MIG (CXCL9) chemokine gene therapy combines with antibody-cytokine fusion protein to suppress growth and dissemination of murine colon carcinoma.

The induction of a CTL response capable of eradicating disseminated tumor metastases and the establishment of a persistent tumor-protective immunity remain major goals of cancer immunotherapy. Here, we demonstrate for the first time that the combination of interleukin 2 (IL-2) targeted to the tumor microenvironment by a recombinant antibody-IL-2 fusion protein (huKS1/4-IL-2) with gene therapy by the murine chemokine MIG (CXCL9) markedly reduced s.c. tumor burden and decisively suppressed dissemination of experimental lung metastases of CT26-KSA colon carcinoma in syngeneic BALB/c mice. This combined therapy significantly prolonged the life span of these mice 3-4-fold by concurrently delivering MIG and IL-2 to the tumor site and thereby achieving chemoattraction of T cells together with their activation. The antitumor effect obtained was mediated predominantly by MHC class I antigen-restricted CD8(+) T cells with help from MHC class II antigen-restricted CD4(+) T lymphocytes. In addition, the MIG chemokine also induced angiostatic effects in the tumor vasculature. Taken together, this combination of MIG chemokine gene therapy with tumor-targeted cytokine IL-2 provides an approach for the rational design of novel cancer immunotherapy modalities.

A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice.

A carcinoembryonic Ag (CEA)-based DNA vaccine encoding both CEA and CD40 ligand trimer achieved effective tumor-protective immunity against murine colon carcinoma in CEA-transgenic mice by activating both naive T cells and dendritic cells. Peripheral T cell tolerance to CEA was broken in a prophylactic model by this novel, dual-function DNA vaccine, whose efficacy was further enhanced by boosts with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2). These conclusions are supported by four lines of evidence. First, a lethal challenge of MC38-CEA-KS Ag murine colon carcinoma cells was for the first time completely rejected in 100% of experimental animals treated by oral gavage of this DNA vaccine carried by attenuated Salmonella typhimurium, followed by five boosts with huKS1/4-IL-2. Second, specific activation of dendritic cells was indicated by their marked up-regulation in expression of costimulatory molecules B7.1 (CD80), B7.2 (CD86), and ICAM-1. Third, a decisive increase over control values was observed in both MHC class I Ag-restricted cytotoxicity of CTLs from successfully vaccinated mice and secretion of proinflammatory cytokines IFN-gamma and IL-12. Fourth, activation of CTLs was augmented, as indicated by up-regulation of activity markers LFA-1, CD25, CD28, and CD69. Taken together, these results suggest that a dual-function DNA vaccine encoding CEA and CD40 ligand trimer combined with tumor-targeted IL-2 may be a promising strategy for the rational development of DNA-based cancer vaccines for future clinical applications.

An oral DNA vaccine against human carcinoembryonic antigen (CEA) prevents growth and dissemination of Lewis lung carcinoma in CEA transgenic mice.

A DNA vaccine encoding human carcinoembryonic antigen (CEA) broke peripheral T-cell tolerance toward this tumor self-antigen expressed by Lewis lung carcinoma stably transduced with CEA in C57BL/6J mice transgenic for CEA. This vaccine, delivered by oral gavage with an attenuated strain of Salmonella typhimurium (SL7207), and boosted with an antibody-IL2 fusion protein, induced tumor-protective immunity mediated by MHC class I antigen-restricted CD8(+) T cells, resulting in eradication of subcutaneous tumors in 100% of mice and prevention of experimental pulmonary metastases in 75% of experimental animals. Both CTL and antigen-presenting dendritic cells were activated as indicated by a decisive increase in their respective activation markers CD2, CD25, CD28 as well as CD48 and CD80. The antitumor effects of this CEA-based DNA vaccine obtained in prophylactic settings, suggest that this approach could lead to the rational design of effective treatment modalities for human lung cancer.

Augmentation of antitumor activity of an antibody-interleukin 2 immunocytokine with chemotherapeutic agents.

PURPOSE: Immune-based therapies, such as the immunocytokine huKS-IL2, exert potent antitumor responses in some animal models by targeting cytokine activity to the tumor microenvironment. We found that certain chemotherapy agents in the appropriate dose and schedule can augment the antitumor activity of huKS-IL2. EXPERIMENTAL DESIGN: Chemotherapy agents were given in a single dose followed 1 day (paclitaxel) or 3 days (cyclophosphamide) later with five daily doses of huKS-IL2 in mice bearing established s.c. tumors, liver metastases, or lung metastases. Tumor models used were CT26/KSA colon, 4T1/KSA mammary, or LLCKSA Lewis lung carcinomas. To measure huKS-IL2 distribution, radiolabeled protein was given to CT26/KSA tumor-bearing mice 1 or 24 h after paclitaxel. huKS-IL2 levels in the tumors were evaluated. RESULTS: Both paclitaxel and cyclophosphamide followed by huKS-IL2 resulted in enhanced antitumor responses compared with either of the treatments alone in the three different tumor models. Results from studies to determine whether the role of the cytotoxic agents in antitumor activity enhancement was related to tumor uptake indicated that a larger fraction of the radiolabeled huKS-IL2 penetrated the tumors when it was administered 24 h after cytotoxic drug "sensitization." CONCLUSION: These data support the idea that prior drug therapy serves to decompress the tumor and lower the diffusion barrier for macromolecules, thus allowing for increased uptake of the huKS-IL2 immunocytokine into the tumor microenvironment. Because the toxicity of the immunocytokine is relatively low at optimal doses, the therapeutic index would likely be greater with the combination treatments.

Targeted interleukin 2 therapy enhances protective immunity induced by an autologous oral DNA vaccine against murine melanoma.

We demonstrate that a mouse-human chimeric anti-ganglioside GD2-interleukin (IL)-2 fusion protein (ch14.18-IL2) substantially amplifies tumor-protective immunity against murine melanoma induced by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to murine melanoma peptide epitopes gp100(25-35) and TRP-2(181-188). This combination therapy led to the complete rejection of a lethal challenge with B78D14 murine melanoma cells in six of eight mice and a marked suppression of s.c. tumor growth in the two remaining animals. The tumor-protective immunity was mediated by MHC class I antigen- restricted CD8(+) T cells together with CD4(+) T cell help, which was required only for tumor cell killing in the effector phase of the immune response. A single oral vaccination with the DNA vaccine, which was carried by attenuated Salmonella typhimurium, was equally as effective as three such vaccinations applied at 2-week intervals. The immunological mechanisms involved in this antitumor effect were suggested by a decisively increased secretion of tumor necrosis factor alpha TNFTnTNa and IFN-gamma from CD4(+) and CD8(+) T cells and a markedly up-regulated expression on CD8(+) T cells of high-affinity IL-2 receptor alpha chain (CD25), costimulatory molecule CD28, and adhesion molecule lymphocyte function-associated antigen-2 (LFA-2/CD2). Additionally, the combination therapy induced increased expression of costimulatory molecules B7.1 and CD48 on murine antigen-presenting cells. Taken together, our results suggest that IL-2 targeted to the tumor microenvironment by a specific antibody-IL-2 fusion protein is a potent enhancer of tumor-protective immunity induced by an oral DNA vaccine that may ultimately enhance the chances of success in its clinical application.

Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice.

Peripheral T-cell tolerance toward human carcinoembryonic self-antigen (CEA) was broken in CEA-transgenic C57BL/6J mice by an oral CEA-based DNA vaccine. This vaccine, delivered by the live, attenuated AroA- strain of Salmonella typhimurium (SL7207), induced tumor-protective immunity mediated by MHC class I-restricted CD8+ T cells. Activation of these T cells was indicated by increased secretion of proinflammatory cytokines IFN-gamma, interleukin (IL)-12 and granulocyte/macrophage-colony stimulating factor, as well as specific tumor rejection and growth suppression in vaccinated CEA-transgenic mice after a lethal challenge with murine MC38 colon carcinoma cells. These tumor cells were double transfected with CEA and the human epithelial cell adhesion molecule (Ep-CAM)/KSA and consequently served as a docking site for a recombinant antibody-IL2 fusion protein (KS1/4-IL2) recognizing KSA. Importantly, the efficacy of the tumor-protective immune response was markedly increased by boosts with this antibody-IL2 fusion protein, resulting in more effective tumor rejection coupled with increased expression of costimulatory molecules B7.2/B7.2 and intercellular adhesion molecule 1 (ICAM-1) on dendritic cells and intensified release of proinflammatory cytokines IFN-gamma, IL-12, and granulocyte/macrophage-colony stimulating factor from T cells of successfully vaccinated CEA-transgenic C57BL/6J mice. Increased T-cell activation mediated by boosts with KS1/4-IL2 fusion protein after tumor cell challenge was further indicated by expanded expression of T-cell activation markers CD25, CD28, CD69, and LFA-1. The application of such CEA-based DNA vaccines and its further improved versions may ultimately prove useful in combination therapies directed against human carcinomas expressing CEA self-antigens.

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.

Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice.

BACKGROUND: Angiogenesis is essential for tumor growth and progression. Therefore, inhibition of angiogenesis is being studied as a new anticancer therapy. Because cytotoxic chemotherapy is more effective on rapidly growing tumors than on slowly growing tumors, it has been assumed that antiangiogenic therapy will also be effective only on rapidly growing, highly vascularized tumors. We compared the effects of two angiogenesis inhibitors, TNP-470 and angiostatin, on slowly growing, poorly vascularized and rapidly growing, highly vascularized human tumors in mice. METHODS: Slowly growing (RT-4) and rapidly growing (MGH-U1) human bladder carcinoma cell lines were grown in severe combined immunodeficiency mice. Established tumors were treated with one of the two angiogenesis inhibitors. Tumor volumes, vascularity, and proliferation indices were determined. The in vitro effects of TNP-470 and of angiostatin on the proliferation of RT-4 and MGH-U1 cells were also investigated. All statistical tests were two-sided. RESULTS: RT-4 and MGH-U1 tumor growth was statistically significantly inhibited by both angiogenesis inhibitors (P<.001). Both inhibitors decreased the blood vessel density in both tumor types but did not alter the in vivo proliferation indices of the tumors. TNP-470, but not angiostatin, marginally decreased the in vitro proliferation of MGH-U1 cells. CONCLUSION: Slowly growing, poorly vascularized tumors in animal models respond as well as rapidly growing, highly vascularized tumors to therapy with the angiogenesis inhibitors TNP-470 and angiostatin.

A fusion inhibitor (FP-21399) for the treatment of human immunodeficiency virus infection: a phase I study.

FP-21399 is a bis(disulfonaphthalene) derivative that prevents human immunodeficiency virus (HIV) infection of uninfected cells by blocking entry of the virus. FP-21399 shows an affinity for lymph nodes. In this phase I study, FP-21399 was administered intravenously over 1 h as a single dose (0.9, 1.7, 2.8, and 4.2 mg/kg) or as a once-weekly infusion (1, 2, and 3 mg/kg) for 4 consecutive weeks to 34 HIV-1 infected patients with CD4(+) cell counts of 50-400 cells/microL. Concomitant antiretroviral therapy was permitted but not required. The most frequent adverse events involved the transient, dose-dependent appearance of drug- or metabolite-related color in the urine and skin. Plasma drug levels were linear with dose. The drug was cleared, with an elimination half-life of 4 h and a terminal half-life of 1.5-2 days; the terminal half-life represented redistribution and clearance from tissues. FP-21399 administered weekly for 4 weeks was well tolerated. Further studies are necessary to define the role of this fusion inhibitor in the treatment of HIV infection.

Melanoma immunotherapy by targeted IL-2 depends on CD4(+) T-cell help mediated by CD40/CD40L interaction.

The induction of tumor-protective immunity against malignancies remains a major challenge in cancer immunotherapy. A novel, humanized anti-ganglioside-GD(2)-IL-2 immunocytokine (hu14.18-IL-2) induced CD8(+) T cells to eradicate established pulmonary metastases of B78-D14 murine melanoma, in a process that required help by CD4(+) T cells and was mediated by the CD40/CD40 ligand (CD40L) interaction. The anti-tumor effect was diminished in mice deficient in CD4(+) T-cells. Three lines of evidence show that CD4(+) T-cell help was mediated by CD40/CD40L interaction but not by endogenous IL-2 production. First, the hu14.18-IL-2-induced anti-tumor response is partially abrogated in C57BL/6J CD40L knockout (KO) mice in contrast to C57BL/6J IL-2 KO animals, in which the immunocytokine was completely effective. Second, partial abrogation of the anti-tumor effect is induced with anti-CD40L antibodies to the same extent as with CD4(+) T-cell depletion. Third, a complete anti-tumor response induced by hu14.18-IL-2 can be reconstituted in C57BL/6J CD40L KO mice by simultaneous stimulation with an anti-CD40 mAb. These results suggest that help provided by CD4(+) T cells via CD40/CD40L interactions in our tumor model is crucial for effective immunotherapy with an IL-2 immunocytokine.

What to do with targeted IL-2.

A common strategy in immunotherapy of cancer is the induction of an increased immunogenicity of syngeneic malignancies. A novel approach to achieve this goal is the targeting of cytokines into the tumor microenvironment with antibody-cytokine fusion proteins, called immunocytokines. This report summarizes therapeutic efficacy and immune mechanisms involved in targeting IL-2 to syngeneic tumors and describes their extended use as a synergistic treatment modality for cancer vaccines and antiangiogenesis. Treatment of established melanoma and colon carcinoma metastases with IL-2 immunocytokines resulted in eradication of disease, followed by a vaccination effect protecting mice from lethal challenges with wild-type tumor cells. In a syngeneic neuroblastoma model, targeted IL-2 elicited effective antitumor responses mediated by NK cells in the absence of a T-cell memory. Interestingly, targeted IL-2 was effective in amplification of memory immune responses previously induced by cancer vaccines. Furthermore, a synergistic effect achieved by combining targeted IL-2-immunotherapy with an antiangiogenic inhibitor of integrin alpha(v)beta(3) extends the potential of this immunotherapeutic strategy in combination with antiangiogenesis as demonstrated in three syngeneic tumor models. Based on these findings, targeted IL-2 may provide an effective tool for the adjuvant treatment of cancer either applied as a single strategy or in combination with cancer vaccines and antiangiogenic strategies.

T cell memory against colon carcinoma is long-lived in the absence of antigen.

Eradication of established colon carcinoma metastases is a major goal for adjuvant immunotherapy of this disease. This was accomplished in a murine model by targeting IL-2 to the tumor microenvironment with a recombinant Ab-IL-2 fusion protein (huKS1/4-IL-2). The generation of a long-lived protective immunity was demonstrated by a 10- to 14-fold increase in CTL precursor (pCTL) frequency and induction of genes encoding Th1 cytokines, followed by the generation of tumor-specific CD8+ T effector cells, some of which differentiated into long-lived T memory cells. The frequency of pCTL correlated with enhanced immune protection against tumor cell challenge, and long-lived T cell memory was maintained in syngeneic SCID mice in the absence of tumor Ag. Tumor cell challenge of these SCID mice, concomitant with a boost of two noncurative doses of huKS1/4-IL-2 fusion protein, resulted in the generation of primed CD8+ T effector cells with concurrent release of Th1 cytokines. These events culminated in the complete rejection of the tumor cell challenge and prevention of pulmonary metastases. Taken together, the data suggest that T cell memory against colon carcinoma can be maintained in the absence of Ag.

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.

Tumor-targeted IL-2 amplifies T cell-mediated immune response induced by gene therapy with single-chain IL-12.

Induction, maintenance, and amplification of tumor-protective immunity after cytokine gene therapy is essential for the clinical success of immunotherapeutic approaches. We investigated whether this could be achieved by single-chain IL-12 (scIL-12) gene therapy followed by tumor-targeted IL-2 using a fusion protein containing a tumor-specific recombinant anti-ganglioside GD(2) antibody and IL-2 (ch14.18-IL-2) in a poorly immunogenic murine neuroblastoma model. Herein, we demonstrate the absence of liver and bone marrow metastases after a lethal challenge with NXS2 wild-type cells only in mice (five of six animals) vaccinated with scIL-12-producing NXS2 cells and given a booster injection of low-dose ch14.18-IL-2 fusion protein. This tumor-protective immunity was effective 3 months after initial vaccination, in contrast to control animals treated with a nonspecific fusion protein or an equivalent mixture of antibody and IL-2. Only vaccinated mice receiving the tumor-specific ch14.18-IL-2 fusion protein revealed a reactivation of CD8(+) T cells and subsequent MHC class I-restricted tumor target cell lysis in vitro. The sequential increase in the usage of TCR chains Vbeta11 and -13 in mouse CD8(+) T cells after vaccination and amplification with ch14.18-IL-2 suggests that the initial polyclonal CD8(+) T cell response is effectively boosted by targeted IL-2. In conclusion, we demonstrate that a successful boost of a partially protective memory T cell immune response that is induced by scIL-12 gene therapy could be generated by tumor-specific targeting of IL-2 with a ch14.18-IL-2 fusion protein. This approach could increase success rates of clinical cancer vaccine trials.

Improving the efficacy of antibody-interleukin 2 fusion proteins by reducing their interaction with Fc receptors.

Fusion proteins between whole antibodies (Abs) and cytokines (immunocytokines) such as interleukin 2 have shown efficacy in several mouse tumor models despite a circulating half-life that is significantly shorter than that of the original Ab. We have examined the potential mechanisms responsible for clearance and shown that an important factor is enhanced binding to Fc receptor (FcR). Improvements in the half-lives of two different immunocytokines were made by changing the isotype of the human heavy chain C region from IgG1 or IgG3 to those with reduced binding to FcR, e.g., IgG4. The same effect could also be achieved through site-directed mutagenesis of the FcR binding site in the IgG1 H chain. In vitro studies using mouse J774 FcR-expressing cells showed increased binding of interleukin 2-based immunocytokines, relative to their corresponding Abs, and that this was reversed in those fusion proteins made with IgG4 or mutated IgG1 H chains. All of the fusion proteins showing reduced FcR binding also had reduced Ab-dependent cellular cytotoxicity activity, as measured in 4-h chromium release assays. A complete loss of complement-dependent cytotoxicity activity was seen with an IgG4-based immunocytokine derived from an IgG1 Ab with potent activity. Despite these reduced effector functions, the IgG4-based immunocytokines with extended circulating half-lives showed equivalent (in the case of severe combined immunodeficiency mouse xenograft models) or better (in the case of syngeneic models) efficacy in mouse tumor models than the original IgG1-based molecules. These novel immunocytokines may show improved efficacy in therapeutic situations where T cell- rather than natural killer- or complement-mediated antitumor mechanisms are involved.

Specific enzyme-linked immunosorbent assays for quantitation of antibody-cytokine fusion proteins.

Preliminary testing has shown in vitro and in vivo that antitumor activity can be obtained with fusion proteins linking tumor-reactive monoclonal antibodies to cytokines, such as granulocyte-macrophage colony-stimulating factor or interleukin 2 (IL-2). Preclinical and clinical testing of these reagents requires their in vitro and in vivo quantitation and pharmacokinetic evaluation. We have focused on the detection of a fusion protein which links one human IL-2 molecule to the carboxy terminus of each heavy chain of the tumor-reactive human-mouse chimeric anti-GD2 antibody, ch14.18. We have developed enzyme-linked immunosorbent assays (ELISAs) to evaluate intact tumor-reactive fusion proteins. By these ELISAs we can reliably measure nanogram quantities of intact ch14.18-IL-2 fusion protein and distinguish the intact protein from its components (ch14.18 and IL-2) in buffer, mouse serum, and human serum with specificity and reproducibility. The measurement of intact ch14.18-IL-2 fusion protein is not confounded by free IL-2 or free ch14.18 when 100 ng or less of total immunoglobulin per ml is used during the assay procedure. Our results indicate that these ELISAs are suitable for preclinical and clinical testing and with slight modifications are applicable to the analysis of a variety of other fusion proteins.

Synergy between an antiangiogenic integrin alphav antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases.

The suppression and eradication of primary tumors and distant metastases is a major goal of alternative treatment strategies for cancer, such as inhibition of angiogenesis and targeted immunotherapy. We report here a synergy between two novel monotherapies directed against vascular and tumor compartments, respectively, a tumor vasculature-specific antiangiogenic integrin alphav antagonist and tumor-specific antibody-interleukin 2 (IL-2) fusion proteins. Simultaneous and sequential combination of these monotherapies effectively eradicated spontaneous liver metastases in a poorly immunogenic syngeneic model of neuroblastoma. This was in contrast to controls subjected to monotherapies with either an antiangiogenic integrin alphav antagonist or antibody-IL-2 fusion proteins, which were only partially effective at the dose levels applied. Furthermore, simultaneous treatments with the integrin alphav antagonist and tumor-specific antibody-IL-2 fusion proteins induced dramatic primary tumor regressions in three syngeneic murine tumor models, i.e., melanoma, colon carcinoma, and neuroblastoma. However, each agent used as monotherapy induced only a delay in tumor growth. A mechanism for this synergism was suggested because the antitumor response was accompanied by a simultaneous 50% reduction in tumor vessel density and a 5-fold increase in inflammatory cells in the tumor microenvironment. Subsequently, tumor necrosis was demonstrated only in animals receiving the combination therapy, but not when each agent was applied as monotherapy. The results suggest that these synergistic treatment modalities may provide a novel and effective tool for future therapies of metastatic cancer.

Construction of phosphorylatable chimeric monoclonal antibody CC49 with a casein kinase I recognition site.

Phosphorylation sites for casein kinase I were introduced into chimeric monoclonal antibody CC49 (MAb-chCC49) by inserting a synthetic fragment (CK1) encoding two casein kinase I phosphorylation sites into an expression vector. The phosphorylation sites were created by incorporating the predicted consensus sequences for phosphorylation by the casein kinase I at the carboxyl terminus of the heavy-chain constant region of the MAb-chCC49. The resultant modified MAb-chCC49 (MAb-chCC49CK1) was expressed and purified. The MAb-chCC49CK1 protein can be phosphorylated by the casein kinase I with [gamma-32P]ATP to high radiospecific activity. The 32P-labeled MAb-chCC49CK1 protein binds to cells expressing TAG-72 antigens. The introduction of phosphorylation sites into MAb provides new reagents for the diagnosis and treatment of cancer. This demonstrates that, as was described for the cAMP-dependent protein kinase site, the casein kinase I recognition site can also be used to introduce phosphorylation sites into proteins.

The Ch14.18-GM-CSF fusion protein is effective at mediating antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in vitro.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is very effective at enhancing antibody-dependent cellular cytotoxicity (ADCC) mediated by granulocytes and monocytes. Recently, a fusion protein consisting of GM-CSF and chimeric human/mouse anti-ganglioside G(D2) antibody Ch14.18 (Ch14.18-GM-CSF) has been generated to improve the effectiveness of immunotherapy by directing GM-CSF to the tumor microenvironment and prolonging its relatively short half-life. In this study, we examined the ability of this fusion protein to enhance the in vitro killing of G(D2)-expressing human neuroblastoma cells by granulocytes and mononuclear cells, as well as by complement. The Ch14.18-GM-CSF fusion protein was equally effective as the combination of equivalent amounts of free Ch14.18 and GM-CSF in mediating the killing of NMB7 neuroblastoma cells by granulocytes from seven of eight neuroblastoma patients. The fusion protein was also equally effective as the combination of Ch14.18 and GM-CSF in mediating ADCC by neuroblastoma patients' mononuclear cells. In addition, the fusion protein was as effective as Ch14.18 alone in directing complement-dependent cytotoxicity against NMB7 cells. Our results demonstrate that the biological activities expressed by ADCC and complement-dependent cytotoxicity of both monoclonal antibody Ch14.18 and GM-CSF are retained by the Ch14.18-GM-CSF fusion protein and lend further support for future clinical trials of this fusion protein in patients with neuroblastoma.