Induction of antitumor immunity using intercellular adhesion molecule 1 (ICAM-1) transfection in mouse glioma cells.

We investigated the role of intercellular adhesion molecule 1 (ICAM-1) expression in tumorigenicity of gliomas and the antitumor effects of glioma cells genetically engineered to express ICAM- 1. Mouse glioma cells transfected with ICAM-1 grew in T-cell deficient mice but not in syngeneic mice. Vaccination with ICAM-1-transfected tumor cells markedly inhibited the growth of subcutaneously inoculated gliomas but not gliomas located in the brain. In vivo antibody ablation study revealed that CD4+ T, CD8+ T and NK cells were all required to produce the antitumor effect of SR/ICAM- 1. In this study, we demonstrated the therapeutic potential of vaccination with ICAM-1-overexpressing tumor cells for the control of the tumor growth.     

Adenovirus-mediated CD40 ligand gene-engineered dendritic cells elicit enhanced CD8(+) cytotoxic T-cell activation and antitumor immunity

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in their activation and is essential for induction of antigen-specific T-cell responses. In the present study, we investigated the efficacy of antitumor immunity induced by vaccination with DCs engineered to express CD40L and pulsed with Mut1 tumor peptide. Our data show that transfection of DCs with recombinant adenovirus AdV-CD40L resulted in activation of DCs with up-regulated expression of proinflammatory cytokines (IL-1beta and IL-12), chemokines (RANTES, IP-10, and MIP-1alpha), and immunologically important cell surface molecules (CD54, CD80, and CD86). Our data also demonstrate that DCs transfected with AdV-CD40L (DC(CD40L)) are able to stimulate enhanced allogeneic T-cell proliferation and Mut1-specific CD8(+) cytotoxic T-cell responses in vitro. Vaccination of mice with Mut1 peptide-pulsed control virus-transfected DC (DC(pLpA)) could only protect mice from challenge of a low dose (0.5 x 10(5) cells per mouse, 8/8 mice), but not a high dose (3 x 10(5) cells per mouse, 0/8 mice) of 3LL tumor cells. However, vaccination of Mut1 peptide-pulsed AdV-CD40L-transfected DC(CD40L) induced an augmented antitumor immunity in vivo by complete protection of mice (8/8) from challenge of both low and high doses of 3LL tumor cells. Thus, DCs engineered to express CD40L by adenovirus-mediated CD40 ligand gene transfer may offer a new strategy in production of DC cancer vaccines.     

Enhanced antitumor effect of oncolytic adenovirus expressing interleukin-12 and B7-1 in an immunocompetent murine model.

PURPOSE: We investigated whether an armed viral platform, where lytic property of a viral infection is coupled to viral-mediated delivery of therapeutic genes, could increase the therapeutic potential of a viral-based therapy. EXPERIMENTAL DESIGN: We generated interleukin (IL)-12-expressing oncolytic adenovirus (YKL-IL-12) and IL-12- and B7-1-expressing (YKL-IL12/B7) oncolytic adenovirus. Therapeutic efficacy of these newly engineered adenoviruses was then evaluated in vivo using an immunocompetent mouse bearing murine melanoma B16-F10 tumors. Overall survival was assessed with the Kaplan-Meier method. The induction of immune cell cytotoxicity was assessed by CTL assay, IFN-gamma enzyme-linked immunospot assay, and immunohistochemical studies. RESULTS: YKL-IL12/B7 oncolytic adenovirus, expressing both IL-12 and B7-1, showed a higher incidence of complete tumor regression compared with the analogous oncolytic adenovirus, YKL-1, or IL-12-expressing, YKL-IL12. Significant survival advantage was also seen in response to YKL-IL12/B7. Moreover, IL-12 and IFN-gamma levels produced in tumors treated with YKL-IL12/B7 was significantly greater than those treated with YKL-IL12. The enhanced survival advantage was mediated by the induction of immune cell cytotoxicity. In agreement with these results, massive infiltration of CD4(+) and CD8(+) T cells into tissues surrounding the necrotic area of the tumor was observed following in situ delivery of YKL-IL12/B7. CONCLUSION: Combination of oncolysis and the enhancement of antitumor immune response by oncolytic adenovirus expressing both IL-12 and B7-1 elicits potent antitumor effect and survival advantage.     

Antitumor immunity after vaccination with B lymphoma cells overexpressing a triad of costimulatory molecules

BACKGROUND: The costimulatory molecules B7-1, intercellular adhesion molecule-1 (ICAM-1), and leukocyte function-associated antigen-3 (LFA-3) play pivotal roles in the activation of T cells. We investigated whether in vivo vaccination with lymphoma cells infected with a recombinant, nonreplicating fowlpox (FP) virus encoding this triad of costimulatory molecules (TRICOM) could stimulate lymphoma-specific immunity. METHODS: TRICOM-infected A20 B lymphoma cells were analyzed for expression of B7-1, ICAM-1, and LFA-3. Mice (10 per group) were vaccinated with irradiated A20 cells infected with either the TRICOM vector or the wild-type FP virus (WT-FP), challenged with live A20 tumor cells, and followed for survival. Mice with established A20 tumors were also treated with irradiated TRICOM-infected A20 cells. Survival curves were compared with the log-rank statistic. The mechanism of the antitumor effect was studied by in vivo depletion of CD4(+) and CD8(+) T cells and in vitro cytotoxicity assays. All statistical tests were two-sided. RESULTS: A20 tumor cells infected with TRICOM expressed high levels of B7-1, ICAM-1, and LFA-3. Mice vaccinated with irradiated TRICOM-infected A20 cells had prolonged survival relative to mice vaccinated with WT-FP-infected cells (80% versus 20% survival at 110 days; P<.001). In mice with established tumors, tumor growth was slower in those treated with TRICOM-infected tumor cells than in those treated with WT-FP-infected cells, and this treatment provided a survival advantage (P<.001). Depletion of CD4(+) or CD8(+) T cells reduced the antitumor immunity provided by the tumor cell-TRICOM vaccine, and lymphocytes from vaccinated mice displayed in vitro cytotoxic activity toward A20 cells. CONCLUSIONS: Increasing expression of costimulatory molecules on B lymphoma cells by infection with a recombinant FP virus encoding B7-1, ICAM-1, and LFA-3 stimulates antitumor immune responses in vivo and may provide a novel strategy for treating patients with B-cell malignancies.     

Both CD4+ and CD8+ T cell epitopes fused to heat shock cognate protein 70 (hsc70) can function to eradicate tumors

Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP-associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant-free manner and is mediated by CD8(+) T cells, indicating that HSP can act as a natural adjuvant and cross-prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8(+) T cell epitope fused to the carboxyl-terminus of hsc70 and demonstrated efficient generation of antigen-specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70-CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4(+) T cell epitope to the amino terminus of the hsc70-CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)-derived 8 mer peptide, OVA(257-264), and 16mer peptide, OVA(265-280), were used as CD8(+) and CD4(+) T cell epitopes, respectively. Vaccination with hsc70-OVA(257-264) generated peptide specific CTL more effectively than a peptide plus incomplete Freund's adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA(265-280) to the amino-terminus of hsc70-OVA(257-264) (OVA(265-280)-hsc70-OVA(257-264)) enhanced the generation of the OVA(257-264)-specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70-OVA(257-264). Our results suggest that fusion of both CD4(+) and CD8(+) T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8(+) T cell epitope alone.     

Immune properties of recombinant vaccinia virus encoding CD154 (CD40L) are determined by expression of virally encoded CD40L and the presence of CD40L protein in viral particles

Expression of costimulatory molecules by recombinant poxviruses is a promising strategy for enhancing therapeutic vaccines. CD40-CD40L interactions are critical for conditioning dendritic cells (DC) and priming T- and B-cell immunity. We constructed a vaccinia virus expressing murine CD40L (rV-CD40L) and studied its immunomodulatory properties in vitro. Direct DC infection with control vaccinia or psoralen/UV-inactivated rV-CD40L stimulated high levels of interleukin 12 (IL-12) release. However, replication-competent rV-CD40L did not stimulate IL-12 under similar conditions. We observed a high level of CD40L protein on purified viral particles and demonstrated that induction of IL-12 by nonreplicating rV-CD40L was blocked by anti-CD40 antibodies suggesting that functional CD40L on viral particles contributed to alterations in IL-12 synthesis.Since cross-presentation of tumor-associated antigens by DC is augmented by viral infection of tumor cells, we infected MC38 murine colon carcinoma cells with rV-CD40L. Infected cells stimulated IL-12 secretion by DC and proliferation of B cells and DX5(+) (NK/NKT) cells through direct CD40-CD40L interaction. A subpopulation of NKT cells expressing CD40 (NK1.1(+), CD3(lo)) appeared to be a major effector population responding to MC38/rV-CD40L. These results highlight the complex immune regulatory effects of rV-CD40L defined by the cumulative effects of CD40L expression, presence of CD40L protein in viral particles, and the replication potential of the virus.     

In vivo antitumor effect of CD40L-transduced tumor cells as a vaccine for B-cell lymphoma

CD40-CD40 ligand (CD40L) interactions play a critical role in the activationof cellular immunity. CD40L enhances the antigen presentation function of CD40-expressing B cells. We have used a murine B-cell lymphoma model (A20) to study the in vivo antitumor effect of the administration of tumor cells transduced with a recombinant adenovirus encoding CD40L (AdvCD40L). After infection with AdvCD40L, A20 tumor cells up-regulate several T-cell costimulatory molecules (CD80, CD86, ICAM-1, and LFA-3) and Fas expression. Animals vaccinated with irradiated tumor cells transduced with AdvCD40L are protected against a lethal dose of parental A20 tumor cells. Animals with pre-existing tumors treated with AdvCD40L-transduced tumor cells display inhibition of the tumor growth, and this treatment confers a survival advantage. In vivo depletion studies demonstrate that both CD4(+) and CD8(+) T cells mediate the antitumor immunity provided by AdvCD40L-transduced tumor cells. These results show that genetic modification of tumor B cells with CD40L can be a useful strategy to promote systemic immunity against B-cell malignancies and provide an in vivo system to allow for additional evaluation and refinement of this approach.     

Recombinant adenoviral vector expressing human wild-type p53, GM-CSF,and B7-1 genes suppresses the growth of glioma in vivo

Malignant gliomas are the most common of primary brain tumors and have been proven incurable with conventional treatments. Evidence have shown that a recombinant adenoviral vector expressing human wild-type p53, granulocyte-macrophage colony-stimulating factor (GM-CSF), and B7-1 genes (BB-102) may have antitumor effects in vitro. In this study, we investigated the effects of BB-102-based vaccine on glioma in vivo. An animal model using nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with human immune system was established. The mice were vaccinated with inactivated U251 glioma cells transduced with BB-102 or adenoviral vector expressing green fluorescence protein (Ad-GFP) as a control and followed by the challenge of live U251 glioma cells. Tumor growth and antitumor responses were measured. Data showed that mice vaccinated with BB-102 had significantly reduced local tumor growth compared to mice with Ad-GFP vaccination or the control group. Histopathological analysis displayed low tumor cell density and significant infiltration of human peripheral blood lymphocytes (HuPBLs) in the tumor tissues of mice transduced with BB-102. Immunohistochemical analysis showed that mutant p53 was not expressed in tumor tissues of mice with BB-102 vaccination, and the expression level of Ki67 was significantly lower in the tumor tissues of the BB-102 group than those in the Ad-GFP group or the control group. Further study demonstrated that mice with BB-102 vaccination had significantly increased total T cell numbers, total T cell proportion, CD4+ T cell proportion, and CD8+ T cell proportion in spleens, as well as higher value of IgG, IgA, and IgE in sera. These data suggest that the recombinant adenoviral vector expressing human wild-type p53, GM-CSF, and B7-1 genes could suppress glioma in NOD/SCID mice model and might be considered as a novel strategy for glioma therapy.     

Induction of potent antitumor immunity by intratumoral injection of interleukin 23-transduced dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in priming immune responses to tumor. Interleukin (IL)-23 can act directly on DC to promote immunogenic presentation of tumor peptide in vitro. Here, we evaluated the combination of bone marrow-derived DC and IL-23 on the induction of antitumor immunity in a mouse intracranial glioma model. DCs can be transduced by an adenoviral vector coding single-chain mouse IL-23 to express high levels of bioactive IL-23. Intratumoral implantation of IL-23-expressing DCs produced a protective effect on intracranial tumor-bearing mice. The mice consequently gained systemic immunity against the same tumor rechallenge. The protective effect of IL-23-expressing DCs was comparable with or even better than that of IL-12-expressing DCs. IL-23-transduced DC (DC-IL-23) treatment resulted in robust intratumoral CD8(+) and CD4(+) T-cell infiltration and induced a specific TH1-type response to the tumor in regional lymph nodes and spleen at levels greater than those of nontransduced DCs. Moreover, splenocytes from animals treated with DC-IL-23 showed heightened levels of specific CTL activity. In vivo lymphocyte depletion experiments showed that the antitumor immunity induced by DC-IL-23 was mainly dependent on CD8(+) T cells and that CD4(+) T cells and natural killer cells were also involved. In summary, i.t. injection of DC-IL-23 resulted in significant and effective systemic antitumor immunity in intracranial tumor-bearing mice. These findings suggest a new approach to induce potent tumor-specific immunity to intracranial tumors. This approach may have therapeutic potential for treating human glioma.     

Interleukin-12-secreting human papillomavirus type 16-transformed cells provide a potent cancer vaccine that generates E7-directed immunity

The development of a vaccine that would be capable of preventing or curing the (pre)cancerous lesions induced by genital oncogenic human papillomaviruses (HPVs) is the focus of much research. Many studies are presently evaluating vaccines based on the viral E6 and E7 oncoproteins, both of which are continually expressed by tumor cells. The success of a cancer vaccine relies, in large part, on the induction of a tumor-specific Th1-type immunity. In this study, we have evaluated the ability of B7-related and/or interleukin-12 (IL-12)-expressing, non-immunogenic murine HPV16-transformed BMK-16/myc cells, to achieve this goal. BMK-16/myc cells engineered to express surface B7-1 or B7-2 molecules remain tumorigenic in syngeneic BALB/c mice, suggesting that expression of these molecules alone is not sufficient to induce tumor regression. In contrast, mice injected with tumor cells engineered to secrete IL-12 remained tumor-free, demonstrating that IL-12 expression is sufficient to induce tumor rejection. IL-12-secreting BMK-16/myc cells were further shown to induce potent and specific long-term tumor resistance, even after irradiation. B7-1 was found to slightly but systematically improve anti-tumor immunity elicited by IL-12-secreting BMK-16/myc cells. Injection of irradiated B7-1/IL-12+ BMK-16/myc cells generates long-lasting, Th1-type, BMK-16/myc-directed immunity in tumor-resistant mice. These mice display a memory-type, E7-specific, cell-mediated immune response, which is potentially significant for clinical applications.     

Autologous lymphocyte responses to adenovirus-B7-1-transduced human cancer cells.

Transfection of the costimulatory molecule B7-1 (CD80) into murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this study was to test autologous lymphocyte responses against freshly resected human cancers infected in vitro with an adenovirus vector expressing the B7-1 molecule (AdB7). Resected tumors (sarcomas, adenocarcinomas, melanomas, and multiple myelomas) were disaggregated into single-cell suspensions and divided into three groups: (a) native, noninfected tumor cells (TM); (b) AdB7-infected, B7-1(+) tumor cells (TMB7); and (c) control Addl70.3-infected, B7-1(-) tumor cells (TMAd). B7-1 expression was verified by flow cytometry. Autologous peripheral blood lymphocytes from these patients were tested for proliferative and cytotoxic activity against the three tumor groups. There was an increased lymphocyte-proliferative response against B7-1(+) tumor cells, particularly in the presence of interleukin-12 (IL-12) or low-dose IL-2. B7-1(+) tumor cells were also killed more efficiently than B7-1(-) tumor cells in natural killer cell-mediated cytotoxicity assays, and this was most significant when lymphocytes had been pretreated with IL-12. Human natural killer cells were found to express CD28, a receptor for B7-1. The high efficiency of AdB7-mediated gene transfer and the augmented B7-1-mediated lymphocyte responses suggest that AdB7 vectors may be effective in human cancer immunotherapy.     

Synergy of vaccine strategies to amplify antigen-specific immune responses and antitumor effects

Several different vaccine strategies have been evaluated and combined in an attempt to amplify T-cell responses toward induction of antitumor immunity. The model tumor antigen used was carcinoembryonic antigen (CEA). While initial T-cell activation studies were conducted in conventional mice, combined vaccine strategy studies and antitumor studies were conducted in transgenic mice in which CEA is expressed in normal gastrointestinal tissue and CEA protein is found in sera. The studies reported here demonstrate: (a) A recombinant avipox (fowlpox, rF) vector expressing the signal 1 (CEA) and the B7-1 costimulatory molecule transgenes (designated rF-CEA/B7-1) is more potent in inducing CEA-specific T-cell responses than rF-CEA; one administration of recombinant fowlpox vector expressing CEA and three different costimulatory molecule transgenes (B7-1, ICAM-1, LFA-3, designated rF-CEA/TRICOM) was more potent in inducing CEA-specific T-cell responses than four vaccinations with rF-CEA or two vaccinations with rF-CEA/B7-1. Moreover, up to four vaccinations with rF-CEA/TRICOM induced greater CEA-specific T-cell responses with each vaccination. (b) A diversified prime and boost strategy using a prime with a recombinant vaccinia vector expressing CEA and the triad of costimulatory molecules (designated rV-CEA/TRICOM) and a boost with rF-CEA/TRICOM was more potent in inducing CEA-specific T-cell responses than the repeated use of rF-CEA/TRICOM alone. (c) The addition of granulocyte macrophage colony-stimulating factor (GM-CSF) to the rF-CEA or rF-CEA/TRICOM vaccinations via the simultaneous administration of a rF-GM-CSF vector enhanced CEA-specific T-cell responses. These strategies (TRICOM/diversified prime and boost/GM-CSF) were combined to treat CEA-expressing carcinoma liver metastases in CEA-transgenic mice; vaccination was initiated 14 days posttumor transplant. Antitumor effects in terms of survival and CD8(+) and CD4(+) responses specific for CEA were also observed in this CEA-transgenic mouse model. These studies demonstrate that the use of cytokines and diversified prime and boost regimens can be combined with the use of recombinant vectors expressing signal 1 and multiple costimulatory molecules to further amplify T-cell responses toward more effective vaccine strategies.     

GM-CSF and B7-1 (CD80) co-stimulatory signals co-operate in the induction of effective anti-tumor immunity in syngeneic mice

B7-1 (CD80) co-stimulatory molecule gene–transduced Lewis lung carcinoma (LLC) cells (LLC/B7 cells) resulted in remarkable loss of tumorigenicity in syngeneic C57BL/6 mice (87.5% rejection) compared to B7-negative, wild-type LLC (LLC/wt) cells (0% rejection). However, mice that had rejected LLC/B7 cells developed almost no systemic immunity protective against challenge with wild-type tumor cells after 4 weeks (11.8% rejection). Enhancement of MHC class I (H-2K^b) expression of LLC/B7 cells with in vitro interferon-γ treatment did not result in enhancement of protective immunity. In vivo depletion assay revealed that abrogation of tumorigenicity in LLC/B7 depended on CD8⁺ T cells but not on CD4⁺ T cells. However, vaccination of C57BL/6 mice with irradiated LLC cells transduced with GM-CSF (LLC/GM) led to the induction of potent, specific immunity against challenge with the LLC/wt cells after 2 weeks (80.8% rejection). Next, we established a double transfectant of LLC cells expressing both B7-1 and GM-CSF (LLC/GM + B7). The tumorigenicity of these clonal cells was also remarkably suppressed (90% rejection) to the same degree as LLC/B7, whereas that of LLC/GM was not suppressed (0% rejection). Interestingly, mice that had rejected LLC/GM+B7 cells developed enhanced protective immunity against challenge with LLC/wt cells after 4 weeks (55.6% rejection) compared to the results of LLC/B7 cells (11.8%). To evaluate whether co-expression of GM-CSF and B7-1 enabled the tumor cells to activate cytotoxic T cells more efficiently than B7-1 alone, we performed an in vitro killing assay. We found that immunization with LLC/GM+B7 cells resulted in a 3-fold stronger cytotoxic response than that with LLC/B7. Our data indicate that co-transfection of the B7-1 co-stimulatory molecule and GM-CSF genes may be more effective for the induction of stronger protective immunity in this experimental system. Int. J. Cancer 73:556–561, 1997. © 1997 Wiley-Liss, Inc.     

Depletion of CD4+CD25+ regulatory T cells enhances interleukin-2-induced antitumor immunity in a mouse model of colon adenocarcinoma

Interleukin 2 (IL)-2 induces antitumor immunity and clinical responses in melanoma and renal cell carcinoma. However, IL-2 also increases the number of CD4(+)CD25(+) regulatory T (Treg) cells that suppress antitumor immune responses. The aim of the present study was to elucidate the effect of depletion of Treg cells on IL-2-induced antitumor immunity. IL-2-transfected mouse colon adenocarcinoma (MC38/IL-2) cells were implanted subcutaneously or intrahepatically into male C57BL/6 mice, and tumor growth and the proportion of tumor-infiltrating lymphocytes with Treg-cell depletion in response to treatment with anti-CD25 monoclonal antibody (PC61) were determined. In mice treated with phosphate-buffered saline, 40-60% of MC38/IL-2 tumors were rejected. In contrast, all MC38/IL-2 tumors were rejected in mice treated with PC61. The number of tumor-infiltrating CD8(+) T cells in mice treated with PC61 was approximately twice that in mice treated with PBS. The numbers of tumor-infiltrating CD4(+) and natural killer cells were also increased significantly. To test the antimetastatic effects of IL-2 treatment in combination with Treg-cell depletion, human recombinant IL-2 (rIL-2) and PC61 were administered to mice implanted with MC38/mock cells in the spleen, and hepatic metastasis was investigated. The average liver weight in mice treated with rIL-2 plus PC61 was 1.04 +/- 0.03 g, less than that in mice treated with rIL-2 (2.04 +/- 0.51 g) or PC61 alone (1.81 +/- 0.38 g). We conclude that IL-2-induced antitumor immunity is enhanced by Treg-cell depletion and is due to expansion of the tumor-infiltrating cytotoxic CD8(+) T-cell population.     

Coexpression of CD40L and CD70 by semiallogenic tumor cells induces anti-tumor immunity

The immune system is potentially qualified to detect and eliminate tumor cells, but various mechanisms developed by tumor cells allow tumor escape. Strategies selected to promote antitumor responses have included genetic modifications of tumor cells to induce expression of costimulatory molecules. Moreover, alloantigens can also act as strong enhancers of the immune response. In this work, we have associated the expression of two costimulatory members of the TNF superfamily, CD40L and CD70 along with an allogenic MHC Class I (H-2K(d)) molecule expression on melanoma cells (B16F10, H-2(b)) to favor the antitumor immune response. B16F10 tumor growth slows significantly when CD40L and CD70 are coexpressed by tumor cells and the association with the allogenic molecule (H-2K(d)) enhances this effect. Growth kinetics of mock and CD40L-CD70-H-2K(d)-expressing B16F10 tumors in immunocompetent versus nu/nu and beige mice suggested that CD8(+) T lymphocytes and NK cells were involved in this antitumor immunity. A delay in mock tumor growth was observed when CD40L-CD70-H-2K(d)-expressing B16F10 cells and mock tumor cells were injected simultaneously and contralaterally. It was also shown that in vivo immunization of immunocompetent mice with CD40L-CD70-H-2K(d) B16F10 tumor cells improved the generation of cytotoxic lymphocytes against the wild-type melanoma cells expressing the syngenic MHC Class I molecule H-2K(b) (B16K1). These observations lay a path for new immunotherapeutic trials using semiallogenic fibroblasts expressing costimulatory molecules and tumor-associated antigens.     

Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fragments for tumor targeting.

Efficient T-cell activation requires two signals. The first signal, which confers specificity, is provided by interaction of the T-cell receptor with peptides presented by MHC molecules. One of the second costimulatory signals is induced by binding of B7 proteins on the surface of antigen-presenting cells to CD28 on the T-cell surface. Expression of B7 molecules on tumor cells can result in the activation of tumor specific T lymphocytes and induce protective antitumor immunity. However, at present such gene-therapeutic approaches are limited by the inability to selectively target B7 gene expression to cancer cells. As an alternative approach we exploited recombinant antibody fragments to localize a costimulatory B7 molecule to the surface of tumor cells. We constructed chimeric proteins that contain in a single polypeptide chain a portion of human B7-2 (CD86) genetically fused to single-chain (sc) Fv antibody domains specific for the tumor-associated antigens epidermal growth factor receptor and the closely related ErbB2 receptor tyrosine kinase. A small recombinant fragment of human CD86 was characterized that corresponds to amino acid residues 1-111 (CD86(111)) of the mature protein. CD86(111) produced in the yeast Pichia pastoris and CD86(111) expressed in bacteria was functionally active and displayed specific binding to B7 counter receptors. Bacterially expressed CD86(111)-scFv fusion proteins specifically localized to the respective target antigens on the surface of tumor cells and markedly enhanced the proliferation of primary T cells when bound to immobilized tumor antigen.     

Regression of intracerebral rat glioma isografts by therapeutic subcutaneous immunization with interferon-gamma, interleukin-7, or B7-1-transfected tumor cells

Progress in the definition of the roles of various costimulators and cytokines in determining the type and height of immune responses has made it important to explore genetically altered tumor cells expressing such molecules for therapeutic immunizations. We have studied the effect of therapeutic subcutaneous (s.c.) immunizations on the growth of preexisting intracerebral brain tumor isografts in the rat. Transfectant glioma cell clones expressing either rat interferon-gamma (IFN-gamma), rat interleukin-7 (IL-7), or rat B7-1 were selected. After irradiation (80 Gy) the clones were used for immunization (administered in up to four s.c. doses in a hind leg over 14-day intervals starting 1 day after the intracranial isografting of the parental tumor). Significant growth inhibition of the intracerebral parental tumors was induced by transfectants expressing IFN-gamma and IL-7, respectively. The strongest effect was observed with IFN-gamma-expressing cells, resulting in cures in 37% of the males and in 100% of the females. Immunization with IL-7 had a similar, strong initial effect, with significantly prolonged survival in the majority of the rats but a lower final cure rate (survival for >150 days). The B7-1-expressing tumor clones induced cures in seven of eight female rats; however, no cures were seen in the male rats. It was also shown that the B7-1-expressing cells were themselves strongly immunogenic in female rats, requiring high cell numbers to result in a progressively growing tumor upon s.c. isografting; this was not the case in male rats. As a whole, the results imply that despite the unfavorable location of intracerebral tumors, therapeutic s.c. immunizations with certain types of genetically altered tumor cells can induce complete regressions with permanent survival and without gross neurological or other apparent signs of brain damage. The present results demonstrate complete regressions when immunizations are initiated shortly after intracranial isografting, when the intracerebral tumor is small.     

In vivo induction of antitumor immunity and protection against tumor growth by injection of CD154-expressing tumor cells

As they should enhance tumor-specific antigen presentation by dendritic cells, tumor cell lines genetically modified to express CD154 molecules have been used in an attempt to induce protective antitumor immunity. Two murine models were used: the major histocompatibility complex (MHC) class I negative melanoma B16F10 and the MHC class I positive mammary adenocarcinoma TS/A. CD154 or mock-transfected B16F10 or TS/A cells were injected subcutaneously into H-2-compatible B6D2 mice. CD154 expression by tumor cells induced a complete rejection (in the TS/A model) or a striking reduction (in the B16F10 model) of modified tumors growth, but also a significant protection against the growth of mock tumor cells injected simultaneously, either mixed with the CD154-expressing tumor cells, or in the other flank of mice. Thirty days after CD154-expressing tumor rejection, splenic lymphocytes from surviving tumor-free mice were able to inhibit tumor proliferation in vitro and significant amounts of IFN-gamma were detected in the sera of these mice. Growth kinetics of mock and CD154-expressing tumors in immunocompetent versus nude mice suggest that T lymphocytes and natural killer cells responses are implicated in this antitumor immunity. The injection of CD154-expressing tumor cell induced an antitumor protective response, both locally and distant from the injection site. The effect was most pronounced in MHC class I expressing TS/A tumor model.     

Bispecific antibody-directed antitumor activity of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus interleukin 2.

Freshly isolated human CD4+ T cells can not respond to recombinant interleukin 2 (rIL-2) because of their lack of p75 IL-2 receptor expression. However, we succeeded in inducing a marked proliferation of purified CD4+ T cells by activation with rIL-2 plus anti-CD3 monoclonal antibody (mAb) cross-linked to a plastic plate. The proliferated CD4+ T cells produced a significant amount of IL-2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4+ T cells activated with anti-CD3 mAb plus rIL-2 revealed a strong cytotoxic activity against Fc receptor (FcR)-positive tumor cells in the presence of anti-CD3 mAb. Moreover, the CD4+ T cells could lyse FcR-negative glioma cells by targeting with bispecific mAb containing anti-CD3 mAb and anti-glioma mAb. Thus, we demonstrated that rIL-2 and immobilized anti-CD3 mAb allowed the rapid generation of human CD4+ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.