The combined activation of positive costimulatory signals with modulation of a negative costimulatory signal for the enhancement of vaccine-mediated T-cell responses

Blockade of CTLA-4 by monoclonal antibodies (mAb) can mediate regression of tumors and increase the efficacy of tumor antigen specific vaccines. Blockade of CTLA-4 has also been shown to significantly increase the avidity of antigen-specific T cells after immunization with live recombinant viral vector based vaccine. Here, we demonstrate a biological synergy between CTLA-4 blockade and active vaccine therapy consisting of recombinant vaccinia and avipox viruses expressing carcinoembryonic antigen (CEA) and three T cell costimulatory molecules to enhance antitumor effects. However, this synergy was very much dependent on the temporal relationship of scheduling of the two agents. We evaluated the strategies in both a foreign antigen model using β-galactosidase as immunogen, and in a “self” antigen model using CEA as immunogen. For antitumor activity the model used consisted of mice transgenic for human CEA and a murine carcinoma cell line transfected with CEA. The enhanced antitumor activity after vaccine and CTLA-4 blockade did not result in any signs of autoimmunity. These studies form a rational basis for the use of vector-based vaccines with anti-CTLA-4 and demonstrate that both enhancement of positive costimulatory signals and inhibition of negative costimulatory signals can be simultaneously exploited. These studies also underscore the importance of “drug” scheduling in vaccine combination therapies.     

Targeting NKT cells and PD-L1 pathway results in augmented anti-tumor responses in a melanoma model

Invariant or Type 1 NKT cells (iNKT cells) are a unique population of lymphocytes that share characteristics of T cells and natural killer (NK) cells. Various studies have shown that positive costimulatory pathways such as the CD28 and CD40 pathways can influence the expansion and cytokine production by iNKT cells. However, little is understood about the regulation of iNKT cells by negative costimulatory pathways. Here, we show that in vivo activation with α-GalCer results in increased cytokine production and expansion of iNKT cells in the absence of programmed cell death ligand-1 (PD-L1, B7-H1, and CD274). To study whether PD-L1 deficiency on NKT cells would enhance antigen-specific T-cell responses, we utilized CD8⁺ OT-1 OVA transgenic T cells. α-GalCer enhanced the expansion and cytokine production of OT-1 CD8⁺ cells after adoptive transfer into wild-type recipients. However, this expansion was significantly enhanced when OT-1 CD8⁺ T cells were adoptively transferred into PD-L1^−/− recipients. To extend these results to a tumor model, we used the B16 melanoma system. PD-L1^−/− mice given dendritic cells loaded with antigen and α-GalCer had a significant reduction in tumor growth and this was associated with increased trafficking of antigen-presenting cells and CD8⁺ T cells to the tumors. These data demonstrate that abrogating PDL1:PD-1 interactions during the activation of iNKT cells amplifies an anti-tumor response when coupled with DC vaccination.     

Safety profile and anti-tumor effects of adoptive immunotherapy using gamma-delta T cells against advanced renal cell carcinoma: a pilot study

Purpose Although various types of immunotherapy have been used to improve the prognosis of patients with advanced renal cell carcinoma (RCC), adoptive immunotherapy using gamma-delta (γδ) T cells has not yet been tried. In this study, we designed a pilot study of adoptive immunotherapy using in vitro activated γδ T cells against advanced RCC to evaluate the safety profile and possible anti-tumor effects of this study. Experimental design Patients with advanced RCC after radical nephrectomy were administered via intravenous infusion in vitro-activated autologous γδ T cells every week or every 2 weeks, 6–12 times, with 70 JRU of teceleukin. Adverse events, anti-tumor effects and immunomonitoring were assessed. The anti-tumor effects were evaluated according to tumor doubling time (DT) by computed tomography (CT) and immunomonitoring was performed by flow cytometric analysis. Results Seven advanced RCC patients were entered in this study. The most common adverse events were fever, general fatigue and elevation of hepatobiliary enzymes, but no severe adverse events were seen. Prolongation of tumor DT was seen in three out of five patients; these three patients showed an increase in the number of γδ T cells in peripheral blood and also a high response to the antigen in vitro. Conclusions The results indicated that adoptive immunotherapy using in vitro-activated autologous γδ T cells was well tolerated and induced anti-tumor effects.     

Expression of costimulatory molecules in the bovine corpus luteum

Background  

Bovine luteal parenchymal cells express class II major histocompatibility complex (MHC) molecules and stimulate class II MHC-dependent activation of T cells in vitro. The ability of a class II MHC-expressing cell type to elicit a response from T cells in vivo is also dependent on expression of costimulatory molecules by the antigen presenting cell and delivery of a costimulatory signal to the T cell. Whether bovine luteal parenchymal cells express costimulatory molecules and can deliver the costimulatory signal is currently unknown.     

Intratumoral delivery of vector mediated IL-2 in combination with vaccine results in enhanced T cell avidity and anti-tumor activity

Systemic IL-2 is currently employed in the therapy of several tumor types, but at the price of often severe toxicities. Local vector mediated delivery of IL-2 at the tumor site may enhance local effector cell activity while reducing toxicity. To examine this, a model using CEA-transgenic mice bearing established CEA expressing tumors was employed. The vaccine regimen was a s.c. prime vaccination with recombinant vaccinia (rV) expressing transgenes for CEA and a triad of costimulatory molecules (TRICOM) followed by i.t. boosting with rF-CEA/TRICOM. The addition of intratumoral (i.t.) delivery of IL-2 via a recombinant fowlpox (rF) IL-2 vector greatly enhanced anti-tumor activity of a recombinant vaccine, resulting in complete tumor regression in 70–80% of mice. The anti-tumor activity was shown to be dependent on CD8⁺ cells and NK1.1⁺. Cellular immune assays revealed that the addition of rF-IL-2 to the vaccination therapy enhanced CEA-specific tetramer⁺ cell numbers, cytokine release and CTL lysis of CEA⁺ targets. Moreover, tumor-bearing mice vaccinated with the CEA/TRICOM displayed an antigen cascade, i.e., CD8⁺ T cell responses to two other antigens expressed on the tumor and not the vaccine: wild-type p53 and endogenous retroviral antigen gp70. Mice receiving rF-IL-2 during vaccination demonstrated higher avidity CEA-specific, as well as higher avidity gp70-specific, CD8⁺ T cells when compared with mice vaccinated without rF-IL-2. These studies demonstrate for the first time that the level and avidity of antigen specific CTL, as well as the therapeutic outcome can be improved with the use of i.t. rF-IL-2 with vaccine regimens.     

Vγ9Vδ2 T cell-mediated recognition of human solid tumors. Potential for immunotherapy of hepatocellular and colorectal carcinomas

Introduction Vγ9Vδ2 T lymphocytes are reported to participate in the anti-tumor immune surveillance in human. They are known to recognize phosphoantigens and molecules expressed on cells undergoing neoplasic transformation. In this study, we investigated phenotype and anti-tumor cytotoxicity of ex vivo expanded Vγ9Vδ2 T cells in view of adoptive immunotherapy. Materials and Methods Experiments were performed with peripheral blood samples from eleven patients [six colorectal carcinoma (CRC), four hepatocellular carcinoma (HCC), one sarcoma] and sixteen healthy donors. Results/Discussion Ex vivo expansion of Vγ9Vδ2 T cells could be achieved by a single dose of phosphoantigen, either bromohydrin pyrophosphate or zoledronate, and supported by exogenous IL-2. After 2 weeks, expanded Vγ9Vδ2 T lymphocytes acquired the effector memory phenotype CD45RA⁻CD45RO^highCD27⁻. They expressed NKG2D and CD161 and the proinflammatory CXCR3 and CCR5 chemokine receptors. Vγ9Vδ2 T cells displayed a strong lytic activity toward a broad panel of tumor cell lines or primary cultures. Interestingly, HCC and CRC primary cells could be lysed by autologous Vγ9Vδ2 T cells whereas autologous normal cells were not sensitive to the lysis. mAbs blocking assays demonstrated that TCR was the most important receptor involved in the lysis of tumor cells. However, NKG2D receptor could deliver a costimulatory signal enhancing the lysis of HCC and CRC tumors expressing MICA/B. Treatment of tumor cells by the mevalonate pathway inhibitor, zoledronate, enhanced the killing of both HCC and CRC. Expansion index of Vγ9Vδ2 T cells was in similar levels in healthy donors or in cancer patients and total expansion was suitable for adoptive immunotherapy. Conclusion These results provide a rationale for the clinical evaluation of Vγ9Vδ2 T lymphocytes in HCC and CRC.     

Costimulation of T-cell proliferation by a chimeric B7-antibody fusion protein

 T cells require at least two signals for activation and clonal expansion. The first signal conferring specificity is initiated by interaction of the T cell receptor with peptide-bearing MHC molecules. The second, costimulatory signal can be provided by cell-surface molecules on antigen-presenting cells such as B7-1 (CD80) and B7-2 (CD86), which interact with CD28 on T cells. To direct the costimulatory B7-2 molecule to the surface of tumor cells we have constructed a chimeric fusion protein, which consists of the extracellular domain of human B7-2 fused to a single-chain antibody domain (scFv) specific for the ErbB2 protein, a type I growth factor receptor overexpressed in a high percentage of human adenocarcinomas. This B7-2₂₂₅-scFv(FRP5) molecule, expressed in the yeast Pichia pastoris and purified from culture supernatants, binds to B7 counter-receptors and to ErbB2. B7-2_225-scFv(FRP5) localizes specifically to the surface of ErbB2-expressing target cells, thereby providing a costimulatory signal, which results in enhanced proliferation of syngeneic T cells. Accepted: 14 October 1997     

Alternate costimulatory molecules in T cell activation: differential mechanisms for directing the immune response

T cells are required for an effective immune response against a wide range of pathogens and for the generation of immunological memory. T cell activation can be divided into two phases: an antigen-specific signal delivered through the T cell antigen receptor, and a costimulatory signal delivered through accessory molecules on the T cell surface. Following activation, T cells differentiate to acquire distinct effector functions depending on the costimulatory signal, cytokine environment, and the pathogen itself. Although CD28 has been identified as the dominant costimulatory molecule, several other molecules have been described as having a costimulatory function. This review will focus on recent evidence for the existence of alternate costimulatory molecules, and the differential roles they might play in the activation, development, and survival of T cells.     

Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition

Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z⁺ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1⁺ or 19z1-CD80⁺ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z⁺ T cells had 10-fold reduced tumor progression compared to those treated with 19z1⁺ or 19z1-CD80⁺ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80⁺ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z⁺ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.     

From ‘Hellstrom Paradox–to anti-adenosinergic cancer immunotherapy

Cancer therapy by endogenous or adoptively transferred anti-tumor T cells is considered complementary to conventional cancer treatment by surgery, radiotherapy or chemotherapy. However, the scope of promising immunotherapeutic protocols is currently limited because tumors can create a “hostile” immunosuppressive microenvironment that prevents their destruction by anti-tumor T cells. There is a possibility to develop better and more effective immunotherapies by inactivating mechanisms that inhibit anti-tumor T cells in the tumor microenvironment and thereby protect cancerous tissues from immune damage. This may be now possible because of the recent demonstration that genetic deletion of immunosuppressive A2A and A2B adenosine receptors (A2AR and A2BR) or their pharmacological inactivation can prevent the inhibition of anti-tumor T cells by the hypoxic tumor microenvironment and as a result facilitate full tumor rejection [Ohta A, Gorelik E, Prasad SJ et al (2006) Proc Natl Acad Sci USA 103(35):13132–13137]. This approach is based on in vivo genetic evidence that A2AR play a critical role in the protection of normal tissues from overactive immune cells in acutely inflamed and hypoxic areas. The observations of much improved T-cell-mediated rejection of tumors in mice with inactivated A2AR strongly suggest that A2AR also protects hypoxic cancerous tissues and that A2AR should be inactivated in order to improve tumor rejection by anti-tumor T cells.     

Electrofusion of a weakly immunogenic neuroblastoma with dendritic cells produces a tumor vaccine.

The absence of surface costimulatory molecules explains in part the lack of an effective anti-tumor immune response in tumor-bearing animals, even though unique tumor antigens may be presented by class I MHC. We determined that the immunogenicity of a murine neuroblastoma, Neuro-2a, which lacks surface costimulatory molecules, could be increased by electrically induced fusion with dendritic cells. Electrofusion induced a higher level of cell fusion than polyethylene glycol, and tumor/dendritic cell heterokaryons expressed high levels of costimulatory molecules. While Neuro-2a was unable to induce the proliferation of syngeneic or allogeneic T cells in vitro, fused cells were able to induce T cell responses both in vitro and in vivo. When fused dendritic tumor cells were used as a cancer vaccine, immunized mice were significantly protected from challenge with Neuro-2a. We propose that electrofusion with patient-derived tumor and dendritic cells may provide a rapid means to produce patient-specific tumor vaccines.     

Fibronectin binding protein A of Staphylococcus aureus can mediate human T lymphocyte adhesion and coactivation

The extracellular matrix protein fibronectin (FN) mediates the adhesion of bacteria as well as T lymphocytes. Mammalian cells express integrins alpha(4)beta(1) and alpha(5)beta(1) as the major FN-binding cell surface receptors. Bacteria such as Staphylococcus aureus, also express FN-binding receptors that are important for adherence to host tissue and initiation of infection. The S. aureus FN-binding protein, FnbpA, has been previously identified, and recombinant proteins that correspond to distinct functional regions of this protein have been made. Three recombinant truncated forms of FnbpA, rFnbpA(37-881), rFnbpA(37-605), and rFnbpA(620-881), were examined for effects on in vitro adhesion and coactivation of human T lymphocytes. These proteins, when coimmobilized with anti-CD3 mAb, activated T lymphocyte proliferation. The coactivation signal generated by the rFnbpA proteins required medium containing serum with FN. Furthermore, the costimulatory signal could be restored in FN-depleted serum when the rFnbpAs were preloaded with soluble FN. Monoclonal Ab blocking studies revealed that integrin alpha(5)beta(1) is the major receptor responsible for the rFnbpA costimulatory signal. Shear flow cell detachment assays confirmed that lymphocytes can bind to FN captured by the rFnbpA proteins. These results suggest that the S. aureus rFnbpA can interact with integrin alpha(5)beta(1) via an FN bridge to mediate adhesion and costimulatory signals to T lymphocytes.     

Characterization of MHC class-I restricted TCRαβ<Superscript>+</Superscript> CD4<Superscript>−</Superscript> CD8<Superscript>−</Superscript> double negative T cells recognizing the gp100 antigen from a melanoma patient after gp100 vaccination

The immune attack against malignant tumors require the concerted action of CD8⁺ cytotoxic T lymphocytes (CTL) as well as CD4⁺ T helper cells. The contribution of T cell receptor (TCR) αβ⁺ CD4⁻ CD8⁻ double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here, we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-γ and TNF. Although lacking the CD8 molecule the gp100-specifc DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2⁺ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo. A. Mackensen and K. Fischer contributed equally to this work and should be considered joint senior authors. This work was supported by the Deutsche Forschungsgemeinschaft (MA 1351/5-1, KFO 146) and NIH grants CA90873, CA102280, 104947 (MIN). Companion paper: “Relationship between CD8-dependent antigen recognition, T cell functional avidity, and tumor cell recognition” by Tamson V. Moore et al. doi: .     

Liposomal vaccines--targeting the delivery of antigen

Vaccines that can prime the adaptive immune system for a quick and effective response against a pathogen or tumor cells, require the generation of antigen (Ag)-specific memory T and B cells. The unique ability of dendritic cells (DCs) to activate na?ve T cells, implies a key role for DCs in this process. The generation of tumor-specific CD8(+) cytotoxic T cells (CTLs) is dependent on both T cell stimulation with Ag (peptide-MHC-complexes) and costimulation. Interestingly, tumor cells that lack expression of T cell costimulatory molecules become highly immunogenic when transfected to express such molecules on their surface. Adoptive immunotherapy with Ag-pulsed DCs also is a strategy showing promise as a treatment for cancer. The use of such cell-based vaccines, however, is cumbersome and expensive to use clinically, and/or may carry risks due to genetic manipulations. Liposomes are particulate vesicular lipid structures that can incorporate Ag, immunomodulatory factors and targeting molecules, and hence can serve as potent vaccines. Similarly, Ag-containing plasma membrane vesicles (PMV) derived from tumor cells can be modified to incorporate a T cell costimulatory molecule to provide both TCR stimulation, and costimulation. PMVs also can be modified to contain IFN-gamma and molecules for targeting DCs, permitting delivery of both Ag and a DC maturation signal for initiating an effective immune response. Our results show that use of such agents as vaccines can induce potent anti-tumor immune responses and immunotherapeutic effects in tumor models, and provide a strategy for the development of effective vaccines and immunotherapies for cancer and infectious diseases.     

A diabetogenic gene prevents T cells from receiving costimulatory signals.

T cell fate following antigen encounter is determined by several intracellular signals generated by the interaction of the T cell with an antigen-presenting cell. In the periphery activation requires T cell receptor signaling (signal one) in combination with costimulatory signals (signal two), usually provided through the cognate interaction of CD28 and B7 molecules. Provision of signal one alone to purified murine peripheral T cells in vitro induces apoptosis or anergy rather than promoting activation. These T cells can be rescued from apoptosis if they are provided with costimulation supplied, for example, by engaging the CD28 co-receptor with an anti-CD28 monoclonal antibody or by adding an exogenous source of interleukin-2. However, a majority of peripheral T cells from autoimmune, diabetes-prone Biobreeding (BB) rats exhibited different responses to these stimuli. T cells from these rats could not be rescued from apoptosis by costimulation. This was not due to the inability of BB-DP T cells to upregulate CD28 and the IL-2 receptor in response to TCR crosslinking. The failure of these costimulatory interactions to rescue BB-DP T cells segregated with the diabetes-susceptibility gene iddm1. Iddm1 in the rat causes peripheral T cell lymphopenia, which is associated with a dramatically shortened peripheral T cell life span. Our results indicate that a diabetogenic gene may contribute to autoimmunity by negating costimulatory signals important for the survival of long-lived peripheral T cells.     

T-cell activation induced by anti-CD3 × anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand

 T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific anibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy. Accepted: 14 October 1997     

Extracellular domain of human 4-1BBL enhanced the function of cytotoxic T-lymphocyte induced by dendritic cell

Interaction of costimulatory molecules and their receptors is crucial for tumor lysate-pulsed dendritic cells (sensitized DC, sDC) to promote T cell activation, clonal expansion and its antitumor immunity. To augment the costimulatory signal may regulate the interaction between DC and cytotoxic T lymphocyte (CTL) and consequently enhance the antitumor response. The costimulatory ligand and receptor pair of 4-1BB/4-1BBL is one of the main factors in the costimulation of CTL. We explored the adjuvant role of a recombinant human 4-1BBL extracellular domain (ex4-1BBL) in modulating CTL activation induced by HepG2 antigen-loaded DC (sDC). The augment effects of sDC in combination with ex4-1BBL on the proliferation, activation, cell survival and cytotoxicity against HepG2 cells of CTL were examined. In the presence of ex4-1BBL, sDC exhibited markedly augmented effects on the above four functions of CTL. These results demonstrate that ex4-1BBL plays an important role in the costimulation pathway for DC-mediated CTL’s activation, which might be a useful adjuvant factor for DC-based cancer biotherapy.     

IL-12 enhances efficacy and shortens enrichment time in cytokine-induced killer cell immunotherapy

Cytokine-induced killer (CIK) cells are T cell derived ex vivo expanded cells with both NK and T cell properties. They exhibit potent anti-tumor efficacy against various malignancies in preclinical models and have proven safe and effective in clinical studies. We combined CIK cell adoptive immunotherapy with IL-12 cytokine immunotherapy in an immunocompetent preclinical breast cancer model. Combining CIK cells with IL-12 increased anti-tumor efficacy in vivo compared to either therapy alone. Combination led to full tumor remission and long-term protection in 75% of animals. IL-12 treatment sharply increased the anti-tumor efficacy of short-term cultured CIK cells that exhibited no therapeutic effect alone. Bioluminescence imaging based in vitro cytotoxicity and in vivo homing assays revealed that short-term cultured CIK cells exhibit full cytotoxicity in vitro, but display different tumor homing properties than fully expanded CIK cells in vivo. Our data suggest that short-term cultured CIK cells can be “educated” in vivo, producing fully expanded CIK cells upon IL-12 administration with anti-tumor efficacy in a mouse model. Our findings demonstrate the potential to improve current CIK cell-based immunotherapy by increasing efficacy and shortening ex vivo expansion time. This holds promise for a highly efficacious cancer therapy utilizing synergistic effects of cytokine and cellular immunotherapy.     

Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule

 The generation of cytotoxic effector T cells requires delivery of two signals, one derived from a specific antigenic epitope and one from a costimulatory molecule. A phase I clinical trial was conducted with a non-replicating canarypoxvirus (ALVAC) constructed to express both human carcinoembryonic antigen (CEA) and the B7.1 costimulatory molecule. This was the first study in cancer patients to determine if the delivery of costimulation with a tumor vaccine was feasible and improved immune responses. Three cohorts of six patients, each with advanced CEA-expressing adenocarcinomas, were treated with increasing doses of an ALVAC-CEA-B7.1 vaccine (4.5 × 10⁶, 4.5 × 10⁷, and 4.5 × 10⁸ plaque-forming units, PFU). Patients were vaccinated by intramuscular injection every 4 weeks for 3 months and monitored for side-effects, tumor growth and anti-CEA immune responses. ALVAC-CEA- B7.1 at doses up to 4.5 × 10⁸ PFU was given without evidence of significant toxicity or autoimmune reactions. Three patients experienced clinically stable disease that correlated with increasing CEA-specific precursor T cells, as shown by in vitro interferon-γ enzyme-linked immunoassay spot tests (ELISPOT). These three patients underwent repeated vaccination resulting in augmented CEA-specific T cell responses. This study represents the first use of costimulation to enhance antitumor vaccines in cancer patients. This approach resulted in CEA-specific immunity associated with stable diseases in three patients. This study also demonstrated that CEA-specific T cell responses could be sustained by repeated vaccinations. Although the number of patients was small, the addition of B7.1 to virus-based vaccines may improve immunological and stable diseases to vaccination against tumor-associated antigens with tolerable toxicity. Received: 6 May 2000 / Accepted: 13 July 2000     

The small molecule TGF-β signaling inhibitor SM16 synergizes with agonistic OX40 antibody to suppress established mammary tumors and reduce spontaneous metastasis

Effective tumor immunotherapy may require not only activation of anti-tumor effector cells, but also abrogation of tumor-mediated immunosuppression. The cytokine TGF-β, is frequently elevated in the tumor microenvironment and is a potent immunosuppressive agent and promoter of tumor metastasis. OX40 (CD134) is a member of the TNF-α receptor superfamily and ligation by agonistic antibody (anti-OX40) enhances effector function, expansion, and survival of activated T cells. In this study, we examined the therapeutic efficacy and anti-tumor immune response induced by the combination of a small molecule TGF-β signaling inhibitor, SM16, plus anti-OX40 in the poorly immunogenic, highly metastatic, TGF-β-secreting 4T1 mammary tumor model. Our data show that SM16 and anti-OX40 mutually enhanced each other to elicit a potent anti-tumor effect against established primary tumors, with a 79% reduction in tumor size, a 95% reduction in the number of metastatic lung nodules, and a cure rate of 38%. This positive treatment outcome was associated with a 3.2-fold increase of tumor-infiltrating, activated CD8⁺ T cells, an overall accumulation of CD4⁺ and CD8⁺ T cells, and an increased tumor-specific effector T cell response. Complete abrogation of the therapeutic effect in vivo following depletion of CD4⁺ and CD8⁺ T cells suggests that the anti-tumor efficacy of SM16+ anti-OX40 therapy is T cell dependent. Mice that were cured of their tumors were able to reject tumor re-challenge and manifested a significant tumor-specific peripheral memory IFN-γ response. Taken together, these data suggest that combining a TGF-β signaling inhibitor with anti-OX40 is a viable approach for treating metastatic breast cancer.