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Immunotherapy with tumor-targeted superantigens (TTS) in combination with docetaxel results in synergistic anti-tumor effects
International Immunopharmacology 9 (2009) 1063–1070
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Immunotherapy with tumor-targeted superantigens (TTS) in combination with docetaxel results in synergistic anti-tumor effects Anette Sundstedt ⁎, Mona Celander, Marie Wallén Öhman, Göran Forsberg, Gunnar Hedlund Active Biotech Research AB, P.O. Box 724, SE-220 07 Lund, Sweden
a r t i c l e
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Article history: Received 20 March 2009 Received in revised form 24 April 2009 Accepted 27 April 2009 Keywords: Tumor-targeted superantigens Immunotherapy Docetaxel Chemotherapy Cancer
a b s t r a c t In this study we explored the possibility of combining immunotherapy against cancer with the well-established cytostatic drug docetaxel. Tumor-targeted superantigens (TTS) utilizes the powerful T cell activating property of a superantigen such as staphylococcal enterotoxin A (SEA) in fusion with an anti-tumor Fab-fragment to target this T cell activity against tumor cells. TTS fusion proteins are efficient in a number of experimental tumor models including the B16 mouse melanoma transfected with a human tumor-associated antigen (GA733-2 or EpCam) recognized by the C215 monoclonal antibody. The distinct mechanisms of action of TTS and docetaxel provide the prerequisites for successful combination treatment. However, as a result of the anti-proliferative properties of cytostatic drugs, chemotherapy may modify TTS induced immune activation during combination treatment. Here we evaluated the anti-tumor effects of combining C215Fab-SEA with docetaxel against B16-C215 tumors growing in the lung of C57Bl/6 mice. Both compounds generated a significant reduction in the number of B16C215 lung tumors when administered alone. Prior treatment with docetaxel at therapeutic doses did not interfere with superantigen induced T cell activation but rather appeared to enhance the response, while simultaneous treatment was suppressive. Combining TTS and docetaxel significantly improved tumor therapy, further reducing the number of lung tumors as compared to mono therapies. Importantly, the combination treatment at timely settings synergistically prolonged long term survival in B16-C215 tumor bearing mice. The results of this study demonstrate that TTS immunotherapy is highly compatible with docetaxel and suggest a significant potential of the combination for human cancer therapy. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Tumor-targeted superantigens (TTS) is a concept for cancer immunotherapy which aims to activate and direct T lymphocytes to attack tumor cells by means of fusion proteins between bacterial superantigens, such as SEA, and Fab-fragments of tumor-reactive monoclonal antibodies. Superantigens are bacterial and viral proteins that share the ability to activate a large number of T lymphocytes. Bacterial superantigens bind to MHC class II molecules as unprocessed proteins and subsequently interact with T cells expressing particular T cell receptor (TCR) Vβ chains [1–3]. Superantigens are efficient inducers of inflammatory cytokine production and cell-mediated cytotoxicity [4–6]. To target the superantigen-induced T cell activity against tumor cells, Fab regions of tumor-reactive monoclonal antibodies have been genetically fused with the superantigen staphylococcal enterotoxin A
Abbreviations: TTS, tumor-targeted superantigens; SEA, staphylococcal enterotoxin A; MHC, major histocompatibility complex; TCR, T cell receptor; IFN, interferon; NSCLC, non-small cell lung cancer; RCC, renal cell carcinoma; CTL, cytotoxic T lymphocyte; SDCC, superantigen-dependent cell-mediated cytotoxicity; Treg, regulatory T cells; MDSC, myeloid-derived suppressor cells; sag, superantigen. ⁎ Corresponding author. Tel.: +46 46 191039; fax: +46 46 191105. E-mail address: [email protected] (A. Sundstedt). 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.04.013
(Fab-SEA) [7,8]. The effectiveness of this therapy has been demonstrated both in syngeneic and xenogeneic tumor models in mice [7–9]. The poorly immunogenic B16 melanoma was transfected with the human colon carcinoma antigen C215 [7] and used to evaluate the anti-tumor effects of C215Fab-SEA fusion protein in a syngeneic lung metastasis model. Treatment with repeated injections of C215Fab-SEA eradicated N90% of lung tumors in mice carrying established B16-C215 melanoma metastases [7,10]. Combining TTS with immune modulators such as IL-2 and IFN-α has also been tested with good results [11,12]. TTS proteins are in clinical development and have shown promising results, e.g. in patients with advanced non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) using anatumomab mafenatox/ABR-214936 [13,14]. Pharmacological proof-of-concept in man was obtained by a series of observations in subsequent phase I clinical studies of the TTS naptumomab estafenatox/ABR-217620, including dose-dependent induction of IL-2 and IFN-γ, selective expansion of TTS-reactive T cells as well as tumor infiltration of T lymphocytes in tumor biopsies from ABR-217620 treated patients [Borghaei et al., Journal of Clinical Oncology, in press]. As a consequence of the engineering process to optimize ABR217620 for human use, the compound does not activate murine T cells and is therefore not therapeutically active in experimental mouse
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models. The prototype drug molecule C215Fab-SEA is fully active in mice. Thus using C215Fab-SEA, which activates murine T cells in a similar fashion as ABR-217620 activates human T lymphocytes, makes it possible to perform pharmacological studies in mice. To further improve TTS-based therapy, we hypothesized that combination treatment with conventional therapies may be the most effective strategy for patient benefit. The goal of chemotherapy is direct cytotoxicity and induction of tumor cell death. Taxanes, which are among the most widely used cancer chemotherapeutic agents, induce anti-neoplastic activity by disrupting microtubular networks. Members of the taxane family, including docetaxel and the closely related compound paclitaxel, have been used in a variety of malignancies, such as lung, breast and prostate cancers [15]. The administration of cytotoxic chemotherapy can result in bone marrow suppression and has been traditionally perceived to have a negative effect on immune function. Recent data suggest, however, that taxanebased chemotherapy may actually exert beneficial immunomodulatory effects through a variety of mechanisms, including cytokine production and T-cell infiltration in tumors [16,17]. To determine the optimal combination tumor treatment regiment using the TTS protein C215Fab-SEA and docetaxel, we first examined the in vivo effects of docetaxel administered at different time points on the function of CD4+ and CD8+ T cells in response to TTS-induced activation. These studies were conducted in non-tumor bearing mice to rule out any indirect effects of docetaxel on the immune system that result from a reduction in tumor size. The results show that prior treatment with docetaxel at therapeutic doses did not interfere with superantigen induced T cell activation but rather appeared to enhance the response. Next, we used the selected combination regiment of docetaxel and TTS in tumor-bearing mice. Combining TTS and docetaxel significantly improved tumor therapy, further reducing the number of lung tumors as compared to mono therapies. Furthermore, TTS and docetaxel therapy synergistically prolonged long term survival in B16-C215 tumor bearing mice suggesting potential clinical benefit for the combined use. 2. Materials and methods 2.1. Animals and treatment Female C57Bl/6 mice were purchased from Taconic Europe A/S (Denmark), and maintained under standardized conditions. The mice were routinely used at the age of 8 to 12 weeks. All studies were approved by the local animal ethical committee. Recombinant SEA and C215Fab-SEA were expressed in E. coli and purified as described elsewhere [18,19]. Mice were given various doses of C215Fab-SEA in 0.2 ml PBS+1% normal mouse serum (NMS) i.v. in the tail vein. Docetaxel (Taxotere®; Sanofi Aventis, Paris, France) was administered i.p. at 1 or 2 mg/injection in 0.2 ml buffer solution (10.9% polysorbate 80, 5.1% ethanol and 0.45% NaCl). The selected dose of docetaxel was previously shown to have an anti-tumor effect on B16 melanoma cells in vivo [20]. 2.2. Reagents Fluorochrome-labeled monoclonal antibodies directed to murine CD4, CD8, TCR-Vβ3 and TCR-Vβ8 were purchased from BD Biosciences (San Jose, CA). 2.3. Cell lines The murine B-cell lymphoma A20 and C215-tranfected murine B16-F10 melanoma (B16-C215) [7] were cultured in R10 medium (RPMI-1640 with Ultraglutamine (BioWhittaker/Lonza, Wokingham, UK); supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA), 1 mM sodium pyruvate, 10 mM HEPES, 0.1 mg/ml
gentamicine sulfate and 50 µM β-mercaptoethanol). In addition, 1 mg/ml G418 (Calbiochem, La Jolla, CA) was added to the medium for culture of B16-C215 cells. 2.4. Cytotoxicity assay Cytotoxicity of spleen cells from treated mice was measured at various effector to target (E:T) ratios in a standard 4-hour 51Cr-release assay [4]. Percent specific cytotoxicity was calculated as: 100 × (cpm experimental release − cpm background)/(cpm total release − cpm background release). 51Cr-labelled A20 target cells were used at 2.5 × 106 cells per well in V-bottomed 96 well microtiter plates. SEA was used at a concentration of 1 µg/ml. 2.5. Analysis by flow cytometry Flow cytometric analysis was performed according to standard settings on a FACSort™ flow cytometer (BD Biosciences). Before incubating splenocytes with specific fluorochrome-labelled antibodies, Fc-receptors were blocked using anti-CD16/CD32 mAb (BD Biosciences). 2.6. Therapy of B16-C215 tumors Groups of 8 C57Bl/6 mice were inoculated i.v. with 1.75 × 105 B16C215 melanoma cells into the tail vein to induce lung tumors. Mice were treated with daily injections of 10 µg C215Fab-SEA on days 3 to 6 and docetaxel was injected i.p. (1 mg/injection) on day 2 or on days 2 and 9. On day 21 mice were sacrificed and the lungs were removed. After fixation in Bouins solution (24% formalin, 71% picric acid solution, and 5% glacial acetic acid) for at least 24 h, the numbers of lung tumors were counted. In the survival experiments, groups of 10 or 20 mice were inoculated with 1.25 × 105 B16-C215 melanoma cells into the tail vein and 10 µg C215Fab-SEA was injected i.v. daily for four days on days 3 to 6 or when using multi-cycle therapy on days 3–6, days 17–20, days 45–48 and on days 59–62. Docetaxel was injected i.p. on day 2 or on days 2 and 9 (1 mg/injection) or on days 7, 21, 35, 49, 63 and 77 (2 mg/ injection) in the multi-cycle experiment. Animals were sacrificed when showing signs of morbidity or otherwise at 90 or 119 days after tumor inoculation. 2.7. Determination of mouse anti-SEA antibodies The concentration of anti-SEA antibodies in the mouse plasma samples was determined by ELISA. Briefly, plates were coated with recombinant SEA. After blocking in BSA, samples and standards of affinity purified mouse anti-SEA antibodies were added followed by a goat anti-mouse IgG antibody (DAKO Denmark A/S, Glostrup, Denmark). Next, a biotinylated rabbit anti-goat IgG antibody (DAKO Denmark A/S) was added, followed by streptavidin conjugated with HRP (DAKO Denmark A/S). The reaction was detected by addition of an enzyme substrate (TMB; 3,3′,5,5′-tetramethylbenzidine) (Bio-Rad, Hercules, CA) and the absorbance was monitored at 450 nm, with 650 nm as a reference wavelength, in an ELISA spectrophotometer. A four-parameter function was adjusted to the obtained concentration/ absorbance values of the standards and the unknown concentration of anti-SEA antibodies in samples was determined from the standard curve. The measuring range was estimated to 0.40–50 ng/ml. The sample dilution was at least 1:100, i.e. the limit of quantification (LOQ) in plasma was 39 ng/ml. 2.8. Statistical methods To evaluate and compare the therapeutic observations, the following statistical analyses were used. Number of lung tumors in the short
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term tumor model was evaluated using Mann–Whitney Rank Sum test (Sigma Stat®). Survival time was analyzed using non-parametric logrank test (GraphPad Prism®). An additional parametric analysis of survival data was performed (PROC LIFEREG in SAS, SAS Institute Inc., Cary, NC), where statistical interactions between treatments were evaluated using model: logðSurvivali Þ = c + β1 TTTSi + β2 TDoc2i + β3 TTTSi TDoc2i + β4 T Doc29i + β5 TDoc29i TTTSi + erroriði = 1;2; N ;nÞ ; where TTS, Doc2 and Doc29 are indicator variables (0 or 1) for TTS treatment days 3–6, docetaxel treatment day 2, or days 2 and 9. The parameters β3 and β5 are 0 in an additive model and greater than 0 in a synergistic model. The hypotheses β3 = 0 and β5 = 0 were tested. The logarithm of anti-SEA antibody levels was analyzed using analysis of variance (PROC MIXED in SAS, SAS Institute Inc., Cary, NC). 3. Results We have previously demonstrated that 3–4 daily injections of C215Fab-SEA efficiently activate CD4+ and CD8+ T cells expressing distinct TCR-Vβ chains to inflammatory cytokine production, massive expansion and induction of potent CTL activity within the CD8+ T cell compartment [10]. This regime also inhibits tumor growth in C215transfected B16 melanoma bearing mice [7,10,21]. 3.1. Timely administration of docetaxel does not inhibit superantigeninduced T cell expansion in spleen Being a cytotoxic drug, docetaxel targets dividing cells by inhibiting the mitotic activity. To investigate the cytostatic activity of docetaxel on TTS induced T cell expansion, mice were given three daily i.v. injections of C215Fab-SEA (10 µg/injection) in combination with one i.p. injection of docetaxel (1 mg/mouse) given on days 1, 2 or 3 of TTS treatment. Mice were sacrificed 48 h after the last C215Fab-SEA treatment and the spleens were removed for analysis of T cell populations. Simultaneous treatment either suppressed or augmented TTS-mediated T cell expansion, depending on the temporal relationship between the drugs. Administration of docetaxel on day 3 of C215Fab-SEA treatment clearly inhibited the expansion of SEA-specific TCR-Vβ3+ T cells (Fig. 1). Interestingly however, docetaxel injected in conjunction with the first C215Fab-SEA injection resulted in increased expansion of CD8+Vβ3+ T cells but not of CD4+Vβ3+ T cells (Fig. 1), which were slightly inhibited also at this time point, indicating that docetaxel may affect various T cell subsets differently. SEA non-responsive TCR-Vβ8+ T cells were only marginally affected by treatment (Fig. 1).
Fig. 2. Effect of simultaneous treatment with C215Fab-SEA and docetaxel on the induction of CTL activity. Mice (3 animals/group) were injected i.v. with 3 daily doses of C215Fab-SEA (10 µg/animal) in combination with i.p. injections of docetaxel (1 mg/ animal) given either on day 3 of TTS treatment (A) or the day before starting the TTS treatment (B). Docetaxel was injected approximately 30 min after C215Fab-SEA administration when given on the same day as C215Fab-SEA. Forty-eight hours after the last C215Fab-SEA injection, spleens were removed and cytotoxic activity (SDCC) against SEA-coated A20 cells was measured in a standard 51Cr release assay at different effector to target (E:T) ratios (mean ± SEM).
Fig. 1. Effect of simultaneous treatment with C215Fab-SEA and docetaxel on the expansion of SEA-specific TCR-Vβ3+ T cells. Mice (3 animals/group) were injected i.v. with 3 daily doses of C215Fab-SEA (10 µg/animal) in combination with i.p. injections of docetaxel (1 mg/animal) given on days 1, 2 or 3 of TTS treatment. Docetaxel was injected approximately 30 min after C215Fab-SEA administration. Forty-eight hours after the last C215Fab-SEA injection, spleens were removed and TCR-Vβ3 specific expansion of CD4 and CD8 T cells (mean ± SEM) was measured by flow cytometry.
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3.2. Docetaxel given before TTS enhances the induction of superantigeninduced effector CTLs, while simultaneous treatment is suppressive
Fig. 3. C215Fab-SEA and docetaxel combination therapy in B16-C215 tumor bearing mice. Groups of eight C57Bl/6 mice were inoculated i.v. with 1.75 × 105 B16-C215 melanoma cells on day 0. Treatment started on day 2 with one i.p. injection of 1 mg/ mouse of docetaxel or vehicle (10.9% polysorbate 80, 5.1% ethanol and 0.45% NaCl). This was followed by four daily i.v. injections on days 3 to 6 of 10 µg/mouse C215Fab-SEA or vehicle (PBS+1% C57Bl/6 mouse serum), and then a second i.p. injection of docetaxel (1 mg/mouse) or vehicle given on day 9. On day 21, mice were sacrificed, the lungs were removed and the number of tumors counted. Statistical analysis was done using Mann– Whitney Rank Sum test.
Induction of perforin-dependent cytotoxic activity within the CD8+ T cell subset by TTS treatment is a central effector mechanism involved in tumor cell killing in vivo [22]. Thus, it was important to determine not only the effect of docetaxel on T cell division but also how the drug influences the killing potential of TTS-activated CTLs. Mice received three daily i.v. injections of C215Fab-SEA (10 µg/injection) in combination with one i.p. injection of docetaxel (1 mg/mouse) given either the day before or on day 3 of TTS treatment. The cytotoxic response of splenocytes was investigated 48 h after the last treatment. Superantigen-dependent cell-mediated cytotoxicity (SDCC) was measured against SEA-coated MHC class II-expressing target cells in a standard 4 h 51Cr-release assay. Three daily injections of C215Fab-SEA resulted in significant cytotoxic activity (Fig. 2). In line with the previous result on T cell expansion, docetaxel given on day 3 of TTS treatment clearly inhibited the SEA-dependent cytotoxic response of splenocytes compared to cells from mice treated with C215Fab-SEA alone (Fig. 2A). In contrast, cytostatic treatment before C215Fab-SEA therapy did not interfere with SEA-mediated activation of CD8+ T cells. Rather, administration of docetaxel the day before TTS treatment resulted in enhanced cytotoxic activity in this T cell subset (Fig. 2B). No significant SDCC was recorded in mice treated with docetaxel alone or vehicle (Fig. 2). Taken together, these results demonstrate that docetaxel is compatible with TTS therapy when given shortly before the first C215Fab-SEA injection.
Fig. 4. Long term survival of B16-C215 tumor bearing mice treated with C215Fab-SEA and docetaxel. Groups of ten C57Bl/6 mice were inoculated i.v. with 1.25 × 105 B16-C215 melanoma cells day 0. Treatment was started on day 2 with one i.p. injection of 1 mg/mouse of docetaxel or vehicle (10.9% polysorbate 80, 5.1% ethanol and 0.45% NaCl). This was followed by four daily i.v. injections on days 3 to 6 of 10 µg/mouse of C215Fab-SEA or vehicle (PBS+1% C57Bl/6 mouse serum), and then a second i.p. injection of docetaxel (1 mg/ mouse) or vehicle given on day 9. Mice were sacrificed when moribund behavior was noticed or otherwise 90 days after tumor inoculation. Survival curves, median survival times and statistical analysis using non-parametric logrank test are displayed.
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3.3. TTS and docetaxel in combination is superior to the mono therapies: inhibition of B16-C215 lung tumors Based on the results above, we next investigated anti-tumor effects against B16-C215 expressing tumor cells of combining C215Fab-SEA and docetaxel. The chosen schedule was to give docetaxel before the TTS protein. We also included treatment with docetaxel after the TTS cycle, administered after the proliferative peak of the T cells, to investigate if the anti-tumor effect could be enhanced. C57Bl/6 mice were inoculated i.v. on day 0 with B16-C215 melanoma cells and the number of lung tumors was determined three weeks later. Docetaxel was injected i.p. on day 2 or on days 2 and 9, in combination with four daily i.v. injections of C215Fab-SEA on days 3 to 6. Mice treated with docetaxel, one or two times, or mice treated with C215Fab-SEA alone had less lung tumors compared to vehicle (Fig. 3). The therapeutic effects of combining C215Fab-SEA and docetaxel was significantly better than C215Fab-SEA alone (P = 0.003 and P b 0.001, respectively) (Fig. 3). In spite of the fact that the combination of TTS with one injection (day 2) of docetaxel showed only few lung tumors, there was a clear tendency of a further reduction in tumor load in the group treated with the two docetaxel injection combination. Also, in contrast to all other treatment groups, 2 of 8 mice in this group were free of tumor at the end of the experiment. The mice were carefully monitored during and after treatment and the therapy was well tolerated. No additional adverse effects were recorded after combination treatment as compared to mono therapies. 3.4. TTS and docetaxel in combination synergistically prolongs long term survival in B16-C215 tumor bearing mice Next we wanted to explore whether the improved tumor therapy by C215Fab-SEA and C215Fab-SEA combination treatment in the short
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term B16-C215 model also translated into long term survival in the same tumor model. When the mice showed signs of morbidity or on day 90, they were sacrificed and autopsies were carried out. Treatment with docetaxel, one or two times, in combination with C215Fab-SEA showed a dramatically prolonged survival time as compared to the mono therapies (Fig. 4). The median survival time was increased from 42 days after C215Fab-SEA to N90 days in the case of combination treatment with 2 injections of docetaxel (days 2 and 9), constituting a significant difference (P b 0.0001) (Fig. 4). Also the combination using one injection of docetaxel significantly prolonged median survival to 66 days (P b 0.0001). Median survival times in the vehicle treated control group and in mice treated with docetaxel alone were 33 and 38 days, respectively. Statistical analysis demonstrated that the therapeutic effect of combining C215Fab-SEA and docetaxel (days 2 and 9) gave rise to significant synergistic anti-tumor responses compared to C215Fab-SEA mono therapy (P b 0.0001), when statistical interactions between treatments were evaluated as described in Materials and methods. Moreover, 7 of 10 animals survived for N90 days following combination treatment (C215Fab-SEA + docetaxel days 2 and 9) while all control mice were lost before day 40 in this low immunogenic tumor model, underlining the potential of combining TTS therapy with docetaxel treatment. Post-mortem analysis of the remaining animals (n = 7) in the combination treatment group on day 90 revealed no visible signs of tumor, indicating that they were tumor free. These findings show that the use of C215Fab-SEA in combination with docetaxel give rise to a synergistic survival advantage over animals treated with docetaxel or C215FabSEA alone. It is concluded that more than additive treatment effects were recorded in the experiments using the combination treatments as compared to mono therapies when evaluating the number of tumors in the lungs which extrapolate to clear synergism in increasing survival.
Fig. 5. Long term survival after multi-cycle treatment with C215Fab-SEA in combination with docetaxel. Groups of twenty C57Bl/6 mice were inoculated i.v. with 1.25 × 105 B16-C215 melanoma cells on day 0. Mice were treated with 10 µg/mouse of C215Fab-SEA or vehicle (PBS+1% C57Bl/6 mouse serum) i.v. with four cycles of four daily i.v. injections on days 3–6, 17–20, 45–48 and 59–62. This was followed by i.p. injections of docetaxel (2 mg/mouse) or vehicle at biweekly intervals on days 7, 21, 35, 49, 63 and 77. Mice were sacrificed when moribund behavior was noticed or otherwise 119 days after tumor inoculation. Survival curves, median survival times and statistical analysis using non-parametric logrank test are displayed.
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3.5. Multi-cycle TTS treatment followed by docetaxel induces long term survival in B16-C215 tumor bearing mice Treatment with repeated cycles of TTS therapy enhances the antitumor effect against B16-C215 melanoma [23]. In addition, the clinical use of TTS protein therapy is based on multi-cycle treatment [13,14] [Borghaei et al., Journal of Clinical Oncology, in press]. Thus, to test combination therapy in a more clinically relevant setting, mice were treated with four cycles of C215Fab-SEA in combination with docetaxel, six times. Using this protocol, C215Fab-SEA treatment increased the median survival from 36 days in the vehicle group to 76 days (Fig. 5). Mice treated with docetaxel alone had a median survival time of 61 days. Again, combination therapy significantly prolonged the survival time compared to C215Fab-SEA mono therapy (P = 0.0005). Twelve out of 20 mice in the combination group were tumor free at the end of the experiment (day 119) compared to 4 out of 20 mice treated with C215Fab-SEA alone. 3.6. Docetaxel inhibits the development of anti-SEA antibodies after repeated TTS treatment One of the limiting factors allowing effective dosing of repeated cycles of TTS therapy is the development of anti-superantigen antibodies [13,14] [Borghaei et al., Journal of Clinical Oncology, in press]. We hypothesized that docetaxel might be able to prevent the induction of a specific antibody response by inhibiting dividing B cells. To determine the influence of docetaxel treatment on the development of anti-superantigen antibodies during multi-cycle TTS treatment, we measured the levels of anti-SEA antibodies in plasma of the surviving mice (day 119) in the experiment showed in Fig. 5. At this time point, 5 mice from the C215Fab-SEA group and 12 mice from the combination group were still alive. As shown in Fig. 6A, docetaxel significantly reduced the development of an anti-SEA antibody response after C215Fab-SEA treatment. To make a more detailed analysis of the development of anti-SEA antibodies, mice were treated as before and the levels of antibodies were measured on days 34 and 76. Again, docetaxel significantly reduced the development of anti-SEA antibodies after C215Fab-SEA treatment (Fig. 6B). Thus, it is likely that inhibition of anti-superantigen antibodies by the add-on docetaxel treatment is one component explaining the significantly improved therapeutic effect of the combination. 4. Discussion Many clinical trials have shown that combinations can be more effective than the single use of any particular modality. However, when it comes to combining immunotherapy and chemotherapy in cancer, it has been widely assumed that cytostatic drugs are immunosuppressive and would counteract the benefits of immunotherapy. In this study we investigated how docetaxel can be administered safely in combination with TTS immunotherapy without inhibiting specific T cell responses. We demonstrate that timely administration of docetaxel does not interfere with TTS induced T cell activation and more importantly that this combination therapy gives rise to strong synergistic anti-tumor effects against B16 tumors growing in the lung. Most anticancer agents, such as cytostatic drugs and radiation, work by affecting or preventing cell division. Because they are nonspecific, all dividing cells are affected and this results in adverse side effects. In an immune response, the requisite lymphocyte expansion phase is characterized by massive proliferation. This proliferation is fundamental and essential to a productive immune response. Thus, a compromised immune system is one of the more common and serious side effects of treating cancer with chemotherapeutic drugs. Immunotherapies involving T lymphocytes, such as TTS or tumor vaccines, depend on a functional immune response. Treatment with TTS is preferably performed in cycles of 3–5 daily injections. The TTS induced
Fig. 6. Effect of docetaxel on the development of anti-SEA antibodies after C215Fab-SEA treatment. (A) Mice were inoculated B16-C215 melanoma cells and treated as described in Fig. 5. At the end of the experiment (day 119), blood samples were drained from vena cava for preparation of plasma from mice that were treated with C215Fab-SEA (n = 5) or C215Fab-SEA and docetaxel (n = 12). Levels of anti-SEA antibodies were measured by ELISA and are displayed as mean ± SEM. (B) C57Bl/6 mice were inoculated i.v. with 1.25 × 105 B16-C215 melanoma cells on day 0. Mice were treated with 10 µg/mouse of C215Fab-SEA or vehicle (PBS+1% C57Bl/6 mouse serum) i.v. with four cycles of four daily injections on days 3–6, 17–20, 45–48 and 59–62. This was followed by i.p. injections of docetaxel (2 mg/mouse) or vehicle at biweekly intervals on days 7, 21, 35, 49, and 63. Blood samples were drained from vena saphena on days 34 and in the case of the combination treatment also on day 76 for preparation of plasma: vehicle (n = 5), C215Fab-SEA (n = 58) or C215Fab-SEA and docetaxel, day 34 (n = 28), day 76 (n = 20). Levels of anti-SEA antibodies were measured by ELISA and are displayed as mean ± SEM.
immune response is immediate and much broader, activating T cells with distinct TCR–Vβ expression, than what is seen when giving a vaccine. Therefore the time frame of immune cell proliferation is limited and, as seen from the data in this study, clearly avoidable in a controlled manner if combining with an anti-proliferative drug such as docetaxel. Despite that cytostatic drugs are anti-proliferative in general and have an inhibitory effect on immune function, several studies have shown that under certain circumstances they can enhance immunity. Chemotherapy may, by simply reducing the tumor mass (debulking), reduce its immunosuppressive properties. As a proof of principle, surgical removal of the primary tumor could reverse tumor-induced
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immune tolerance even in mice bearing metastatic breast cancer [24]. Chemotherapy can cause immunogenic cancer cell stress or death and hence mediate a sort of cancer vaccination effect [25], and may further stimulate the immune system by causing lymphopenia followed by homeostatic proliferation of immune effectors that may be particularly active in the anti-cancer response. Animal studies have demonstrated that lymphoablation enhances the effect of adoptively transferred tumor-specific CD8+ T cells [26]. It can be anticipated that treatment with docetaxel before the TTS treatment may cause an enhanced expansion of superantigen activated effector cells due to mechanisms equal to what has been demonstrated with lymphoablation. In fact treating with docetaxel immediately before or on the first day of a TTS cycle resulted in our studies in enhanced T cell cytotoxicity and enhanced CD8+ T cell expansion, respectively (Figs. 1 and 2). Garnett et al. [27] showed that docetaxel enhanced IFN-γ production by CD8+ T cells. Furthermore, they showed that docetaxel combined with a recombinant viral vaccine increased specific T-cell responses to antigen in the vaccine as well as to cascade antigens derived from the tumor, and was superior to either agent alone at reducing tumor burden. In agreement with the results in our study, they showed that the outcome of the combination treatment was dependent of the timely relationship of docetaxel treatment and immune stimulus. Among the series of causes to tumor induced immune suppression, regulatory T cells (Treg) have recently been recognized as one of the most important. Some cytostatic agents, in particular cyclophosphamide, can partially deplete or transiently inactivate tumorprotective Treg [28]. On the contrary to what was found using cyclophosphamide, Garnett et al. show that docetaxel does not inhibit the function of Treg [27]. Therefore, modulation of B16 tumor or TTS induced Treg by docetaxel is unlikely. The myeloid-derived suppressor cells (MDSC) are another type of suppressive regulatory cells proven to be important in tumor immunity [29]. MDSC being a subpopulation of activated granulocytes [30] may well be decreased in number by docetaxel having a documented dramatic effect on granulocytes. One of the major advantages to use bacterial superantigens in TTS comes with the characteristics of them being the most powerful T cell activators known. A disadvantage that goes with the bacterial heritage of the protein though, is the immunogenicity and the production of antibodies directed towards the superantigens. Dosing of ABR214936 to cancer patients was performed according to an algorithm recognizing the anti-superantigen (anti-sag) antibody titers [13]. With the new improved TTS ABR-217620, being less antigenic and immunogenic [31], dosing of patients can be done without adjusting for preformed anti-sag antibodies [Borghaei et al., Journal of Clinical Oncology, in press]. Nonetheless, it was shown that anti-sag antibody responses were detected in most patients. Here, we demonstrate that using an anti-proliferative cytostatic drug, in this case docetaxel, immediately after a full TTS cycle significantly inhibited the anti-sag antibody production in the experimental mouse model used in this study. In accordance with these results, the titers of anti-sag antibodies recorded after treatment in the clinical trial using the combination of ABR-217620 and docetaxel in patients with NSCLC were significantly lower as compared to the trial with ABR-217620 alone [Borghaei et al., Journal of Clinical Oncology, in press]. It should be emphasized that other cancer types like breast and prostate cancer where the use of docetaxel is established, also express the 5T4 antigen [32] which is the target for ABR-217620. In this study, we have shown that the timely combination of TTS immunotherapy and the cytostatic drug docetaxel results in synergistic anti-tumor effects. Furthermore, this combination inhibited the production of antibodies binding to TTS. Together with the promising clinical results obtained using ABR-217620 in combination with docetaxel in patients with NSCLC [Borghaei et al., Journal of Clinical Oncology, in press], the impressive anti-tumor efficacy in a preclinical model shown in this study prompt for further clinical evaluation of
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ABR-217620 in combination with docetaxel and other cytostatic antitumor modalities. Acknowledgements We thank Anneli Nilsson, Jonas Tuvesson, and Jan Nilsson for excellent technical assistance, and Örjan Nordle for statistical analyses. References [1] Kappler J, Kotzin B, Herron L, Gelfand EW, Bigler RD, Boylston A, et al. V beta-specific stimulation of human T cells by staphylococcal toxins. Science 1989;244:811–3. [2] White J, Herman A, Pullen AM, Kubo R, Kappler JW, Marrack P. The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 1989;56:27–35. [3] Janeway Jr CA, Yagi J, Conrad PJ, Katz ME, Jones B, Vroegop S, et al. T-cell responses to Mls and to bacterial proteins that mimic its behavior. Immunol Rev 1989;107:61–88. [4] Dohlsten M, Lando PA, Hedlund G, Trowsdale J, Kalland T. Targeting of human cytotoxic T lymphocytes to MHC class II-expressing cells by staphylococcal enterotoxins. Immunology 1990;71:96–100. [5] Dohlsten M, Sundstedt A, Bjorklund M, Hedlund G, Kalland T. Superantigeninduced cytokines suppress growth of human colon-carcinoma cells. Int J Cancer 1993;54:482–8. [6] Hedlund G, Dohlsten M, Lando PA, Kalland T. Staphylococcal enterotoxins direct and trigger CTL killing of autologous HLA-DR+ mononuclear leukocytes and freshly prepared leukemia cells. Cell Immunol 1990;129:426–34. [7] Dohlsten M, Abrahmsen L, Bjork P, Lando PA, Hedlund G, Forsberg G, et al. Monoclonal antibody-superantigen fusion proteins: tumor-specific agents for Tcell-based tumor therapy. Proc Natl Acad Sci U S A 1994;91:8945–9. [8] Forsberg G, Ohlsson L, Brodin T, Bjork P, Lando PA, Shaw D, et al. Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen. Br J Cancer 2001;85:129–36. [9] Lando PA, Dohlsten M, Ohlsson L, Kalland T. Tumor-reactive superantigens suppress tumor growth in humanized SCID mice. Int J Cancer 1995;62:466–71. [10] Rosendahl A, Hansson J, Sundstedt A, Kalland T, Dohlsten M. Immune response during tumor therapy with antibody-superantigen fusion proteins. Int J Cancer 1996;68:109–13. [11] Sogaard M, Ohlsson L, Kristensson K, Rosendahl A, Sjoberg A, Forsberg G, et al. Treatment with tumor-reactive Fab-IL-2 and Fab-staphylococcal enterotoxin A fusion proteins leads to sustained T cell activation, and long-term survival of mice with established tumors. Int J Oncol 1999;15:873–82. [12] Sundstedt A, Celander M, Hedlund G. Combining tumor-targeted superantigens with interferon-alpha results in synergistic anti-tumor effects. Int Immunopharmacol 2008;8:442–52. [13] Shaw DM, Connolly NB, Patel PM, Kilany S, Hedlund G, Nordle O, et al. A phase II study of a 5T4 oncofoetal antigen tumour-targeted superantigen (ABR-214936) therapy in patients with advanced renal cell carcinoma. Br J Cancer 2007;96:567–74. [14] Cheng JD, Babb JS, Langer C, Aamdal S, Robert F, Engelhardt LR, et al. Individualized patient dosing in phase I clinical trials: the role of escalation with overdose control in PNU-214936. J Clin Oncol 2004;22:602–9. [15] Montero A, Fossella F, Hortobagyi G, Valero V. Docetaxel for treatment of solid tumours: a systematic review of clinical data. Lancet Oncol 2005;6:229–39. [16] Chan OT, Yang LX. The immunological effects of taxanes. Cancer Immunol Immunother 2000;49:181–5. [17] Mason K, Staab A, Hunter N, McBride W, Petersen S, Terry N, et al. Enhancement of tumor radioresponse by docetaxel: involvement of immune system. Int J Oncol 2001;18:599–606. [18] Dohlsten M, Hedlund G, Akerblom E, Lando PA, Kalland T. Monoclonal antibodytargeted superantigens: a different class of anti-tumor agents. Proc Natl Acad Sci U S A 1991;88:9287–91. [19] Abrahmsen L, Dohlsten M, Segren S, Bjork P, Jonsson E, Kalland T. Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A. EMBO J 1995;14:2978–86. [20] Bissery MC, Guenard D, Gueritte-Voegelein F, Lavelle F. Experimental antitumor activity of taxotere (RP 56976, NSC 628503), a taxol analogue. Cancer Res 1991;51:4845–52. [21] Dohlsten M, Hansson J, Ohlsson L, Litton M, Kalland T. Antibody-targeted superantigens are potent inducers of tumor-infiltrating T lymphocytes in vivo. Proc Natl Acad Sci U S A 1995;92:9791–5. [22] Rosendahl A, Kristensson K, Hansson J, Riesbeck K, Kalland T, Dohlsten M. Perforin and IFN-gamma are involved in the antitumor effects of antibody-targeted superantigens. J Immunol 1998;160:5309–13. [23] Rosendahl A, Kristensson K, Hansson J, Ohlsson L, Kalland T, Dohlsten M. Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth. Int J Cancer 1998;76:274–83. [24] Danna EA, Sinha P, Gilbert M, Clements VK, Pulaski BA, Ostrand-Rosenberg S. Surgical removal of primary tumor reverses tumor-induced immunosuppression despite the presence of metastatic disease. Cancer Res 2004;64:2205–11. 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[27] Garnett CT, Schlom J, Hodge JW. Combination of docetaxel and recombinant vaccine enhances T-cell responses and antitumor activity: effects of docetaxel on immune enhancement. Clin Cancer Res 2008;14:3536–44. [28] Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, et al. CD4+ CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol 2004;34:336–44. [29] Serafini P, Meckel K, Kelso M, Noonan K, Califano J, Koch W, et al. Phosphodiesterase-5 inhibition augments endogenous antitumor immunity by reducing myeloidderived suppressor cell function. J Exp Med 2006;203:2691–702.
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International Immunopharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i n t i m p
Immunotherapy with tumor-targeted superantigens (TTS) in combination with docetaxel results in synergistic anti-tumor effects Anette Sundstedt ⁎, Mona Celander, Marie Wallén Öhman, Göran Forsberg, Gunnar Hedlund Active Biotech Research AB, P.O. Box 724, SE-220 07 Lund, Sweden
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Article history: Received 20 March 2009 Received in revised form 24 April 2009 Accepted 27 April 2009 Keywords: Tumor-targeted superantigens Immunotherapy Docetaxel Chemotherapy Cancer
a b s t r a c t In this study we explored the possibility of combining immunotherapy against cancer with the well-established cytostatic drug docetaxel. Tumor-targeted superantigens (TTS) utilizes the powerful T cell activating property of a superantigen such as staphylococcal enterotoxin A (SEA) in fusion with an anti-tumor Fab-fragment to target this T cell activity against tumor cells. TTS fusion proteins are efficient in a number of experimental tumor models including the B16 mouse melanoma transfected with a human tumor-associated antigen (GA733-2 or EpCam) recognized by the C215 monoclonal antibody. The distinct mechanisms of action of TTS and docetaxel provide the prerequisites for successful combination treatment. However, as a result of the anti-proliferative properties of cytostatic drugs, chemotherapy may modify TTS induced immune activation during combination treatment. Here we evaluated the anti-tumor effects of combining C215Fab-SEA with docetaxel against B16-C215 tumors growing in the lung of C57Bl/6 mice. Both compounds generated a significant reduction in the number of B16C215 lung tumors when administered alone. Prior treatment with docetaxel at therapeutic doses did not interfere with superantigen induced T cell activation but rather appeared to enhance the response, while simultaneous treatment was suppressive. Combining TTS and docetaxel significantly improved tumor therapy, further reducing the number of lung tumors as compared to mono therapies. Importantly, the combination treatment at timely settings synergistically prolonged long term survival in B16-C215 tumor bearing mice. The results of this study demonstrate that TTS immunotherapy is highly compatible with docetaxel and suggest a significant potential of the combination for human cancer therapy. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Tumor-targeted superantigens (TTS) is a concept for cancer immunotherapy which aims to activate and direct T lymphocytes to attack tumor cells by means of fusion proteins between bacterial superantigens, such as SEA, and Fab-fragments of tumor-reactive monoclonal antibodies. Superantigens are bacterial and viral proteins that share the ability to activate a large number of T lymphocytes. Bacterial superantigens bind to MHC class II molecules as unprocessed proteins and subsequently interact with T cells expressing particular T cell receptor (TCR) Vβ chains [1–3]. Superantigens are efficient inducers of inflammatory cytokine production and cell-mediated cytotoxicity [4–6]. To target the superantigen-induced T cell activity against tumor cells, Fab regions of tumor-reactive monoclonal antibodies have been genetically fused with the superantigen staphylococcal enterotoxin A
Abbreviations: TTS, tumor-targeted superantigens; SEA, staphylococcal enterotoxin A; MHC, major histocompatibility complex; TCR, T cell receptor; IFN, interferon; NSCLC, non-small cell lung cancer; RCC, renal cell carcinoma; CTL, cytotoxic T lymphocyte; SDCC, superantigen-dependent cell-mediated cytotoxicity; Treg, regulatory T cells; MDSC, myeloid-derived suppressor cells; sag, superantigen. ⁎ Corresponding author. Tel.: +46 46 191039; fax: +46 46 191105. E-mail address: [email protected] (A. Sundstedt). 1567-5769/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2009.04.013
(Fab-SEA) [7,8]. The effectiveness of this therapy has been demonstrated both in syngeneic and xenogeneic tumor models in mice [7–9]. The poorly immunogenic B16 melanoma was transfected with the human colon carcinoma antigen C215 [7] and used to evaluate the anti-tumor effects of C215Fab-SEA fusion protein in a syngeneic lung metastasis model. Treatment with repeated injections of C215Fab-SEA eradicated N90% of lung tumors in mice carrying established B16-C215 melanoma metastases [7,10]. Combining TTS with immune modulators such as IL-2 and IFN-α has also been tested with good results [11,12]. TTS proteins are in clinical development and have shown promising results, e.g. in patients with advanced non-small cell lung cancer (NSCLC) and renal cell carcinoma (RCC) using anatumomab mafenatox/ABR-214936 [13,14]. Pharmacological proof-of-concept in man was obtained by a series of observations in subsequent phase I clinical studies of the TTS naptumomab estafenatox/ABR-217620, including dose-dependent induction of IL-2 and IFN-γ, selective expansion of TTS-reactive T cells as well as tumor infiltration of T lymphocytes in tumor biopsies from ABR-217620 treated patients [Borghaei et al., Journal of Clinical Oncology, in press]. As a consequence of the engineering process to optimize ABR217620 for human use, the compound does not activate murine T cells and is therefore not therapeutically active in experimental mouse
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models. The prototype drug molecule C215Fab-SEA is fully active in mice. Thus using C215Fab-SEA, which activates murine T cells in a similar fashion as ABR-217620 activates human T lymphocytes, makes it possible to perform pharmacological studies in mice. To further improve TTS-based therapy, we hypothesized that combination treatment with conventional therapies may be the most effective strategy for patient benefit. The goal of chemotherapy is direct cytotoxicity and induction of tumor cell death. Taxanes, which are among the most widely used cancer chemotherapeutic agents, induce anti-neoplastic activity by disrupting microtubular networks. Members of the taxane family, including docetaxel and the closely related compound paclitaxel, have been used in a variety of malignancies, such as lung, breast and prostate cancers [15]. The administration of cytotoxic chemotherapy can result in bone marrow suppression and has been traditionally perceived to have a negative effect on immune function. Recent data suggest, however, that taxanebased chemotherapy may actually exert beneficial immunomodulatory effects through a variety of mechanisms, including cytokine production and T-cell infiltration in tumors [16,17]. To determine the optimal combination tumor treatment regiment using the TTS protein C215Fab-SEA and docetaxel, we first examined the in vivo effects of docetaxel administered at different time points on the function of CD4+ and CD8+ T cells in response to TTS-induced activation. These studies were conducted in non-tumor bearing mice to rule out any indirect effects of docetaxel on the immune system that result from a reduction in tumor size. The results show that prior treatment with docetaxel at therapeutic doses did not interfere with superantigen induced T cell activation but rather appeared to enhance the response. Next, we used the selected combination regiment of docetaxel and TTS in tumor-bearing mice. Combining TTS and docetaxel significantly improved tumor therapy, further reducing the number of lung tumors as compared to mono therapies. Furthermore, TTS and docetaxel therapy synergistically prolonged long term survival in B16-C215 tumor bearing mice suggesting potential clinical benefit for the combined use. 2. Materials and methods 2.1. Animals and treatment Female C57Bl/6 mice were purchased from Taconic Europe A/S (Denmark), and maintained under standardized conditions. The mice were routinely used at the age of 8 to 12 weeks. All studies were approved by the local animal ethical committee. Recombinant SEA and C215Fab-SEA were expressed in E. coli and purified as described elsewhere [18,19]. Mice were given various doses of C215Fab-SEA in 0.2 ml PBS+1% normal mouse serum (NMS) i.v. in the tail vein. Docetaxel (Taxotere®; Sanofi Aventis, Paris, France) was administered i.p. at 1 or 2 mg/injection in 0.2 ml buffer solution (10.9% polysorbate 80, 5.1% ethanol and 0.45% NaCl). The selected dose of docetaxel was previously shown to have an anti-tumor effect on B16 melanoma cells in vivo [20]. 2.2. Reagents Fluorochrome-labeled monoclonal antibodies directed to murine CD4, CD8, TCR-Vβ3 and TCR-Vβ8 were purchased from BD Biosciences (San Jose, CA). 2.3. Cell lines The murine B-cell lymphoma A20 and C215-tranfected murine B16-F10 melanoma (B16-C215) [7] were cultured in R10 medium (RPMI-1640 with Ultraglutamine (BioWhittaker/Lonza, Wokingham, UK); supplemented with 10% fetal bovine serum (Fisher Scientific, Pittsburgh, PA), 1 mM sodium pyruvate, 10 mM HEPES, 0.1 mg/ml
gentamicine sulfate and 50 µM β-mercaptoethanol). In addition, 1 mg/ml G418 (Calbiochem, La Jolla, CA) was added to the medium for culture of B16-C215 cells. 2.4. Cytotoxicity assay Cytotoxicity of spleen cells from treated mice was measured at various effector to target (E:T) ratios in a standard 4-hour 51Cr-release assay [4]. Percent specific cytotoxicity was calculated as: 100 × (cpm experimental release − cpm background)/(cpm total release − cpm background release). 51Cr-labelled A20 target cells were used at 2.5 × 106 cells per well in V-bottomed 96 well microtiter plates. SEA was used at a concentration of 1 µg/ml. 2.5. Analysis by flow cytometry Flow cytometric analysis was performed according to standard settings on a FACSort™ flow cytometer (BD Biosciences). Before incubating splenocytes with specific fluorochrome-labelled antibodies, Fc-receptors were blocked using anti-CD16/CD32 mAb (BD Biosciences). 2.6. Therapy of B16-C215 tumors Groups of 8 C57Bl/6 mice were inoculated i.v. with 1.75 × 105 B16C215 melanoma cells into the tail vein to induce lung tumors. Mice were treated with daily injections of 10 µg C215Fab-SEA on days 3 to 6 and docetaxel was injected i.p. (1 mg/injection) on day 2 or on days 2 and 9. On day 21 mice were sacrificed and the lungs were removed. After fixation in Bouins solution (24% formalin, 71% picric acid solution, and 5% glacial acetic acid) for at least 24 h, the numbers of lung tumors were counted. In the survival experiments, groups of 10 or 20 mice were inoculated with 1.25 × 105 B16-C215 melanoma cells into the tail vein and 10 µg C215Fab-SEA was injected i.v. daily for four days on days 3 to 6 or when using multi-cycle therapy on days 3–6, days 17–20, days 45–48 and on days 59–62. Docetaxel was injected i.p. on day 2 or on days 2 and 9 (1 mg/injection) or on days 7, 21, 35, 49, 63 and 77 (2 mg/ injection) in the multi-cycle experiment. Animals were sacrificed when showing signs of morbidity or otherwise at 90 or 119 days after tumor inoculation. 2.7. Determination of mouse anti-SEA antibodies The concentration of anti-SEA antibodies in the mouse plasma samples was determined by ELISA. Briefly, plates were coated with recombinant SEA. After blocking in BSA, samples and standards of affinity purified mouse anti-SEA antibodies were added followed by a goat anti-mouse IgG antibody (DAKO Denmark A/S, Glostrup, Denmark). Next, a biotinylated rabbit anti-goat IgG antibody (DAKO Denmark A/S) was added, followed by streptavidin conjugated with HRP (DAKO Denmark A/S). The reaction was detected by addition of an enzyme substrate (TMB; 3,3′,5,5′-tetramethylbenzidine) (Bio-Rad, Hercules, CA) and the absorbance was monitored at 450 nm, with 650 nm as a reference wavelength, in an ELISA spectrophotometer. A four-parameter function was adjusted to the obtained concentration/ absorbance values of the standards and the unknown concentration of anti-SEA antibodies in samples was determined from the standard curve. The measuring range was estimated to 0.40–50 ng/ml. The sample dilution was at least 1:100, i.e. the limit of quantification (LOQ) in plasma was 39 ng/ml. 2.8. Statistical methods To evaluate and compare the therapeutic observations, the following statistical analyses were used. Number of lung tumors in the short
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term tumor model was evaluated using Mann–Whitney Rank Sum test (Sigma Stat®). Survival time was analyzed using non-parametric logrank test (GraphPad Prism®). An additional parametric analysis of survival data was performed (PROC LIFEREG in SAS, SAS Institute Inc., Cary, NC), where statistical interactions between treatments were evaluated using model: logðSurvivali Þ = c + β1 TTTSi + β2 TDoc2i + β3 TTTSi TDoc2i + β4 T Doc29i + β5 TDoc29i TTTSi + erroriði = 1;2; N ;nÞ ; where TTS, Doc2 and Doc29 are indicator variables (0 or 1) for TTS treatment days 3–6, docetaxel treatment day 2, or days 2 and 9. The parameters β3 and β5 are 0 in an additive model and greater than 0 in a synergistic model. The hypotheses β3 = 0 and β5 = 0 were tested. The logarithm of anti-SEA antibody levels was analyzed using analysis of variance (PROC MIXED in SAS, SAS Institute Inc., Cary, NC). 3. Results We have previously demonstrated that 3–4 daily injections of C215Fab-SEA efficiently activate CD4+ and CD8+ T cells expressing distinct TCR-Vβ chains to inflammatory cytokine production, massive expansion and induction of potent CTL activity within the CD8+ T cell compartment [10]. This regime also inhibits tumor growth in C215transfected B16 melanoma bearing mice [7,10,21]. 3.1. Timely administration of docetaxel does not inhibit superantigeninduced T cell expansion in spleen Being a cytotoxic drug, docetaxel targets dividing cells by inhibiting the mitotic activity. To investigate the cytostatic activity of docetaxel on TTS induced T cell expansion, mice were given three daily i.v. injections of C215Fab-SEA (10 µg/injection) in combination with one i.p. injection of docetaxel (1 mg/mouse) given on days 1, 2 or 3 of TTS treatment. Mice were sacrificed 48 h after the last C215Fab-SEA treatment and the spleens were removed for analysis of T cell populations. Simultaneous treatment either suppressed or augmented TTS-mediated T cell expansion, depending on the temporal relationship between the drugs. Administration of docetaxel on day 3 of C215Fab-SEA treatment clearly inhibited the expansion of SEA-specific TCR-Vβ3+ T cells (Fig. 1). Interestingly however, docetaxel injected in conjunction with the first C215Fab-SEA injection resulted in increased expansion of CD8+Vβ3+ T cells but not of CD4+Vβ3+ T cells (Fig. 1), which were slightly inhibited also at this time point, indicating that docetaxel may affect various T cell subsets differently. SEA non-responsive TCR-Vβ8+ T cells were only marginally affected by treatment (Fig. 1).
Fig. 2. Effect of simultaneous treatment with C215Fab-SEA and docetaxel on the induction of CTL activity. Mice (3 animals/group) were injected i.v. with 3 daily doses of C215Fab-SEA (10 µg/animal) in combination with i.p. injections of docetaxel (1 mg/ animal) given either on day 3 of TTS treatment (A) or the day before starting the TTS treatment (B). Docetaxel was injected approximately 30 min after C215Fab-SEA administration when given on the same day as C215Fab-SEA. Forty-eight hours after the last C215Fab-SEA injection, spleens were removed and cytotoxic activity (SDCC) against SEA-coated A20 cells was measured in a standard 51Cr release assay at different effector to target (E:T) ratios (mean ± SEM).
Fig. 1. Effect of simultaneous treatment with C215Fab-SEA and docetaxel on the expansion of SEA-specific TCR-Vβ3+ T cells. Mice (3 animals/group) were injected i.v. with 3 daily doses of C215Fab-SEA (10 µg/animal) in combination with i.p. injections of docetaxel (1 mg/animal) given on days 1, 2 or 3 of TTS treatment. Docetaxel was injected approximately 30 min after C215Fab-SEA administration. Forty-eight hours after the last C215Fab-SEA injection, spleens were removed and TCR-Vβ3 specific expansion of CD4 and CD8 T cells (mean ± SEM) was measured by flow cytometry.
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3.2. Docetaxel given before TTS enhances the induction of superantigeninduced effector CTLs, while simultaneous treatment is suppressive
Fig. 3. C215Fab-SEA and docetaxel combination therapy in B16-C215 tumor bearing mice. Groups of eight C57Bl/6 mice were inoculated i.v. with 1.75 × 105 B16-C215 melanoma cells on day 0. Treatment started on day 2 with one i.p. injection of 1 mg/ mouse of docetaxel or vehicle (10.9% polysorbate 80, 5.1% ethanol and 0.45% NaCl). This was followed by four daily i.v. injections on days 3 to 6 of 10 µg/mouse C215Fab-SEA or vehicle (PBS+1% C57Bl/6 mouse serum), and then a second i.p. injection of docetaxel (1 mg/mouse) or vehicle given on day 9. On day 21, mice were sacrificed, the lungs were removed and the number of tumors counted. Statistical analysis was done using Mann– Whitney Rank Sum test.
Induction of perforin-dependent cytotoxic activity within the CD8+ T cell subset by TTS treatment is a central effector mechanism involved in tumor cell killing in vivo [22]. Thus, it was important to determine not only the effect of docetaxel on T cell division but also how the drug influences the killing potential of TTS-activated CTLs. Mice received three daily i.v. injections of C215Fab-SEA (10 µg/injection) in combination with one i.p. injection of docetaxel (1 mg/mouse) given either the day before or on day 3 of TTS treatment. The cytotoxic response of splenocytes was investigated 48 h after the last treatment. Superantigen-dependent cell-mediated cytotoxicity (SDCC) was measured against SEA-coated MHC class II-expressing target cells in a standard 4 h 51Cr-release assay. Three daily injections of C215Fab-SEA resulted in significant cytotoxic activity (Fig. 2). In line with the previous result on T cell expansion, docetaxel given on day 3 of TTS treatment clearly inhibited the SEA-dependent cytotoxic response of splenocytes compared to cells from mice treated with C215Fab-SEA alone (Fig. 2A). In contrast, cytostatic treatment before C215Fab-SEA therapy did not interfere with SEA-mediated activation of CD8+ T cells. Rather, administration of docetaxel the day before TTS treatment resulted in enhanced cytotoxic activity in this T cell subset (Fig. 2B). No significant SDCC was recorded in mice treated with docetaxel alone or vehicle (Fig. 2). Taken together, these results demonstrate that docetaxel is compatible with TTS therapy when given shortly before the first C215Fab-SEA injection.
Fig. 4. Long term survival of B16-C215 tumor bearing mice treated with C215Fab-SEA and docetaxel. Groups of ten C57Bl/6 mice were inoculated i.v. with 1.25 × 105 B16-C215 melanoma cells day 0. Treatment was started on day 2 with one i.p. injection of 1 mg/mouse of docetaxel or vehicle (10.9% polysorbate 80, 5.1% ethanol and 0.45% NaCl). This was followed by four daily i.v. injections on days 3 to 6 of 10 µg/mouse of C215Fab-SEA or vehicle (PBS+1% C57Bl/6 mouse serum), and then a second i.p. injection of docetaxel (1 mg/ mouse) or vehicle given on day 9. Mice were sacrificed when moribund behavior was noticed or otherwise 90 days after tumor inoculation. Survival curves, median survival times and statistical analysis using non-parametric logrank test are displayed.
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3.3. TTS and docetaxel in combination is superior to the mono therapies: inhibition of B16-C215 lung tumors Based on the results above, we next investigated anti-tumor effects against B16-C215 expressing tumor cells of combining C215Fab-SEA and docetaxel. The chosen schedule was to give docetaxel before the TTS protein. We also included treatment with docetaxel after the TTS cycle, administered after the proliferative peak of the T cells, to investigate if the anti-tumor effect could be enhanced. C57Bl/6 mice were inoculated i.v. on day 0 with B16-C215 melanoma cells and the number of lung tumors was determined three weeks later. Docetaxel was injected i.p. on day 2 or on days 2 and 9, in combination with four daily i.v. injections of C215Fab-SEA on days 3 to 6. Mice treated with docetaxel, one or two times, or mice treated with C215Fab-SEA alone had less lung tumors compared to vehicle (Fig. 3). The therapeutic effects of combining C215Fab-SEA and docetaxel was significantly better than C215Fab-SEA alone (P = 0.003 and P b 0.001, respectively) (Fig. 3). In spite of the fact that the combination of TTS with one injection (day 2) of docetaxel showed only few lung tumors, there was a clear tendency of a further reduction in tumor load in the group treated with the two docetaxel injection combination. Also, in contrast to all other treatment groups, 2 of 8 mice in this group were free of tumor at the end of the experiment. The mice were carefully monitored during and after treatment and the therapy was well tolerated. No additional adverse effects were recorded after combination treatment as compared to mono therapies. 3.4. TTS and docetaxel in combination synergistically prolongs long term survival in B16-C215 tumor bearing mice Next we wanted to explore whether the improved tumor therapy by C215Fab-SEA and C215Fab-SEA combination treatment in the short
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term B16-C215 model also translated into long term survival in the same tumor model. When the mice showed signs of morbidity or on day 90, they were sacrificed and autopsies were carried out. Treatment with docetaxel, one or two times, in combination with C215Fab-SEA showed a dramatically prolonged survival time as compared to the mono therapies (Fig. 4). The median survival time was increased from 42 days after C215Fab-SEA to N90 days in the case of combination treatment with 2 injections of docetaxel (days 2 and 9), constituting a significant difference (P b 0.0001) (Fig. 4). Also the combination using one injection of docetaxel significantly prolonged median survival to 66 days (P b 0.0001). Median survival times in the vehicle treated control group and in mice treated with docetaxel alone were 33 and 38 days, respectively. Statistical analysis demonstrated that the therapeutic effect of combining C215Fab-SEA and docetaxel (days 2 and 9) gave rise to significant synergistic anti-tumor responses compared to C215Fab-SEA mono therapy (P b 0.0001), when statistical interactions between treatments were evaluated as described in Materials and methods. Moreover, 7 of 10 animals survived for N90 days following combination treatment (C215Fab-SEA + docetaxel days 2 and 9) while all control mice were lost before day 40 in this low immunogenic tumor model, underlining the potential of combining TTS therapy with docetaxel treatment. Post-mortem analysis of the remaining animals (n = 7) in the combination treatment group on day 90 revealed no visible signs of tumor, indicating that they were tumor free. These findings show that the use of C215Fab-SEA in combination with docetaxel give rise to a synergistic survival advantage over animals treated with docetaxel or C215FabSEA alone. It is concluded that more than additive treatment effects were recorded in the experiments using the combination treatments as compared to mono therapies when evaluating the number of tumors in the lungs which extrapolate to clear synergism in increasing survival.
Fig. 5. Long term survival after multi-cycle treatment with C215Fab-SEA in combination with docetaxel. Groups of twenty C57Bl/6 mice were inoculated i.v. with 1.25 × 105 B16-C215 melanoma cells on day 0. Mice were treated with 10 µg/mouse of C215Fab-SEA or vehicle (PBS+1% C57Bl/6 mouse serum) i.v. with four cycles of four daily i.v. injections on days 3–6, 17–20, 45–48 and 59–62. This was followed by i.p. injections of docetaxel (2 mg/mouse) or vehicle at biweekly intervals on days 7, 21, 35, 49, 63 and 77. Mice were sacrificed when moribund behavior was noticed or otherwise 119 days after tumor inoculation. Survival curves, median survival times and statistical analysis using non-parametric logrank test are displayed.
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3.5. Multi-cycle TTS treatment followed by docetaxel induces long term survival in B16-C215 tumor bearing mice Treatment with repeated cycles of TTS therapy enhances the antitumor effect against B16-C215 melanoma [23]. In addition, the clinical use of TTS protein therapy is based on multi-cycle treatment [13,14] [Borghaei et al., Journal of Clinical Oncology, in press]. Thus, to test combination therapy in a more clinically relevant setting, mice were treated with four cycles of C215Fab-SEA in combination with docetaxel, six times. Using this protocol, C215Fab-SEA treatment increased the median survival from 36 days in the vehicle group to 76 days (Fig. 5). Mice treated with docetaxel alone had a median survival time of 61 days. Again, combination therapy significantly prolonged the survival time compared to C215Fab-SEA mono therapy (P = 0.0005). Twelve out of 20 mice in the combination group were tumor free at the end of the experiment (day 119) compared to 4 out of 20 mice treated with C215Fab-SEA alone. 3.6. Docetaxel inhibits the development of anti-SEA antibodies after repeated TTS treatment One of the limiting factors allowing effective dosing of repeated cycles of TTS therapy is the development of anti-superantigen antibodies [13,14] [Borghaei et al., Journal of Clinical Oncology, in press]. We hypothesized that docetaxel might be able to prevent the induction of a specific antibody response by inhibiting dividing B cells. To determine the influence of docetaxel treatment on the development of anti-superantigen antibodies during multi-cycle TTS treatment, we measured the levels of anti-SEA antibodies in plasma of the surviving mice (day 119) in the experiment showed in Fig. 5. At this time point, 5 mice from the C215Fab-SEA group and 12 mice from the combination group were still alive. As shown in Fig. 6A, docetaxel significantly reduced the development of an anti-SEA antibody response after C215Fab-SEA treatment. To make a more detailed analysis of the development of anti-SEA antibodies, mice were treated as before and the levels of antibodies were measured on days 34 and 76. Again, docetaxel significantly reduced the development of anti-SEA antibodies after C215Fab-SEA treatment (Fig. 6B). Thus, it is likely that inhibition of anti-superantigen antibodies by the add-on docetaxel treatment is one component explaining the significantly improved therapeutic effect of the combination. 4. Discussion Many clinical trials have shown that combinations can be more effective than the single use of any particular modality. However, when it comes to combining immunotherapy and chemotherapy in cancer, it has been widely assumed that cytostatic drugs are immunosuppressive and would counteract the benefits of immunotherapy. In this study we investigated how docetaxel can be administered safely in combination with TTS immunotherapy without inhibiting specific T cell responses. We demonstrate that timely administration of docetaxel does not interfere with TTS induced T cell activation and more importantly that this combination therapy gives rise to strong synergistic anti-tumor effects against B16 tumors growing in the lung. Most anticancer agents, such as cytostatic drugs and radiation, work by affecting or preventing cell division. Because they are nonspecific, all dividing cells are affected and this results in adverse side effects. In an immune response, the requisite lymphocyte expansion phase is characterized by massive proliferation. This proliferation is fundamental and essential to a productive immune response. Thus, a compromised immune system is one of the more common and serious side effects of treating cancer with chemotherapeutic drugs. Immunotherapies involving T lymphocytes, such as TTS or tumor vaccines, depend on a functional immune response. Treatment with TTS is preferably performed in cycles of 3–5 daily injections. The TTS induced
Fig. 6. Effect of docetaxel on the development of anti-SEA antibodies after C215Fab-SEA treatment. (A) Mice were inoculated B16-C215 melanoma cells and treated as described in Fig. 5. At the end of the experiment (day 119), blood samples were drained from vena cava for preparation of plasma from mice that were treated with C215Fab-SEA (n = 5) or C215Fab-SEA and docetaxel (n = 12). Levels of anti-SEA antibodies were measured by ELISA and are displayed as mean ± SEM. (B) C57Bl/6 mice were inoculated i.v. with 1.25 × 105 B16-C215 melanoma cells on day 0. Mice were treated with 10 µg/mouse of C215Fab-SEA or vehicle (PBS+1% C57Bl/6 mouse serum) i.v. with four cycles of four daily injections on days 3–6, 17–20, 45–48 and 59–62. This was followed by i.p. injections of docetaxel (2 mg/mouse) or vehicle at biweekly intervals on days 7, 21, 35, 49, and 63. Blood samples were drained from vena saphena on days 34 and in the case of the combination treatment also on day 76 for preparation of plasma: vehicle (n = 5), C215Fab-SEA (n = 58) or C215Fab-SEA and docetaxel, day 34 (n = 28), day 76 (n = 20). Levels of anti-SEA antibodies were measured by ELISA and are displayed as mean ± SEM.
immune response is immediate and much broader, activating T cells with distinct TCR–Vβ expression, than what is seen when giving a vaccine. Therefore the time frame of immune cell proliferation is limited and, as seen from the data in this study, clearly avoidable in a controlled manner if combining with an anti-proliferative drug such as docetaxel. Despite that cytostatic drugs are anti-proliferative in general and have an inhibitory effect on immune function, several studies have shown that under certain circumstances they can enhance immunity. Chemotherapy may, by simply reducing the tumor mass (debulking), reduce its immunosuppressive properties. As a proof of principle, surgical removal of the primary tumor could reverse tumor-induced
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immune tolerance even in mice bearing metastatic breast cancer [24]. Chemotherapy can cause immunogenic cancer cell stress or death and hence mediate a sort of cancer vaccination effect [25], and may further stimulate the immune system by causing lymphopenia followed by homeostatic proliferation of immune effectors that may be particularly active in the anti-cancer response. Animal studies have demonstrated that lymphoablation enhances the effect of adoptively transferred tumor-specific CD8+ T cells [26]. It can be anticipated that treatment with docetaxel before the TTS treatment may cause an enhanced expansion of superantigen activated effector cells due to mechanisms equal to what has been demonstrated with lymphoablation. In fact treating with docetaxel immediately before or on the first day of a TTS cycle resulted in our studies in enhanced T cell cytotoxicity and enhanced CD8+ T cell expansion, respectively (Figs. 1 and 2). Garnett et al. [27] showed that docetaxel enhanced IFN-γ production by CD8+ T cells. Furthermore, they showed that docetaxel combined with a recombinant viral vaccine increased specific T-cell responses to antigen in the vaccine as well as to cascade antigens derived from the tumor, and was superior to either agent alone at reducing tumor burden. In agreement with the results in our study, they showed that the outcome of the combination treatment was dependent of the timely relationship of docetaxel treatment and immune stimulus. Among the series of causes to tumor induced immune suppression, regulatory T cells (Treg) have recently been recognized as one of the most important. Some cytostatic agents, in particular cyclophosphamide, can partially deplete or transiently inactivate tumorprotective Treg [28]. On the contrary to what was found using cyclophosphamide, Garnett et al. show that docetaxel does not inhibit the function of Treg [27]. Therefore, modulation of B16 tumor or TTS induced Treg by docetaxel is unlikely. The myeloid-derived suppressor cells (MDSC) are another type of suppressive regulatory cells proven to be important in tumor immunity [29]. MDSC being a subpopulation of activated granulocytes [30] may well be decreased in number by docetaxel having a documented dramatic effect on granulocytes. One of the major advantages to use bacterial superantigens in TTS comes with the characteristics of them being the most powerful T cell activators known. A disadvantage that goes with the bacterial heritage of the protein though, is the immunogenicity and the production of antibodies directed towards the superantigens. Dosing of ABR214936 to cancer patients was performed according to an algorithm recognizing the anti-superantigen (anti-sag) antibody titers [13]. With the new improved TTS ABR-217620, being less antigenic and immunogenic [31], dosing of patients can be done without adjusting for preformed anti-sag antibodies [Borghaei et al., Journal of Clinical Oncology, in press]. Nonetheless, it was shown that anti-sag antibody responses were detected in most patients. Here, we demonstrate that using an anti-proliferative cytostatic drug, in this case docetaxel, immediately after a full TTS cycle significantly inhibited the anti-sag antibody production in the experimental mouse model used in this study. In accordance with these results, the titers of anti-sag antibodies recorded after treatment in the clinical trial using the combination of ABR-217620 and docetaxel in patients with NSCLC were significantly lower as compared to the trial with ABR-217620 alone [Borghaei et al., Journal of Clinical Oncology, in press]. It should be emphasized that other cancer types like breast and prostate cancer where the use of docetaxel is established, also express the 5T4 antigen [32] which is the target for ABR-217620. In this study, we have shown that the timely combination of TTS immunotherapy and the cytostatic drug docetaxel results in synergistic anti-tumor effects. Furthermore, this combination inhibited the production of antibodies binding to TTS. Together with the promising clinical results obtained using ABR-217620 in combination with docetaxel in patients with NSCLC [Borghaei et al., Journal of Clinical Oncology, in press], the impressive anti-tumor efficacy in a preclinical model shown in this study prompt for further clinical evaluation of
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