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CCR Translation for the Article from Comparative Impact of Trastuzumab and Cyclophosphamide on HER-2–Positive Human Breast Cancer Xenografts

Purpose: Metastatic breast cancer (MBC) response to allogeneic lymphocytes requires donor T-cell engraftment and is limited by graft-versus-host disease (GVHD). In mice, type-II–polarized T cells promote engraftment and modulate GVHD, whereas type-I–polarized T cells mediate more potent graft-versus-tumor (GVT) effects. This phase I translational study evaluated adoptive transfer of ex vivo costimulated type-I/type-II (T1/T2) donor T cells with T-cell–depleted (TCD) allogeneic stem cell transplantation (AlloSCT) for MBC.Experimental Design: Patients had received anthracycline, taxane, and antibody therapies, and been treated for metastatic disease and a human leukocyte antigen (HLA)-identical–sibling donor. Donor lymphocytes were costimulated ex vivo with anti-CD3/anti-CD28 antibody–coated magnetic beads in interleukin (IL)-2/IL-4–supplemented media. Patients received reduced intensity conditioning, donor stem cells and T1/T2 cells, and monitoring for toxicity, engraftment, GVHD, and tumor response; results were compared with historical controls, identically treated except for T1/T2 product infusions.Results: Mixed type-I/type-II CD4+ T cells predominated in T1/T2 products. Nine patients received T1/T2 cells at dose level 1 (5 × 106 cells/kg). T-cell donor chimerism reached 100% by a median of 28 days. Seven (78%) developed acute GVHD. At day +28, five patients had partial responses (56%) and none had MBC progression; thereafter, two patients had continued responses. Donor T-cell engraftment and tumor responses appeared faster than in historical controls, but GVHD rates were similar and responders progressed early, often following treatment of acute GVHD.Conclusion: Allogeneic T1/T2 cells were safely infused with TCD-AlloSCT, appeared to promote donor engraftment, and may have contributed to transient early tumor responses. Clin Cancer Res; 17(21); 6878–87. ©2011 AACR.

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Purpose: Metronomic chemotherapy is a minimally toxic and frequently effective new treatment strategy that is beginning to show promising phase II clinical trial results, particularly for metastatic breast cancer when combined with various molecularly targeted antitumor agents. Here, we assessed a treatment strategy that uses trastuzumab plus daily oral metronomic cyclophosphamide on metastatic Her-2–positive human breast cancer models.Experimental Design: Treatments were initiated on orthotopic transplanted primary tumors as well as established visceral metastatic disease of two independent Her-2–positive breast cancer models, both independently derived from the human MDA-MB-231 breast cancer cell line. Outcome was assessed by noninvasive measurements of tumor cell–secreted human choriogonadotropin in the urine as a surrogate marker of relative tumor burden, or by whole body bioluminescent imaging, in addition to prolongation of survival.Results: Orthotopic primary tumors responded to trastuzumab monotherapy with significant growth delays, whereas minimal antitumor effect was observed when mice with metastatic disease were treated. Nevertheless, trastuzumab showed a benefit in this latter setting when combined with metronomic low-dose cyclophosphamide as assessed by prolongation of survival. This benefit was similar to trastuzumab plus maximum tolerated dose cyclophosphamide, but was associated with lesser toxicity.Conclusions: Trastuzumab combined with metronomic cyclophosphamide may be an effective long-term maintenance strategy for the treatment of Her-2–positive metastatic breast cancer. (Clin Cancer Res 2009;15(20):6358–66)

Graphical abstract, Highlights • 3-Monochloropropane-1,2-diol (3-MCPD) and its fatty acid esters have the potential to induce nephrotoxicity. • We used an in vitro cellular model of human proximal tubule cells to test the effects of 3-MCPD compound exposures on transporter gene expression. • 3-MCPD-related nephrotoxicity could be associated with indirect modes of action relating to aquaporin homeostasis., 3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in some infant formula products and other foods in the United States. Although rodent studies have demonstrated that 3-MCPD and its palmitic esters have the potential to induce nephrotoxicity, our recent human cell culture studies using the human renal proximal tubule cell line HK-2 have not strongly supported this finding. Considering this disparity, we sought to examine whether changes in transporter gene expression on proximal tubule cells could be modulated by these compounds and allow us to glean mechanistic information on a possible indirect path to proximal tubule injury in vivo. If fundamental processes like water and solute transport could be disrupted by 3-MCPD compounds, then a new avenue of toxicity could be further explored in both infant and adult models. In our current study, we used HK-2 cells as an in vitro cellular model of human proximal tubule cells to investigate the effects of low (10 μM) and high (100 μM) 3-MCPD compound exposures to these cells for 24 hours (h) on the expression of 20 transporter genes that are known to be relevant to proximal tubules. Although we detected consistent upregulation of AQP1 expression at the RNA transcript level following HK-2 treatment with both low and high doses of several ester-bound 3-MCPD compounds, these increases were not associated with statistically significant elevations in their protein expression levels. Moreover, we observed a lack of modulation of other members of the AQP protein family that are known to be expressed by human proximal tubule cells. Overall, our study suggests the possibility that 3-MCPD-related nephrotoxicity could be associated with indirect modes of action relating to aquaporin homeostasis, but additional studies with other human-derived models would be pertinent to further explore these findings and to better understand transporter expression differences under different stages of proximal tubule development.

The renal proximal tubule cell line, human kidney 2 (HK-2), recapitulates many of the functional cellular and molecular characteristics of differentiated primary proximal tubule cells. These features include anchorage dependence, gluconeogenesis capability, and sodium-dependent sugar transport. In order to ascertain how well HK-2 cells can reliably reveal the toxicological profile of compounds having a potential to cause proximal tubule injury in vivo, we sought to evaluate the effects of known proximal tubule toxicants using the HK-2 cell line. We selected 20 pure nephrotoxic compounds that included chemotherapeutic drugs, antibiotics, and heavy metal-containing compounds and evaluated their ability to induce HK-2 cell injury relative to 10 innocuous pure compounds or cell culture media alone. We performed a comprehensive set of in vitro cellular toxicological assays to evaluate cell viability, oxidative stress, mitochondrial integrity, and a specific biomarker of renal injury, Kidney Injury Molecule 1. For each of our selected compounds, we were able to establish a reproducible profile of toxicological outcomes. We compared our results to those described in peer-reviewed publications to understand how well the HK-2 cellular model agrees with overall in vivo rat or human toxicological outcomes. This study begins to address the question of how well in vitro data generated with HK-2 cells can mirror in vivo animal and human outcomes.

Classic toxicology studies often utilize in vivo animal models. Newer approaches employing in vitro organ-specific cellular models have been developed in recent years to help accelerate the speed and reduce the cost of traditional toxicology testing. Toward the goal of supporting in vitro cellular model research with a regulatory application in mind, we have developed a 'designer' human kidney cell line called HK2-Vi that can fluorescently measure the cytotoxicity of potential toxins on proximal tubule cell viability in a direct exposure in vitro model. HK2-Vi was designed to be a reagent-less kinetic assay that can yield data on short- or long-term cell viability after toxin exposure. To generate HK2-Vi, we used monocistronic lentiviral transduction methods to genetically engineer a human kidney cell line called HK-2 to stably co-express two transgenes. The first is Perceval HR, which encodes a fluorescent biosensor of both cytosolic ATP and ADP and the second is pHRed, which encodes a biosensor of cytosolic pH. Relative levels of cellular ATP and ADP effectively serve as a reliable and robust indicator of cell viability. Because the fluorescence Perceval HR is pH-dependent, we co-expressed the pHRed genetic biosensor to correct for variations in pH if necessary. Heterogenous populations of transduced renal cells were enriched by flow cytometry before monoclonal cellular populations were isolated by cell culture methods. A single clonal population of co-transduced cells expressing both Perceval HR and pHRed was selected to be HK2-Vi. This established cell line can now serve as a tool for in vitro toxicology testing and the methods described herein serve as a model for developing designer cell lines derived from other organs.

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in the U.S. food supply, detected in infant formula. In vivo rodent model studies have identified a variety of possible adverse outcomes from 3-MCPD exposure including renal effects like increased kidney weights, tubular hyperplasia, kidney tubular necrosis, and chronic progressive nephropathy. Given the lack of available in vivo toxicological assessments of 3-MCPD in humans and the limited availability of in vitro human cell studies, the health effects of 3-MCPD remain unclear. We used in vitro human proximal tubule cells represented by the HK-2 cell line to compare short- and long-term consequences to continuous exposure to this compound. After periodic lengths of exposure (0-100 mM) ranging from 1 to 16 days, we evaluated cell viability, mitochondrial integrity, oxidative stress, and a specific biomarker of proximal tubule injury, Kidney Injury Molecule-1 (KIM-1). Overall, we found that free 3-MCPD was generally more toxic at high concentrations or extended durations of exposure, but that its overall ability to induce cell injury was limited in this in vitro system. Further experiments will be needed to conduct a comprehensive safety assessment in infants who may be exposed to 3-MCPD through consumption of infant formula, as human renal physiology changes significantly during development.

A routine, selective and sensitive ultra-high performance liquid chromatography-electrospray ionisation tandem triple quadrupole mass spectrometry (UHPLC-ESI-MS/MS) method was developed and validated for the quantification of 3-monochloropropane-1,2-diol (3-MCPD) in Caco-2 cell transport buffer (FaSSIF-V2, the second version of a fasted state simulated intestinal fluid) and receiving buffer (HBSS, Hank’s balanced salt solution). The method involves measuring deuterated 3-MCPD (3-MCPD-d5) as internal standard (IS) during the entire analytical procedure to obtain precise and accurate results. The separation was performed on a Poroshell 120 HILIC column (2.7 µm, 3.0 × 50 mm) at a flow rate of 0.3 mL/min using water (containing 0.025% acetic acid) and acetonitrile (containing 0.025% acetic acid) as the mobile phases. Mass spectrometric detection was operated in dynamic multiple reaction monitoring (dMRM) in negative ion mode. The method exhibited high sensitivity. The limits of detection (LOD) for 3-MCPD in FaSSIF-V2 and HBSS were 0.012 and 0.014 µmol/L, and the limits of quantification (LOQ) were 0.039 and 0.045 µmol/L, respectively. Satisfactory results were observed for linearity (R2 > 0.999), intra-day precision (RSD% RSD% RSD%

3-Monochloropropane-1,2-diol (3-MCPD) is a food processing contaminant in the U.S. food supply, detected in infant formula. In vivo rodent model studies have identified a variety of possible adverse outcomes from 3-MCPD exposure including renal effects like increased kidney weights, tubular hyperplasia, kidney tubular necrosis, and chronic progressive nephropathy. Given the lack of available in vivo toxicological assessments of 3-MCPD in humans and the limited availability of in vitro human cell studies, the health effects of 3-MCPD remain unclear. We used in vitro human proximal tubule cells represented by the HK-2 cell line to compare short- and long-term consequences to continuous exposure to this compound. After periodic lengths of exposure (0-100 mM) ranging from 1 to 16 days, we evaluated cell viability, mitochondrial integrity, oxidative stress, and a specific biomarker of proximal tubule injury, Kidney Injury Molecule-1 (KIM-1). Overall, we found that free 3-MCPD was generally more toxic at high concentrations or extended durations of exposure, but that its overall ability to induce cell injury was limited in this in vitro system. Further experiments will be needed to conduct a comprehensive safety assessment in infants who may be exposed to 3-MCPD through consumption of infant formula, as human renal physiology changes significantly during development.

Carboxymethyl starches are added to food products for thickening or tablet binding/filling purposes. Although they lack toxicity, their synthesis creates the chemical byproduct diglycolic acid (DGA), which is difficult to eliminate and whose toxicity is in question. A rare case of an accidental direct exposure to extremely high concentrations of DGA in a person revealed that DGA has the potential to be toxic to several organs, with the kidneys and liver being the most affected organs. Given that DGA is present in our food supply as a chemical byproduct of carboxymethyl starch food additives, we sought to perform in vitro testing of its potential hepatotoxicity to help complement a recent in vivo rat acute dose-response study that also tested for the potential hepatotoxic effects of daily DGA ingestion by oral gavage over a period of 28 days. Using the HepG2/C3A cellular in vitro model, we tested how escalating doses of DGA exposure over 24 h could induce hepatotoxicity. Both in vitro and in vivo testing systems revealed that DGA is indeed a hepatotoxin once a certain exposure threshold is reached. The concordance of these models highlights the utility of in vitro testing to support and help predict in vivo findings.

The acute oral toxicity of diglycolic acid (DGA) was evaluated. Groups of female rats (n = 8 rats/group) received 28 consecutive daily single doses of 0.3, 1.0, 3.0, 10.0, 30.0, 100.0 or 300.0 mg DGA/kg body weight by gastric intubation. One group of animals served as vehicle control. Tissues and blood serum were collected at necropsy on day 29. Select organs were weighed and fixed in formalin for histopathological analysis. Animals from the 300 mg/kg bw dose group were removed from the study after 5 consecutive days of treatment as a consequence of adverse treatment related effects. The animals in the remaining treatment groups survived the exposure period. No adverse clinical signs were observed throughout the exposure period in the surviving animals. No significant differences from controls were observed for feed and fluid consumption or body weight gain in the surviving animals. Lesions were observed in the kidneys, liver, stomach, intestine, thymus, spleen and bone marrow in rats from the 300 mg/kg dose group and signs of renal tubular regeneration were observed only in the 100 mg/kg dose group. These results suggest that high levels of pure DGA would need to be consumed before renal and other forms of organ toxicity are observed.

Graphical abstract, Highlights • Diglycolic acid (DGA) is an indirect food additive. • DGA was tested for renal cell toxicity in vitro using HK-2 cells. • Evaluation of toxicity included cellular and mitochondrial effects. • In vitro data are highly concordant with in vivo outcomes., Diglycolic acid (DGA) is present in trace amounts in our food supply and is classified as an indirect food additive linked with the primary GRAS food additive carboxymethyl cellulose (CMC). Carboxymethyl starches are used as a filler/binder excipient in dietary supplement tablets and a thickening ingredient in many other processed foods. We sought to utilize the human proximal tubule HK-2 cell line as an in vitro cellular model system to evaluate its acute nephrotoxicity of DGA. We found that DGA was indeed toxic to HK-2 cells in all in vitro assays in our study, including a highly sensitive Luminex assay that measures levels of an in vitro biomarker of kidney-specific toxicity, Kidney Injury Molecule 1 (KIM-1). Interestingly, in vitro KIM-1 levels also correlated with in vivo KIM-1 levels in urine collected from rats treated with DGA by daily oral gavage. The use of in vitro and in vivo models towards understanding the effectiveness of an established in vitro system to predict in vivo outcomes would be particularly useful in rapidly screening compounds that are suspected to be unsafe to consumers. The merit of the HK-2 cell model in predicting human toxicity and accelerating the process of food toxicant screening would be especially important for regulatory purposes. Overall, our study not only revealed the value of HK-2 in vitro cell model for nephrotoxicity evaluation, but also uncovered some of the mechanistic aspects of the human proximal tubule injury that DGA may cause.

Chloropropanols are chemical contaminants that can be formed during industrial processing of foods, such as lipids used in commercially available infant and toddler formula in the USA. Many researchers have studied the most common chloropropanol contaminant, 3-monochloropropane-1,2-diol (3-MCPD), as well as its lipid ester derivatives. A plethora of toxicological outcomes have been described in vivo, including effects on the heart, nervous system, reproductive organs, and kidneys. To better understand the concordance of some of these effects to in vitro outcomes, we focused our research on using an in vitro cellular model to investigate whether the proximal tubule cells of the kidney would be vulnerable to the effects of free 3-MCPD and nine of its common esters in commercial formula. Using the established human kidney proximal tubule cell line, HK-2, we performed 24-h treatments using 3-MCPD and nine mono- or di-esters derived from palmitate, oleate, and linoleate. By directly exposing HK-2 cells at treatment doses ranging from 0 to 100 μM, we could evaluate their effects on cell viability, mitochondrial health, reactive oxygen species (ROS) production, and other endpoints of toxicity. Since chloropropanols reportedly inhibit cellular metabolism through interference with glycolysis, we also tested the extent of this mechanism. Overall, we found mild but statistically significant evidence of cytotoxicity at the highest tested treatment concentrations, which were also associated with mitochondrial dysfunction and transient perturbations in cellular metabolism. Based on these findings, further studies will be required to better understand the effects of these compounds under conditions that are more physiologically relevant to human infant and toddler proximal tubules in order to mimic their exposure to chloropropanol-containing foods.

Adhatoda zeylanica is a dietary supplement ingredient present in several types of dietary supplements, including weight loss, respiratory relief, and immune regulating products. Due to its reported wide range of uses in folk medicine, it was hypothesized that it may have the potential to target multiple organs and lead to a range of toxicity features. As a preliminary evaluation of the safety of this herbal ingredient, an investigation into its effects on the kidney was sought. An in vitro study of its potential nephrotoxicity using the HK-2 human proximal tubule cell line in a variety of functional indicators was performed to capture both general forms of cellular toxicity as well as ones that are specific to proximal tubules. A. zeylanica was only capable of inducing detrimental short-term toxicity to HK-2 cells at relatively high treatment concentrations when exposed directly to the cells. The lack of acute and potent toxicity of A. zeylanica under our experimental conditions calls for further studies to better define its toxicant threshold and establish safe dosage levels. Keywords: Kidney proximal tubule, Adhatoda zeylanica, Nephrotoxicity, HK-2

3-chloro-1,2-propanediol (3-MCPD) and 3-MCPD esters are contaminants present in a variety of processed foods, including infant formulas. Toxicological data are unavailable in humans, but rodent studies have demonstrated renal and testicular toxicity from 3-MCPD and 3-MCPD esters. There is evidence that 3-MCPD esters are hydrolyzed in the digestive system, releasing 3-MCPD that would be absorbed and induce damage. We assessed absorption and metabolism of 3-MCPD and three 3-MCPD monoesters, 1-oleoyl (1-Ol), 1-linoleoyl (1-Li) and 1-palmitoyl (1-Pa) commonly found in U.S. infant formula using differentiated Caco-2 cells. After 1-hour incubation, all three monoesters released free 3-MCPD and free fatty acids (FFA) into Caco-2 cell supernatants. Free 3-MCPD had a high apparent permeability (Papp = 30.36 ± 1.31 cm/s × 10−6) suggesting that it is freely diffusible and highly absorbed by intestinal epithelium. 1-Li released 3–4-fold more 3-MCPD than 1-Ol and 1-Pa over 1 h, suggesting that this variable release rates might contribute to the overall in vivo exposure to 3-MCPD. None of the monoesters or FFA were detected in basolateral supernatants, suggesting that these compounds do not cross the intestinal wall without further transformation. In summary, this study provides relevant data to advance knowledge of in vivo intestinal absorption and metabolism of 3-MCPD monoesters.

In experimental models, ex vivo induced T-cell rapamycin resistance occurred independent of T helper 1 (Th1)/T helper 2 (Th2) differentiation and yielded allogeneic CD4+ T cells of increased in vivo efficacy that facilitated engraftment and permitted graft-versus-tumor effects while minimizing graft-versus-host disease (GVHD). To translate these findings, we performed a phase 2 multicenter clinical trial of rapamycin-resistant donor CD4+ Th2/Th1 (T-Rapa) cells after allogeneic-matched sibling donor hematopoietic cell transplantation (HCT) for therapy of refractory hematologic malignancy. T-Rapa cell products, which expressed a balanced Th2/Th1 phenotype, were administered as a preemptive donor lymphocyte infusion at day 14 post-HCT. After T-Rapa cell infusion, mixed donor/host chimerism rapidly converted, and there was preferential immune reconstitution with donor CD4+ Th2 and Th1 cells relative to regulatory T cells and CD8+ T cells. The cumulative incidence probability of acute GVHD was 20% and 40% at days 100 and 180 post-HCT, respectively. There was no transplant-related mortality. Eighteen of 40 patients (45%) remain in sustained complete remission (range of follow-up: 42-84 months). These results demonstrate the safety of this low-intensity transplant approach and the feasibility of subsequent randomized studies to compare T-Rapa cell-based therapy with standard transplantation regimens. This trial was registered at www.cancer.gov/clinicaltrials as #NCT 00077480.

A recent surge in the use of dietary supplements, including herbal remedies, necessitates investigations into their safety profiles. “Dream herb,”Calea zacatechichi, has long been used in traditional folk medicine for a variety of purposes and is currently being marketed in the US for medicinal purposes, including diabetes treatment. Despite the inherent vulnerability of the renal system to xenobiotic toxicity, there is a lack of safety studies on the nephrotoxic potential of this herb. Additionally, the high frequency of diabetes-associated kidney disease makes safety screening ofC. zacatechichifor safety especially important. We exposed human proximal tubule HK-2 cells to increasing doses of this herb alongside known toxicant and protectant control compounds to examine potential toxicity effects ofC. zacatechichirelative to control compounds. We evaluated both cellular and mitochondrial functional changes related to toxicity of this dietary supplement and found that even at low doses evidence of cellular toxicity was significant. Moreover, these findings correlated with significantly elevated levels of nephrotoxicity biomarkers, lending further support for the need to further scrutinize the safety of this herbal dietary supplement.

The applicability of immunotherapy would be dramatically broadened to a greater number of recipients if direct “off-the-shelf” products could be engineered to engender functionally potent immune responses against true “self”-tumor antigens. This would obviate the need for ex vivo culture of dendritic cells or T cells on a patient-by-patient basis, for example. The carcinoembryonic antigen (CEA) is a glycoprotein expressed in normal gut epithelium that is up-regulated in the majority of colon cancers, non-small cell lung cancers, and half of all breast cancers. Such properties make CEA an excellent and important target for cancer immunotherapy. In this study, we show stabilization of 14-day established s.c. mGC4CEA tumors in human CEA (huCEA) transgenic mice following two direct low-dose injections of 0.15 × 106 transducing units of a lentiviral vector (LV) that directs expression of huCEA (LV-huCEA). This stabilization result was reproducible and detailed analyses including antibody assays, multiplex cytokine analyses on unstimulated splenocytes, lymph node cell characterizations, tetramer staining, and immunofluorescence staining of tumor sections showed that this outcome correlated with both a cellular and humoral immune response. Similar tumor outcomes were not seen when mice were vaccinated with a control LV that engineered expression of enGFP only. The long-term potency of this vaccination strategy was also studied and revealed the requirement for maintenance of tumor antigen-specific immunity for efficient tumor control. These data support the use of direct injections of low doses of LV-huCEA for enhancement of tumor immunotherapy directed against CEA. [Mol Cancer Ther 2009;8(3):OF692–11]

Gene therapy strategies may accelerate the development of prophylactic immunotherapy against cancer. We synthesized a lentiviral (LV) vector encoding a kinase-deficient form of erbB2 (erbB2tr) to transduce murine dendritic cells (DCs) efficiently. Murine erbB2 models a clinically relevant tumor-associated self-antigen; its human homolog (HER-2/neu) is overexpressed in breast cancer and in 80% of metastatic prostate cancers. Following one infection, approximately 47% of DCs overexpressed erbB2tr. To determine whether low doses of transduced DCs could protect mice from prostate cancer cells, we performed prime/boost vaccinations with 2 x 10(3) or 2 x 10(5) erbB2tr-transduced DCs. Six weeks after vaccination, mice were simultaneously bilaterally challenged with the aggressive RM-1 prostate cancer cell line and an erbB2tr-expressing variant (RM-1-erbB2tr). Whereas control mice developed both tumors, all recipients of 2 x 10(5) erbB2tr-transduced DCs developed only wild-type RM-1 tumors. One-third of mice vaccinated with just 2 x 10(3) erbB2tr-transduced DCs also demonstrated erbB2tr-specific tumor protection. Protection against RM-1-erbB2tr tumors was associated with sustained levels of anti-erbB2tr antibody production and also correlated with erbB2tr-specific Th1 cytokine secretion. Depletion of CD4(+), CD8(+), or natural killer (NK) cells prior to tumor challenge underscored their role in mediating tumor protection. We conclude that administration of DCs expressing a self-antigen through efficient LV-based gene transfer activates cellular and humoral immunity, protecting host animals against specific tumor challenge.

Purpose: We hypothesized that lymphoid-selective host conditioning and subsequent adoptive transfer of sirolimus-resistant allogeneic T cells (T-Rapa), when combined with high-dose sirolimus drug therapy in vivo, would safely achieve antitumor effects while avoiding GVHD. Experimental Design: Patients (n = 10) with metastatic renal cell carcinoma (RCC) were accrued because this disease is relatively refractory to high-dose conditioning yet may respond to high-dose sirolimus. A 21-day outpatient regimen of weekly pentostatin (P; 4 mg/m2/dose) combined with daily, dose-adjusted cyclophosphamide (C; ≤200 mg/d) was designed to deplete and suppress host T cells. After PC conditioning, patients received matched sibling, T-cell–replete peripheral blood stem cell allografts, and high-dose sirolimus (serum trough target, 20–30 ng/mL). To augment graft-versus-tumor (GVT) effects, multiple T-Rapa donor lymphocyte infusions (DLI) were administered (days 0, 14, and 45 posttransplant), and sirolimus was discontinued early (day 60 posttransplant). Results: PC conditioning depleted host T cells without neutropenia or infection and facilitated donor engraftment (10 of 10 cases). High-dose sirolimus therapy inhibited multiple T-Rapa DLI, as evidenced by stable mixed donor/host chimerism. No antitumor responses were detected by RECIST criteria and no significant classical acute GVHD was observed. Conclusions: Immune-selective PC conditioning represents a new approach to safely achieve alloengraftment without neutropenia. However, allogeneic T cells generated ex vivo in sirolimus are not resistant to the tolerance-inducing effects of in vivo sirolimus drug therapy, thereby cautioning against use of this intervention in patients with refractory cancer. Clin Cancer Res; 21(19); 4312–20. ©2015 AACR.

In order to design and validate a method to identify virulence genes of Salmonella typhimurium using DNA microarray, a protocol was developed to label the isolated bacterial DNA directly and to use PCR amplification of limited numbers of genes to validate the hybridization signals. Therefore, a DNA microarray chip of 71 virulence genes of S. typhimurium was developed and evaluated using 10 isolates. Each gene was represented by 65 bp oligonucleotide probes (oligoprobes) and immobilized on the surface of chemically modified slides. Whole DNA genomes were digested with Hin f1 and Sau 3AI, labeled with a fluorescent tag of Cy3 and then hybridized. The presence of virulence genes in 10 strains of S. typhimurium was established by measuring a fluorescent signal above the background noise of the chip. PCR amplification of 10 genes ( orgA , ORF319, ttrB, rmbA, misL, spi4F, spi4H, spi4N, rRNA, and purR ) of S. typhimurium was used as a standard to verify the confidence level of the DNA microarray chip. In conclusion, using PCR amplification to increase the confidence level of the microarray hybridization data was successful.

Background: Renal and hepatotoxicity of diglycolic acid (DGA) has been described with in vitro cellular models as well as in vivo animal and human systems. The possibility of DGA being toxic

Background aims. We completed a phase II clinical trial evaluating rapamycin-resistant allogeneic T cells (T-rapa) and now have evaluated a T-rapa product manufactured in 6 days (T-rapa6) rather than 12 days (T-Rapa12). Methods. Using gene expression microarrays, we addressed our hypothesis that the two products would express a similar phenotype. The products had similar phenotypes using conventional comparison methods of cytokine secretion and surface markers. Results. Unsupervised analysis of 34,340 genes revealed that T-rapa6 and T-rapa12 products clustered together, distinct from culture input CD4 þ T cells. Statistical analysis of T-rapa6 products revealed differential expression of 19.3% of genes (n ¼ 6641) compared with input CD4 þ cells; similarly, 17.8% of genes (n ¼ 6147) were differentially expressed between T-rapa12 products and input CD4 þ cells. Compared with input CD4 þ cells, T-rapa6 and T-rapa12 products were similar in terms of up-regulation of major gene families (cell cycle, stress response, glucose catabolism, DNA metabolism) and down-regulation (inflammatory response, immune response, apoptosis, transcriptional regulation). However, when directly compared, T-rapa6 and T-rapa12 products showed differential expression of 5.8% of genes (n ¼ 1994; T-rapa6 vs. T-rapa12). Conclusions. Second-generation T-rapa6 cells possess a similar yet distinct gene expression profile relative to first-generation T-rapa12 cells and may mediate differential effects after adoptive transfer.

Purpose: Metastatic breast cancer (MBC) response to allogeneic lymphocytes requires donor T-cell engraftment and is limited by graft-versus-host disease (GVHD). In mice, type-II–polarized T cells promote engraftment and modulate GVHD, whereas type-I–polarized T cells mediate more potent graft-versus-tumor (GVT) effects. This phase I translational study evaluated adoptive transfer of ex vivo costimulated type-I/type-II (T1/T2) donor T cells with T-cell–depleted (TCD) allogeneic stem cell transplantation (AlloSCT) for MBC. Experimental Design: Patients had received anthracycline, taxane, and antibody therapies, and been treated for metastatic disease and a human leukocyte antigen (HLA)-identical–sibling donor. Donor lymphocytes were costimulated ex vivo with anti-CD3/anti-CD28 antibody–coated magnetic beads in interleukin (IL)-2/IL-4–supplemented media. Patients received reduced intensity conditioning, donor stem cells and T1/T2 cells, and monitoring for toxicity, engraftment, GVHD, and tumor response; results were compared with historical controls, identically treated except for T1/T2 product infusions. Results: Mixed type-I/type-II CD4+ T cells predominated in T1/T2 products. Nine patients received T1/T2 cells at dose level 1 (5 × 106 cells/kg). T-cell donor chimerism reached 100% by a median of 28 days. Seven (78%) developed acute GVHD. At day +28, five patients had partial responses (56%) and none had MBC progression; thereafter, two patients had continued responses. Donor T-cell engraftment and tumor responses appeared faster than in historical controls, but GVHD rates were similar and responders progressed early, often following treatment of acute GVHD. Conclusion: Allogeneic T1/T2 cells were safely infused with TCD-AlloSCT, appeared to promote donor engraftment, and may have contributed to transient early tumor responses. Clin Cancer Res; 17(21); 6878–87. ©2011 AACR.

Purpose: Metronomic chemotherapy is a minimally toxic and frequently effective new treatment strategy that is beginning to show promising phase II clinical trial results, particularly for metastatic breast cancer when combined with various molecularly targeted antitumor agents. Here, we assessed a treatment strategy that uses trastuzumab plus daily oral metronomic cyclophosphamide on metastatic Her-2–positive human breast cancer models. Experimental Design: Treatments were initiated on orthotopic transplanted primary tumors as well as established visceral metastatic disease of two independent Her-2–positive breast cancer models, both independently derived from the human MDA-MB-231 breast cancer cell line. Outcome was assessed by noninvasive measurements of tumor cell–secreted human choriogonadotropin in the urine as a surrogate marker of relative tumor burden, or by whole body bioluminescent imaging, in addition to prolongation of survival. Results: Orthotopic primary tumors responded to trastuzumab monotherapy with significant growth delays, whereas minimal antitumor effect was observed when mice with metastatic disease were treated. Nevertheless, trastuzumab showed a benefit in this latter setting when combined with metronomic low-dose cyclophosphamide as assessed by prolongation of survival. This benefit was similar to trastuzumab plus maximum tolerated dose cyclophosphamide, but was associated with lesser toxicity. Conclusions: Trastuzumab combined with metronomic cyclophosphamide may be an effective long-term maintenance strategy for the treatment of Her-2–positive metastatic breast cancer. (Clin Cancer Res 2009;15(20):6358–66)

Historically, the use of mouse models of metastatic disease to evaluate anticancer therapies has been hampered because of difficulties in detection and quantification of such lesions without sacrificing the mice, which in turn may also be dictated by institutional or ethical guidelines. Advancements in imaging technologies have begun to change this situation. A new method to non-invasively measure tumor burden, as yet untested to monitor spontaneous metastases, is the use of transplanted tumors expressing secretable human β-chorionic gonadotropin (β-hCG) that can be measured in urine. We describe examples of β-hCG–transfected tumor cell lines for evaluating the effect of different therapies on metastatic disease, which in some cases involved monitoring tumor growth for >100 days. We used β-hCG–tagged mouse B16 melanoma and erbB-2/Her-2–expressing human breast cancer MDA-MB-231 models, and drug treatments included metronomic low-dose cyclophosphamide chemotherapy with or without a vascular endothelial growth factor receptor 2–targeting antibody (DC101) or trastuzumab, the erbB-2/Her-2–targeting antibody. Both experimental and spontaneous metastasis models were studied; in the latter case, an increase in urine β-hCG always foreshadowed the development of lung, liver, brain, and kidney metastases. Metastatic disease was unresponsive to DC101 or trastuzumab monotherapy treatment, as assessed by β-hCG levels. Our results also suggest that β-hCG levels may be set as an end point for metastasis studies, circumventing guidelines, which have often hampered the use of advanced disease models. Collectively, our data indicates that β-hCG is an effective noninvasive preclinical marker for the long term monitoring of untreated or treated metastatic disease. [Mol Cancer Ther 2008;7(10):3452–9]

alphabetaTCR(+)CD4(-)CD8(-) double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress antigen-specific immune responses mediated by CD8(+) and CD4(+) T cells in mice and humans. In this study, we developed a system to expand DN Treg cells for transplantation therapy that exclusively uses recipient-derived immune cells and confers a high degree of safety as the protocol does not involve the direct injection of lentiviral vectors. Recipient-derived dendritic cells (DCs) were transduced with lentiviral vectors that express major histocompatibility complex class I L(d) antigen (LV-L(d)), which is expressed by the donor graft but is allogeneic to the graft recipient. LV-L(d)-transduced mature DCs (mDCs) were able to expand effectively both FcRgamma(-/-) and FcRgamma(+/+) DN T cells. After expansion with LV-L(d)-transduced mDCs, only the FcRgamma(+/+) DN Treg cells maintained their ability to suppress CD8(+) T cells in vitro. In addition, adoptive transfer of the FcRgamma(+/+) ex vivo expanded DN Treg cells significantly prolonged the survival of L(d+) skin grafts. This study is the first description of successful ex vivo expansion of antigen-specific DN Treg cells using genetically modified syngeneic DCs for adoptive immunotherapy and demonstrates that although FcRgamma(-/-) DN T cells can be expanded, they do not gain regulatory ability.

The immune system uses a process known as 'immunosurveillance' to help prevent the outgrowth of tumors. In cancer immunotherapy, a major goal is for immunity against tumor-associated antigens to be generated or strengthened in patients. To achieve this goal, several approaches have been tested, including the use of highly potent antigen-presenting cells called dendritic cells (DCs), which can activate T cells efficiently. Presentation of peptides derived from tumor antigens on the surface of DCs can stimulate strong antitumor immunity. Using recombinant viral vectors encoding tumor-associated antigens, DCs can be engineered efficiently to express sustained levels of tumor-antigen peptides. This review discusses the effectiveness of virally transduced DCs in treating tumors and generating antigen-specific T-cell responses. It covers mouse and nonhuman primate studies, preclinical in vitro human cell experiments and clinical trials.

Purpose: Acquired resistance to trastuzumab (Herceptin) is common in patients whose breast cancers show an initial response to the drug. The basis of this acquired resistance is unknown, hampering strategies to delay or treat such acquired resistance, due in part to the relative lack of appropriate in vivo tumorigenic models.Experimental Design: We derived an erbB-2–positive variant called 231-H2N, obtained by gene transfection from the highly tumorigenic erbB-2/HER2–negative human breast cancer cell line, MDA-MB-231. Unlike MDA-MB-231, the 231-H2N variants was sensitive to trastuzumab both in vitro and especially in vivo, thus allowing selection of variant resistant to drug treatment in the latter situation after showing an initial response.Results: The growth of established orthotopic tumors in severe combined immunodeficient mice was blocked for 1 month by trastuzumab, after which rapid growth resumed. These relapsing tumors were found to maintain resistance to trastuzumab, both in vitro and in vivo. We evaluated various therapeutic strategies for two purposes: (a) to delay such tumor relapses or (b) to treat acquired trastuzumab resistance once it has occurred. With respect to the former, a daily oral low-dose metronomic cyclophosphamide regimen was found to be particularly effective. With respect to the latter, an anti–epidermal growth factor receptor antibody (cetuximab) was effective as was the anti–vascular endothelial growth factor (anti-VEGF) antibody bevacizumab, which was likely related to elevated levels of VEGF detected in trastuzumab-resistant tumors.Conclusions: Our results provide a possible additional rationale for combined biological therapy using drugs that target both erbB-2/HER2 and VEGF and also suggest the potential value of combining less toxic metronomic chemotherapy regimens not only with targeted antiangiogenic agents but also with other types of drug such as trastuzumab.

471 Lymphoma progression remains an obstacle after allogeneic HCT, particularly after non-myeloablative conditioning in patients with high-risk histology (non-indolent; Kahl et al; Blood, 2007) and chemotherapy refractory disease (Bishop et al; Cancer, 2010). In murine models, rapamycin-resistant T cells favorably modulated the balance between GVHD, graft rejection, and GVT effects. To translate this, we conducted a multi-center phase II trial (NCT0074490) of T-Rapa cells infused as a pre-emptive DLI after low-intensity allogeneic HCT; here, we report overall outcome of patients with high-risk lymphoma diagnoses, many of whom were chemotherapy refractory. T-Rapa cells were manufactured by ex vivo culture of donor CD4+ T cells using CD3/CD28 co-stimulation and IL-4, IL-2, and rapamycin for 12-days (T-Rapa12) or 6-days (T-Rapa6), and administered (2.5 × 107 cells/kg) at d14 post-HCT. Both populations of T-Rapa cells expressed a mixed Th2/Th1 phenotype with minimal T-Reg content. Patients (n=42) received outpatient-intensity chemotherapy (typically, EPOCH-FR) until CD4 count was < 200 cells/μl, and then received an HLA-matched sibling mobilized allograft and GVHD prophylaxis of cyclosporine plus short-course sirolimus (to d14 post-HCT); conditioning consisted of fludarabine (120 mg/m2) and cyclophosphamide (1200 mg/m2). Table I details the high-risk diagnoses, pre-treatment history (median of 4 prior regimens), remission status at time of HCT (7/42 [17%] in CR), and presence of chemotherapy refractory disease (stable or progressive disease to prior therapy: 23/42 pts, 55%). T-Rapa cell infusion was relatively safe, with no engraftment syndrome or d100 TRM; incidences of grade II-IV acute, late acute, and classical chronic GVHD were 17%, 34%, and 36%, respectively. Initial mixed donor/host chimerism converted to predominate donor chimerism after T-Rapa cell DLI (Table I). Overall median survival probability at 24 months post-HCT is 85.7% for patients with chemotherapy sensitive disease vs. 39.1% for patients with chemotherapy refractory disease ([Fig. 1][1]; p=0.0008). All deaths were due to progressive disease except for 2 infection-related deaths at days 162 and 359 post-HCT; all surviving patients are in CR. Survival was not statistically significantly influenced by DLI type (T-Rapa12 vs. T-Rapa6) or histology-type (NHL vs. HD). Pre-emptive DLI using donor T-Rapa cells after low-intensity conditioning is safe and very effective in patients with high-risk lymphoma diagnoses and chemotherapy sensitive disease; for such patients, future randomized trials should compare low-intensity T-Rapa cell therapy to other transplantation regimens. For patients with high-risk lymphoma diagnoses and chemotherapy-refractory disease, we are evaluating a modification to the current platform that incorporates high-dose sirolimus therapy. | Patient Characteristics | Chimerism Results[c][2] | CD3 | CD15 | |:----------------------------------:| ----------------------- | ---------------------- | ------------------ | ----------- | | Number of Patients | n = 42 | Day 14 post-HCT | 57 (12-97) | 46 (8-94) | | Age (median, range) | 44 (23-68) | Day 28 post-HCT | 77 (37-100) | 76 (29-98) | | Sex (male/female) | (26/16) | | | | | # of Prior Therapies (mean, range) | 4 (1-6) | Day 100 post-HCT | 89 (51-100) | 93 (35-100) | | Histology[a][3] | n=26 total (62%) | Survival Results[d][4] | 24 Mo. Surv. Prob. | | NHL Category | n=12 | ChemoSens/T-R12 | 78.6% (n=7) | | DLBCL | n=5 | ChemoSens/T-R6 | 85.7% (n=12) | | DLBCL-tr | n=2 | ChemoRefr/T-R12 | 41.7% (n=11) | | DLBCL-EBV | n=3 | ChemoRefr/T-R6 | 36.4% (n=12) | | PlasmacytoidDC | n=3 | All NHL | 57.4% (n=26) | | T Cell | n=16 total (38%) | All HD/Grey Zone | 68.8% (n=16) | | HD Category | n=12 | | | | HD | n=4 | | | | Grey Zone | | | | | Chemo. Response[b][5] | 23/42 (55%) | | | | Refractory (SD/PD) | 19/42 (45%) | | | | Sensitive (PR/CR) | | | | | Complete Remission | 7/42 (17%) | | | | At Time of HCT | | | | * DLBCL, diffuse large B cell lymphoma; DLBCL-tr, transformed; DLBCL-EBV, Epstein Barr Virus related; Plasmacytoid DC, dendritic cell; HD, Hodgkins Disease; SD, stable disease; PD, progressive disease; PR/CR is partial or complete response. * [↵][6]a Grey Zone Lymphoma and Hodgkins Disease were pooled for statistical analyses. * [↵][7]b Chemotherapyresponse, as determined after last regimen prior to study entry. * [↵][8]c Chimerism determined by VNTR-PCR at days 14, 28, and 100 post-HCT (mean and range of values shown); CD3, T cell chimerism; CD15, myeloid cell chimerism. * [↵][9]d Survival by Kaplan-Meier analysis; 24 month median survival probability values shown; T-R12 and T-R6 indicates recipient of T-Rapa cells manufactured for 12 days vs. 6 days. Table I ![Figure][10] Disclosures: Levine: TxCell: Consultancy, Membership on an entity's Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June: Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties. Mato: Celgene, Milennium, Genentech, Seattle Genetics: Speakers Bureau. [1]: #F1 [2]: #fn-4 [3]: #fn-2 [4]: #fn-5 [5]: #fn-3 [6]: #xref-fn-2-1 [7]: #xref-fn-3-1 [8]: #xref-fn-4-1 [9]: #xref-fn-5-1 [10]: pending:yes

Abstract 352 Ex-vivo expansion of murine donor CD4+ T cells using co-stimulation, IL-4, and rapamycin generated a T cell population (T-rapa cells) that beneficially modulated GVHD, graft rejection, and GVT effects. We thus conducted a clinical trial to evaluate T-rapa cell infusion after HLA-matched sibling allogeneic HCT. In one trial arm, T-rapa cell infusion (2.5 × 107 cells/kg; d 14 post-HCT) safely accelerated alloengraftment after low-intensity host conditioning, as evidenced by: conversion of mixed chimerism to predominant donor chimerism in the majority of patients by d 100 post-HCT and a low rate of grade II to IV acute GVHD (10%; 4/40 cases). This abstract characterizes the T-rapa clinical products (n=48), particularly with respect to the balance of Th1, Th2, and Treg cells and the magnitude of effector function (cytokine secretion); this latter aspect is relevant because in animal models, T cells of limited differentiation mediate increased in vivo effects upon adoptive transfer. Transplant donors underwent steady-state leukapheresis. CD4+ T cells were then purified (Miltenyi.. CliniMACS) and expanded in Lifecell.. bags for 12 days using co-stimulation (anti-CD3, anti-CD28 coated magnetic beads) and media containing rhIL-2, rhIL-4, and rapamycin. T-rapa products contained minimal cells of naive phenotype (CD45RA+ cells: 2 ± 0.4%) and were comprised of both central memory cells (CCR7+: 28 ± 2%) and effector memory cells (CCR7−: 67 ± 2%). Phenotyping assays were performed on T-rapa clinical products at d 12 of culture (time of cell product infusion); in addition, to assess phenotype stability, assays were performed after an additional 6 days of culture after co-stimulation without IL-4 and rapamycin (“day 18”). For comparison, four separate CD4+ T cell culture conditions were established from each of n=8 normal donors. For these control cultures, CD4+ T cells were co-stimulated and propagated for 12 days in tissue culture flasks using: (1) IL-2, IL-4 (“Th2”); (2) IL-2, IL-4 plus rapamycin (“T-rapa”); (3) IL-2, IFN-a (“Th1”); and (4) IL-2, IFN-a plus rapamycin (“Th1-rapa”). Phenotyping results are shown in Fig. 1. In the four flask cultures, there was modest skewing of the Th2/Th1 balance, as indicated by relatively comparable expression of the Th2 transcription factor GATA-3 and the Th1 transcription factor T-bet by intra-cellular flow cytometry (Fig. 1A). In marked contrast, day 12 T-rapa clinical products expressed a highly polarized Th2/Th1 balance (GATA-3/T-bet ratio of 28 ± 9 [mean ± SEM]); this Th2/Th1 balance was relatively stable after additional culture without IL-4 and rapamycin (“Day 18”). The median frequency of transcription factor expression was 11.5% for GATA-3 (range: 3–37%), 5.1% for T-bet (range: 0–18%), and < 1% expression of the Treg transcription factor FoxP3 (range: 0–0.7%). T-rapa clinical products were evaluated for cytokine secretion in response to co-stimulation at day 12 and day 18 of culture; supernatants were tested for Th2 cytokines (Fig. 1B) and Th1/Th17 cytokines (Fig. 1C). Day 12 T-rapa clinical products secreted each Th2 cytokine measured. The magnitude (pg/ml) of T-rapa cell Th2 cytokine secretion was approximately 2-log reduced relative to control Th2 cells; day 18 T-rapa cell secretion of Th2 cytokines was increased relative to day 12 values, but still reduced relative to control Th2 cells. Day 12 T-rapa clinical products did not secrete IL-17 and secreted low levels of IFN-g, IL-2, and TNF-a (1-3 log reduced relative to Th1 control cells). Day 18 T-rapa cell secretion of IFN-g and TNF-a was increased relative to day 12 values, but still reduced relative to control Th1 cells. Remarkably, day 18 T-rapa cell secretion of IL-2 was greatly diminished relative to day 12 values, and IL-17 secretion remained at minimal levels. In conclusion, T-rapa cell clinical products are comprised of a balance of Th2 and Th1 effector CD4+ T cells, with minimal contamination from Treg or Th17 cells. The T-rapa cell clinical products possessed limited differentiation plasticity and secreted low levels of Th2 and Th1 cytokines. Adoptive transfer of a balance of minimally differentiated and fixed polarity donor Th2/Th1 cells represents a novel approach to safely accelerate alloengraftment after low-intensity conditioning. Disclosures: No relevant conflicts of interest to declare.

Abstract 521 Ex-vivo culture of murine donor CD4+ T cells using rapamycin, co-stimulation, and IL-4 yielded a defined T cell population (T-rapa cells) that favorably modulated the balance between GVHD, graft rejection, and GVT effects. To translate these findings, we conducted a multi-center clinical trial (NCT0074490) to evaluate T-rapa cell therapy after allogeneic HCT. T-rapa cells were manufactured by ex vivo culture of donor CD4+ T cells using CD3/CD28 co-stimulation in media containing IL-4, IL-2, and rapamycin. T-rapa cells had a mixed Th2/Th1 phenotype with minimal Treg content (intra-cellular flow, n=48 products; median transcription factor expression: 11.5% [GATA-3], 5.1% [T-bet], and 0.1% [FoxP3]). Median T-rapa cell cytokine secretion (pg/ml; re-stimulation at harvest) was 1.3 [IL-4], 20.6 [IL-5], 9.7 [IL-10], 23.7 [IL-13], 34.7 [IFN-g], and 17.1 [IL-2]. Patients received an HLA-matched sibling, T cell-replete, G-CSF mobilized allograft, and GVHD prophylaxis of cyclosporine plus short-course sirolimus (to d14 post-HCT). Two protocol arms evaluated T-rapa cell therapy after induction chemotherapy and outpatient, low-intensity preparative chemotherapy (Table I). First, patients (n=25) were accrued to arm A to evaluate T-rapa infusion at d +14 post-HCT; subsequently, accrual was initiated to arm B (n=25) to evaluate T-rapa infusion on d0 of HCT. Arm A was then expanded to n=40 patients. Patients accrued to arms A and B were similar for recipient age, high-risk malignancy diagnosis, chemotherapy refractoriness, and prior regimen number (Table I). Most recipients were not in remission at the time of HCT. High-risk NHL was the most frequent diagnosis (25/65 patients), followed by non-high-risk NHL (11/65), AML/MDS (8/65), myeloma (7/65), CLL (6/65), Hodgkin's disease (5/65), and CML (3/65). Arm A and B recipients had similar mean donor myeloid cell chimerism at d +14, +28, and +100 (arm A, 43%, 74%, and 89%; arm B, 50%, 62%, and 84%). At d +14, arm A and B recipients also had mixed donor T cell chimerism (mean values, 60% in each arm; Table I). At d +28 and +100, T cell chimerism increased in arm A to 80% and 89%; in arm B, these values increased to only 67% and 69%. Four recipients on arm B had < 10% donor T cell chimerism at d +100; in contrast, the lowest donor T cell chimerism value observed at d +100 on arm A was 36%. T-rapa therapy on arm A was relatively safe as there was: no engraftment syndrome, a 10% rate of acute grade II to IV GVHD, a 67% incidence of chronic GVHD, and no transplant-related mortality (Table I). On arm A, 37.5% (15/40) of recipients are in sustained complete remission, with a median survival probability of 63.6% at 24 months post-HCT. Therefore, pre-emptive donor lymphocyte infusion with ex-vivo manufactured T-rapa cells that express a balanced Th2/Th1 effector phenotype represents a novel approach to safely accelerate alloengraftment and harness allogeneic GVT effects after low-intensity conditioning.Table IArm AArm BLow-Intensity Regimen Induction Chemotherapy1EPOCH-FREPOCH-FR 2Terminal Chemotherapy3Flu (120 mg/m2)EPOCH-FRCy (1200 mg/m2)T-Rapa Cell TimingD +14 post-HCTD 0 of HCTPatient Characteristics & of Patients Accrued4025 Age (median, range)55 (25–67)51 (32–66) & of Prior Regimens3 (1–6)3 (1–8) High-Risk Malignancy65% (26/40)52% (13/25) Chemotherapy Refractory50% (20/40)48% (12/25) CR (at time of HCT)25% (10/40)8% (2/25)% Donor T Cell ChimerismMean Median (Range)Mean Median (Range)Day 14 post-HCT6061(8–97)6060(4–100)Day 28 post-HCT8089(27–100)6773(10–100)Day 100 post-HCT8993(36–100)6982(0–100)Clinical Results Engraftment Syndrome0% (0/40)0% (0/25) Acute GVHD10% (4/40)23% (5/22) Chronic GVHD67% (22/33)75% (15/20) Complete Remission38% (15/40)28% (7/25) Transplant-related Mortality0% (0/40)0% (0/25) Percent Survival65% (26/40)40% (10/25) Median Survival27.5 mo11.2 mo Survival Prob. at 24 mo63.6%44.0%1EPOCH-FR, EPOCH with fludarabine (Flu) and rituximab.2Terminal (preparative) chemotherapy administered one week prior to HCT.3Flu/Cy [cyclophosphamide] doses are total doses, given over 4 days (Cy dose is 75% lower than 4800 mg/m2 “reduced-intensity” Cy dose). Disclosures: No relevant conflicts of interest to declare.

Anti-tumor immunotherapy is difficult to achieve in vivo in part due to naturally occurring peripheral tolerance. Our laboratory has developed a potent immunotherapy strategy that uses unusually low doses of dendritic cells (DCs) to break self-tolerance in a mouse model of prostate cancer. We constructed a lentiviral vector (LV) encoding a truncated form of the true self-antigen murine erbB2 (LV/erbB2tr). This protein is the murine form of HER-2/neu, a tumor associated antigen (TAA) upregulated in 20% of primary prostate tumors and 80% of metastatic cases. With LV/erbB2tr, we achieved highly efficient gene transfer into DCs that were generated from donor murine bone marrow, as up to 47% of DCs over-expressed erbB2tr following one infection (MOI of 3). Similar transduction levels were achieved using a control enGFP LV. The purity of transduced DCs was confirmed by flow cytometry for CD11c, CD80, CD86, and I-Ab expression. We then tested the ability of two low dose inoculations of these transduced DCs to overcome self-tolerance to erbB2 and protect against specific challenge in a bilateral tumor model. Mice were vaccinated twice, two weeks apart by administration of either 2 × 105 or 2 ×103 cells (i.p.). Six weeks later, the mice were injected on one hind flank with the highly aggressive murine tumor cell line RM1 and on the opposite flank with erbB2tr-transduced RM1 (RM1-erbB2tr). We saw complete protection from RM1-erbB2tr tumors in 100% of the mice receiving 2 × 105 erbB2tr-transduced DCs (n=6). Control RM1 tumors were not rejected. No systemic auto-immune responses to the self-antigen were observed, as all mice survived and the procedure was well tolerated. Furthermore, our strategy was effective even using 100-fold fewer DCs in the inoculations. In the mice receiving 2 ×103 erbB2tr-transduced DCs, 33% (2/6 animals) were completely protected from developing RM1-erbB2tr tumors and 2 others showed near-complete protection. At regular points throughout the experiment, serum levels of anti-erbB2tr antibodies were measured. In mice receiving the 2 ×105 DC dose, a peak antibody response was seen 1 week after the second immunization (4.6-fold above controls). Long-term antibody responses were detected beyond 6-weeks post-vaccination. T cell responses are being assessed by cytokine (IFN-γ, IL-2, IL-4, IL-5, and IL-10) secretion assays. Cytokine specificity data are pending. We speculated that the antigen-specific anti-tumor effect might have been modulated, in part, by apoptosis of regulatory T (Treg) cells that help maintain peripheral tolerance. Indeed, CD4+CD25+Foxp3+ Treg cells were consistently reduced in mice vaccinated with erbB2tr-transduced DCs, as shown by flow cytometry. Together these experiments show that we can overcome tolerance to an endogenously-expressed TAA, even using very low doses of erbB2-transduced DCs. Future studies will focus on testing our DC therapy in mice with established prostate tumors for a more clinically applicable model.

We describe a novel animal model of nonmyeloablative bone marrow transplantation (BMT) using the purine analog pentostatin. Other cohorts of mice received another purine analog, fludarabine, which we and others have previously evaluated in nonmyeloablative murine models. We evaluated pentostatin for its ability to (1) operate synergistically with cyclophosphamide to induce host T cell depletion; (2) induce host T cell suppression, as defined by modulation of cytokine secretion in vitro and abrogation of host-versus-graft reactivity in vivo; (3) constrain host T cell recovery post-therapy; and (4) prevent the rejection of T cell-depleted, fully major histocompatibility complex-mismatched bone marrow allografts. Relative to single-agent regimens, combination regimens with pentostatin and cyclophosphamide (PC) and with fludarabine and cyclophosphamide (FC) worked synergistically to deplete host CD4(+) and CD8(+) T cells. PC and FC regimens were developed that yielded similar levels of host T cell and myeloid cell depletion. In the setting of these generally comparable states of host T cell and myeloid cell depletion, the PC regimen was found to be highly immunosuppressive, as evidenced by a reduced host T cell capacity to secrete interleukin-2 and interferon-γ in vitro, to mediate host-versus-graft reactivity in vivo, and to recover numerically and functionally during a 2-week observation period after chemotherapy. Finally, using B6 hosts treated with the 14-day chemotherapy regimens, the PC regimen more consistently prevented the rejection of BALB/c T cell-depleted allografts compared with the FC regimen (rate of alloengraftment, 14/15 [93%] of PC-treated recipients vs 8/14 [57%] of FC-treated recipients; P.05); similar results were observed using an 8-day conditioning regimen. These data suggest that host T cell suppression, distinct from T cell depletion, may be a critical determinant of engraftment after purine analog-based regimens and also may be preferentially attained by the use of pentostatin.