone. Mice treated with doxorubicin alone had an increase in tumor size of 139 +/- 29% over untreated controls (P2 less than 0.05) on Day 28 following tumor inoculation and none were cured. In contrast, mice treated with doxorubicin plus 4 or 6 micrograms rhTNF exhibited early reductions in tumor size such that on Day 28 the average tumor areas were decreased by 66 +/- 34% (P2 less than 0.05) and 73 +/- 1% (P2 less than 0.02) of untreated controls with cure rates of 29% and 43% (P2 less than 0.02), respectively. However, the combination of 6 micrograms rhTNF plus doxorubicin led to substantial lethal toxicity with only 29% of mice surviving treatment. 5-Fluorouracil alone resulted in an increase in tumor area of 164% (P2 less than 0.05) over that of untreated controls on Day 28 following tumor inoculation.(ABSTRACT TRUNCATED AT 400 WORDS)