Intratumoral injection of IL-12 plasmid DNA--results of a phase I/IB clinical trial.

Effective eradication of established tumor and generation of a lasting systemic immune response are the goals of cancer immunotherapy. The objective of this phase IB study was to assess the safety and toxicity of treatment to metastatic tumor underlying the skin with the DNA encoding interleukin-12 (IL-12). This treatment strategy allowed the patient's own tumor to serve as a source of autologous antigen in the tumor microenvironment. We proposed that IL-12 protein produced by the transfected cells would result in the generation of both a local and systemic antitumor response. The tumor was treated with either three or six intratumoral injections of plasmid containing IL-12 DNA. This treatment strategy resulted in no significant local or systemic toxicity. The treatment did not result in an increase in serum IL-12 protein. The size of the treated lesion decreased significantly (greater than 30%) in five of the 12 patients. However, nontreated subcutaneous lesions or other disease did not decrease in size.

Effective particle-mediated vaccination against mouse melanoma by coadministration of plasmid DNA encoding Gp100 and granulocyte-macrophage colony-stimulating factor.

Particle-mediated gene delivery was used to immunize mice against melanoma. Mice were immunized with a plasmid cDNA coding for the human melanoma-associated antigen, gp100. Murine B16 melanoma, stably transfected with human gp100 expression plasmid, was used as a tumor model. Particle-mediated delivery of gp100 plasmid into the skin of naïve mice resulted in significant protection from a subsequent tumor challenge. Co-delivery of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) expression plasmid together with the gp100 plasmid consistently resulted in a greater level of protection from tumor challenge. The inclusion of the GM-CSF plasmid with the gp100 DNA vaccine allowed a reduction in the gp100 plasmid dose required for antitumor efficacy. Protection from tumor challenge was achieved with as little as 62.5 ng of gp100 DNA per vaccination. Tumor protection induced by the gp100 + GM-CSF gene combination was T cell mediated, because it was abrogated in vaccinated mice treated with anti-CD4 and anti-CD8 monoclonal antibodies. In addition, administration of the gp100 + GM-CSF DNA vaccine to mice bearing established 7-day tumors resulted in significant suppression of tumor growth. These results indicate that inclusion of GM-CSF DNA augments the efficacy of particle-mediated vaccination with gp100 DNA, and this form of combined gp100 + GM-CSF DNA vaccine warrants clinical evaluation in melanoma patients.

The level of MHC class I expression on murine adenocarcinoma can change the antitumor effector mechanism of immunocytokine therapy.

The huKS1/4-IL2 fusion protein, directed against the human epithelial cell adhesion molecule (huEpCAM) has been shown to induce a strong CD8+ T-cell-dependent, natural killer (NK) cell-independent, antitumor response in mice bearing the huEp-CAM-transfected CT26 colon cancer CT26-EpCAM. Here we investigate the effectiveness of huKS1/4-IL2 against CT26-Ep21.6, a subclone of CT26-EpCAM, expressing low levels of MHC class I. In vitro antibody-dependent cellular cytotoxicity (ADCC) assays in the presence of huKS1/4-IL2 demonstrate that murine NK cells from spleen and blood can kill CT26-Ep21.6 significantly better than they kill CT26-EpCAM. NK-mediated ADCC of CT26-EpCAM can be enhanced by blocking the murine NK cell-inhibitory receptor, Ly-49C. A potent in vivo antitumor effect was observed when BALB/c mice bearing experimental metastases of CT26-Ep21.6 were treated with huKS1/4-IL2. The depletion of NK cells during huKS1/4-IL2 treatment significantly reduced the antitumor effect against CT26-Ep21.6. Together our in vitro and in vivo data in the huEp-CAM-transfected CT26 models indicate that the amount of MHC class I expressed on the tumor target cell plays a critical role in the in vivo antitumor mechanism of huKS1/4-IL2 immunotherapy. A low MHC class I level favors NK cells as effectors, whereas a high level of MHC class I favors T cells as effectors. Given the heterogeneity of MHC class I expression seen in human tumors and the prevailing T-cell suppression in many cancer patients, the observation that huKS1/4-IL2 has the potential to effectively activate an NK cell-based antitumor response may be of potential clinical relevance.

Interleukin 12 gene transfer into skin distant from the tumor site elicits antimetastatic effects equivalent to local gene transfer.

We have reported that particle-mediated interleukin 12 (IL-12) gene transfer into the skin overlying the local tumor inhibits systemic metastases. To further characterize this effect, we compared the antitumor and antimetastatic effects of IL-12 cDNA delivered at the local tumor site versus at a site distant from the primary tumor, in a spontaneous metastasis model of LLC-F5 tumor. Local IL-12 gene delivery into the skin overlying the intradermal tumor (local IL-12 treatment) on days 7, 9, and 11 after tumor implantation resulted in the most suppression of the growth of the primary LLC-F5 tumor, whereas IL-12 gene transfer into the skin distant from the tumor (distant IL-12 treatment) was less effective. In contrast, both local IL-12 and distant IL-12 treatment, followed by tumor excision, inhibited lung metastases to a similar extent, resulting in significantly extended survival of test mice. The results of in vivo studies using depleting anti-asialo GM1 antibody and anti-CD4/anti-CD8 monoclonal antibodies, or neutralizing anti-interferon gamma (IFN-gamma) monoclonal antibody demonstrated that natural killer (NK) cells, CD8(+) T cells, and IFN-gamma contributed to the antimetastatic effects in both treatment groups. Furthermore, the levels of mRNA expression of vascular endothelial growth factor and matrix methalloproteinase 9 at the tumor microenvironment were suppressed after both local and distant IL-12 treatment. These results suggest that the current particle-mediated IL-12 gene delivery in the spontaneous LLC-F5 metastasis model can confer antimetastatic activities, irrespective of the gene transfection site, via a combination of several mechanisms involving CD8(+) T cells, NK cells, IFN-gamma, and antiangiogenesis.

Anti-CD40 antibody induces antitumor and antimetastatic effects: the role of NK cells.

We assessed the effect of the stimulatory anti-CD40 Ab on NK cell activation in vivo and the therapeutic potential of activated NK cells in tumor-bearing mice. Single-dose i.p. injection of the anti-CD40 Ab resulted in production of IL-12 and IFN-gamma in vivo, followed by a dramatic increase in NK cell cytolytic activity in PBLs. NK cell activation by anti-CD40 Ab was also observed in CD40 ligand knockout mice. Because NK cells express CD40 ligand but not CD40, our results suggest that NK activation is mediated by increased cytokine production upon CD40 ligation of APCs. Treatment of tumor-bearing mice with anti-CD40 Ab resulted in substantial antitumor and antimetastatic effects in three tumor models. Depletion of NK cells with anti-asialo GM1 Ab reduced or abrogated the observed antitumor effects in all the tested models. These results indicate that a stimulatory CD40 Ab indirectly activates NK cells, which can produce significant antitumor and antimetastatic effects.

Interleukin-12 gene therapy of a weakly immunogenic mouse mammary carcinoma results in reduction of spontaneous lung metastases via a T-cell-independent mechanism.

In our previous studies using gene gun-mediated delivery of interleukin 12 (IL-12) cDNA in vivo, we observed T-cell-mediated regression of established murine tumors and demonstrated the induction of systemic immunity in test animals. In this study, we further characterized the antitumoral and anti-metastatic effect of this gene therapy approach by employing two murine metastatic mammary tumor models: the immunogenic TS/A adenocarcinoma and the weakly immunogenic 4T1 adenocarcinoma. In the TS/A model, gene transfer into the skin overlying an established intradermal tumor with an IL-12 cDNA expression vector resulted in complete tumor regression in 50% of mice followed by the development of immunological memory. In contrast, the growth of the intradermal 4T1 tumors was not affected by the IL-12 gene therapy protocol. However, this treatment resulted in a substantial reduction of spontaneous metastases in the lungs of 4T1 tumor-bearing mice and significantly prolonged their survival time. T cells were not required for this anti-metastatic effect, because it was also observed in nude mice and in mice depleted of CD4+ and CD8+ T cells. Tumor-draining lymph node cells obtained from 4T1 tumor-bearing mice treated with IL-12 cDNA exhibited increased natural killer (NK) activity and produced enhanced levels of interferon-gamma (IFN-gamma) compared with similar mice treated with luciferase cDNA. In addition, in vivo depletion of NK cells or neutralization of IFN-gamma resulted in partial suppression of the anti-metastatic effect of IL-12 gene therapy, suggesting the involvement of both NK cells and IFN-gamma in this effect.

In vivo particle-mediated gene transfer for cancer therapy.

During the past several years, particle-mediated delivery techniques have been developed as a nonviral technology for gene transfer (1-7). For mammalian somatic tissues, this technology, popularly known as the gene gun method, has been shown effective for transfection of skin, liver, pancreas, muscle, spleen, and other organs in vivo (3,4), brain, mammary, and leukocyte primary cultures or tissue explants ex vivo (2,5-7), and a wide range of cell lines in vitro (3,6,7). In this chapter, we describe the general principles, mechanisms, protocols, and uses of the particle-mediated gene transfer technology for in vivo gene transfer, mainly into skin tissues. Specific applications of this technology to basic studies in molecular biology as well as to gene therapy and genetic immunization against cancer are addressed.

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1beta converting enzyme cDNA.

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1beta converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-gamma. First, we compared the levels of IFN-gamma secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-gamma production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-gamma levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8(+) T cell-mediated and partially IFN-gamma-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8(+) T cells and IFN-gamma-dependent pathways.

Pharmacokinetics and stability of the ch14.18-interleukin-2 fusion protein in mice.

The fusion protein formed from ch14.18 and interleukin-2 (ch14.18-IL-2), shown to exhibit antitumor efficacy in mouse models, consists of IL-2 genetically linked to each heavy chain of the ch14.18 chimeric anti-GD2 monoclonal antibody. The purpose of this study was to determine the pharmacokinetics of ch14.18-IL-2 in mice and assess its stability in murine serum. Following i.v. injection, the fusion protein was found to have a terminal half-life of 4.1 h. Detection of IL-2 following injection of the ch14.18-IL-2 fusion protein showed a similar half-life, indicating that the fusion protein prolongs the circulatory half-life of IL-2. Detection of human IgG1 following injection of ch14.18-IL-2 showed a terminal half-life of 26.9 h. These data suggested that the native fusion protein is being altered in vivo, resulting in a somewhat rapid loss of detectable IL-2, despite prolonged circulation of its immunoglobulin components. In vitro incubation of the ch14.18-IL-2 fusion protein in pooled mouse serum at 37 degrees C for 48 h resulted in a loss of its IL-2 component, as detected in enzyme-linked immunosorbent assay systems and in proliferation assays. Polyacrylamide gel electrophoresis and Western blot analysis of the fusion protein incubated in mouse serum at 37 degrees C indicated that the ch14.18-IL-2 is cleaved, resulting in a loss of the 67-kDa band (representing the IL-2 linked to the IgG1 heavy chain) and the detection of a band of more than 50 kDa, slightly heavier than the IgG1 heavy chain itself. This suggests that the fusion protein is being cleaved in vitro within the IL-2 portion of the molecule. These studies show that (1) ch14.18-IL-2 prolongs the circulatory half-life of IL-2 (compared to that of soluble IL-2) and (2) the in vivo clearance of the fusion protein occurs more rapidly than the clearance of the ch14.18 antibody itself, possibly reflecting in vivo cleavage within the IL-2 portion of the molecule, resulting in loss of IL-2 activity.

Gene gun-mediated IL-12 gene therapy induces antitumor effects in the absence of toxicity: a direct comparison with systemic IL-12 protein therapy.

Using three murine tumor models, we compared the antitumor efficacy and certain physiological effects of an in vivo interleukin-12 (IL-12) gene therapy protocol and a systemic IL-12 protein therapy protocol. An IL-12 cDNA gene construct was administered in situ into skin tissue via gene gun delivery, and recombinant IL-12 protein was administered subcutaneously at a dose of 1 microgram/mouse/treatment. Both treatment regimes induced a comparable level of regression of established intradermal MethA sarcomas. In B16 melanoma and P815 mastocytoma models, antitumor efficacy of IL-12 protein therapy appeared to be slightly higher than that of IL-12 gene therapy; however, the protein therapy protocol in this comparative study resulted in a high level of mortality of mice. It was also demonstrated that IL-12 gene therapy, in contrast to the IL-12 protein therapy, was not associated with weight loss, splenomegaly, increased Ly6 antigen expression in the spleen, or visible signs of toxicity, such as fur ruffling and lethargy. Moreover, serum levels of interferon-gamma (IFN-gamma) induced in response to IL-12 gene therapy were 300-1000 times lower than those induced by the systemic IL-12 protein administration. Together, these results suggest that gene gunmediated in vivo delivery of IL-12 cDNA may be considered as a safer alternative to IL-12 protein therapy for certain human cancers.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted by cDNA-transfected tumor cells induces a more potent antitumor response than exogenous GM-CSF.

Clinical cancer gene therapy trials have generally focused on the transfer of cytokine cDNA to tumor cells ex vivo and with the subsequent vaccination of the patient with these genetically altered tumor cells. This approach results in high local cytokine concentrations that may account for the efficacy of this technique in animal models. We hypothesized that the expression of certain cytokines by tumor cells would be a superior immune stimulant when compared with local delivery of exogenous cytokines. Granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a nonviral expression vector was inserted into MDA-MB-231 (human breast cancer), M21 (human melanoma), B16 (murine melanoma), and P815 (mastocytoma) cells by particle-mediated gene transfer. The ability of transfected tumor cells to generate a tumor-specific immune response was evaluated in an in vitro mixed lymphocyte-tumor cell assay and in an in vivo murine tumor protection model. Peripheral blood lymphocytes cocultured with human GM-CSF-transfected tumor cells were 3- to 5-fold more effective at lysis of the parental tumor cells than were peripheral blood lymphocytes incubated with irradiated tumor cells and exogenous human GM-CSF. Mice immunized with murine GM-CSF-transfected irradiated B16 murine melanoma cells or P815 mastocytoma cells were protected from subsequent tumor challenge, whereas mice immunized with the nontransfected tumors and cutaneous transfection of murine GM-CSF cDNA at the vaccination site developed tumors more frequently. The results indicate that GM-CSF protein expressed in human and murine tumor cells is a superior antitumor immune stimulant compared with exogenous GM-CSF in the tumor microenvironment.

Cytokine gene therapy of cancer using gene gun technology: superior antitumor activity of interleukin-12.

We compared the antitumor effect of several transgene expression plasmids encoding specific cytokines, including interleukin-2 (IL-2), IL-4, IL-6, IL-12, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF), following gene gun-mediated DNA delivery into the epidermis overlying an established intradermal murine tumor. IL-12 gene therapy was much more effective than treatment with any other tested cytokine gene for induction of tumor regression. Strong activation of antitumor immunity in response to IL-12 gene therapy was evidenced by an augmented CD8+ T cell-mediated cytolytic activity in the draining lymph nodes of tumor-bearing mice. Furthermore, following the IL-12 gene therapy protocol, test mice were able to eradicate not only the treated but also the untreated solid tumors at distant sites. This systemic antitumor effect of IL-12 gene therapy was not associated with visible signs of toxicity or significantly elevated systemic levels of IFN-gamma. These results show that gene gun-mediated in vivo delivery of IL-12 cDNA clearly distinguishes itself from the other cytokine gene therapy approaches tested in parallel, suggesting that this delivery system may be employed as an efficient model for comparative studies of in vivo cytokine gene therapy. The results also suggest that the current IL-12 gene therapy strategy may provide a safer alternative to IL-12 protein therapy for clinical treatment of cancers.

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

Neutrophils are essential for resolution of primary and secondary infection with Listeria monocytogenes.

The role for neutrophils in the resolution of primary and secondary infection with Listeria monocytogenes was studied. The results show that although control mice started to clear Listeria from their spleens and livers between days 2 and 4 of sublethal primary infection and eradicated bacteria in 2 weeks, mice given a specific granulocyte-depleting antibody (RB6-8C5) on days 4 or 6 of infection developed lethal listeriosis. Likewise, treatment of immunized mice with RB6-8C5 monoclonal antibody abolished their acquired ability to resolve a lethal challenge infection. The results demonstrate that neutrophils are necessary for the resolution of secondary and primary Listeria infection.

[Effect of total exogenous hyperthermia on the activity of natural killer cells]. / Vliianie obshchei ékzogennoi gipertermii na aktivnost' estestvennykh killernykh kletok.

Murine experiments were undertaken to study the impact of acute and chronic exogenous hyperthermia on the functional activity of natural killers. Single hyperthermia of the animals up to 42 degrees C and thermal shock stages were shown to be followed by suppressed activities of natural killer cells. Daily hyperthermia at 43-44 degrees C for 20 min during 3, 5, and 10 days was characterized by the depressed functional activity of natural killers. Hyperthermia for 20 and 30 days revealed no changes in the activity of natural killer cells. It can be assumed that there is a decrease in antitumor responses of the body in acute hyperthermia and in early chronic hyperthermia.

Gamma delta T cells and acute primary Toxoplasma gondii infection in mice.

A murine model of Toxoplasma gondii infection was used to investigate whether gamma delta T cells are required to resist primary acute toxoplasmosis. The surface phenotype of peritoneal exudate and spleen cells obtained from T. gondii-infected mice was examined using anti-gamma delta T cell-specific reagents and flow cytometry. Survival of mice depleted of gamma delta T cells was also followed during the acute phase of toxoplasmosis. Numbers of gamma delta T cells did not increase in spleen or peritoneal exudates of mice infected with T. gondii. Moreover, infected mice depleted of gamma delta T cells survived as long as untreated infected mice. These results indicate that gamma delta T cells do not play an important role in host defense against T. gondii infection in mice.

Evidence that gamma delta T cells play a limited role in resistance to murine listeriosis.

A role for alpha beta and gamma delta T cells in protection against primary and secondary infection with Listeria monocytogenes was studied. The results show that mice depleted of either gamma delta T cells with 3A10 monoclonal antibody (mAb), or alpha beta T cells with anti-CD4 plus anti-CD8 mAb, or both types of T cells, remained capable of controlling Listeria multiplication during the first 4 days of primary sublethal infection. Moreover, mice depleted of either or both types of T cells also remained capable of resolving primary infection, although the absence of alpha beta T cells, but not gamma delta T cells, caused resolution to be slower. Likewise, Listeria-immune mice depleted of either alpha beta or gamma delta T cells remained capable of resolving secondary infection with a large inoculum of L. monocytogenes, although depletion of alpha beta T cells, and to a much lesser extent gamma delta T cells, resulted in early exacerbation of infection. However, immune mice depleted of both types of T cells lost their ability to resist a lethal Listeria challenge. Taken together, the results show that whereas neither type of T cell is needed for resistance to sublethal primary listeriosis, alpha beta T cells may act in concert with gamma delta T cells in protecting mice against lethal secondary infection. In addition, the results indicate that the role of gamma delta T cells in anti-Listeria resistance is much less important than the role of alpha beta T cells, and can be demonstrated mainly in the absence of alpha beta T cells.

Failure of anti-T cell monoclonal antibodies to prevent acute tumor allograft rejection is associated with their inability to deplete or inactivate T cells at the site of rejection.

The ability of anti-T cell mAb therapy to prevent acute rejection of a tumor allograft was studied. Injection of both anti-CD4 and anti-CD8 rat Ig2b mAbs 1 day before intraperitoneal implantation of P815 tumor cells into AB6F1 mice prevented tumor rejection in most of the mice. However, injection of the same mAbs on day 8 of tumor growth, i.e., 2 days before the onset of tumor rejection, failed to prevent rapid elimination of P815 cells from the peritoneal cavity. Flow cytometric analysis revealed that whereas splenic T cells remained depleted in these mice for up to 6 days after mAb injection, peritoneal T cells returned to control levels in 4-6 days. Moreover, a significant number of rat IgG+ cells were found in the peritoneal cavity 2 days after mAb administration, thus demonstrating that T cells were not depleted but coated with the mAbs at that time. The antitumor activity of the mAb-coated T cells was not impaired, in that CD8+ peritoneal cells from mice rejecting allogeneic P815 tumor on day 10, and rat IgG+ peritoneal cells taken from mice 2 days after giving them anti-CD8 mAb on day 8, were similarly highly cytotoxic against P815 cells in vitro. Taken together, the results demonstrate that the failure of anti-T cell mAb therapy to prevent the acute rejection of allogeneic P815 tumor was associated with resistance of T cells at the site of rejection to the action of anti-T cell mAbs in vivo.

Evidence for a significant role of CD4+ T cells in adoptive immunity to Listeria monocytogenes in the liver.

Although the ability of CD8+ T cells to adoptively immunize mice against Listeria monocytogenes in the spleen is well established, the role of different T-cell subsets in anti-bacterial protection in the liver, a major target of Listeria infection, remains unclear. Therefore, the ability of sorted CD4+ and CD8+ T cells to adoptively immunize mice against a L. monocytogenes infection in the liver was studied. The results show that positively sorted CD4+ T cells from day 7 Listeria-immune mice were as effective as sorted CD8+ cells in transferring significant anti-Listeria protection in the liver. Similar findings were obtained when CD4+ and CD8+ T cells, negatively selected by antibody-induced complement-mediated depletion in vitro, were used for adoptive transfer. CD8+ T cells, however, were more efficient than CD4+ T cells in transferring protection in the spleen. Taken together, the results show that CD4+ T cells are at least as protective as CD8+ T cells against a L. monocytogenes infection in the liver, thereby arguing against the view that CD4+ T cells are of limited importance in adoptive immunity against listeriosis.

Elimination of CD4+ T cells in mice bearing an advanced sarcoma augments the antitumor action of interleukin-2.

Experiments were undertaken to determine whether the depletion of CD4+ T cells from mice bearing an advanced immunogenic SA-1 sarcoma would result in an enhanced ability of interleukin-2 (IL-2) to cause tumor regression. The results show that whereas IL-2 therapy given as a 5-day course starting on day 10 of tumor growth caused complete regression of the tumor, it failed to cause regression if started on day 15 of tumor growth. However, in mice depleted of CD4+ T cells by treatment with anti-CD4 monoclonal antibody (mAb), IL-2 therapy started on day 15 resulted in appreciable tumor regression in most animals, and the therapeutic effect was greatly increased if two consecutive courses of anti-CD4 mAb and IL-2 therapy were given. On the other hand, treatment with anti-CD4 mAb alone had no effect on tumor growth. It was shown that the therapeutic action of combination therapy with anti-CD4 mAb and IL-2 was mediated by CD8+ T cells, because the therapeutic effect was completely ablated in mice depleted of CD8+ T cells with anti-CD8 mAb. Taken together these results suggest that, at a late stage of growth of an immunogenic tumor, depletion of CD4+ T cells can enhance the antitumor effect of IL-2 therapy by releasing CD(8+)-T-cell-mediated immunity from T-cell-mediated suppression.