Twenty-seven subjects with squamous cell cancer of the head and neck received the neoadjuvant IRX-2 immunotherapy regimen prior to surgery in a Phase 2 trial. Pretreatment tumor biopsies were compared with the primary tumor surgical specimens for lymphocyte infiltration, necrosis and fibrosis, using hematoxylin and eosin stain and immunohistochemistry in 25 subjects. Sections were examined by three pathologists. Relative to pretreatment biopsies, increases in lymphocyte infiltration (LI) were seen using H and E or immunohistochemistry. CD3+ CD4+ T cells and CD20+ B cells were primarily found in the peritumoral stroma and CD3+ CD8+ T cells and CD68+ macrophages were mainly intratumoral. LI in the surgical specimens were associated with reductions in the primary tumor size. Improved survival at 5 years was correlated with high overall LI in the tumor specimens. Neoadjuvant IRX-2 immunotherapy regimen may restore immune responsiveness presumably by mobilizing tumor infiltrating effector lymphocytes and macrophages into the tumor.

Background. Cellular immune suppression is observed in head and neck squamous cell cancer (HNSCC) and contributes to poor prognosis. Restoration of immune homeostasis may require primary cell-derived cytokines at physiologic doses. An immunotherapy regimen containing a biologic, with multiple-active cytokine components, and administered with cytoxan, zinc, and indomethacin was developed to modulate cellular immunity.Methods. Study methods were designed to determine the safety and efficacy of a 21-day neoadjuvant immunotherapy regimen in a phase 2 trial that enrolled 27 therapy-naive patients with stage II to IVa HNSCC. Methods included safety, clinical and radiologic tumor response, disease-free survival (DFS), overall survival (OS), and tumor lymphocytic infiltrate (LI) data collection.Results. Acute toxicity was minimal. Patients completed neoadjuvant treatment without surgical delay. By independent radiographic review, 83% had stable disease during treatment. OS was 92%, 73%, and 69% at 12, 24, and 36 months, respectively. Histologic analysis suggested correlation between survival and tumor LI.Conclusion. Immunotherapy regimen was tolerated. Survival results are encouraging. (C) 2011 Wiley Periodicals, Inc. Head Neck 33: 1666-1674, 2011

Objectives IRX-2 is a novel immunotherapeutic containing physiologic quantities of several cytokines which protects human T lymphocytes from tumor-induced or drug-induced apoptosis. Here, we investigate the mechanisms responsible for IRX-2-mediated protection of T lymphocytes exposed to tumor-derived microvesicles (TMV). Methods Jurkat cells or primary human T cells ± IRX-2 were co-incubated with TMV and then examined by flow cytometry or Western blots for expression of molecules regulating cell survival (FLIP, Bcl-2, Bcl-xL, Mcl-1) or death (Fas, caspase 8, caspase 9, Bax, Bid). ANX V binding, caspase activation or cytochrome c release were also measured ± cycloheximide (CHX) or ± the Akt-specific inhibitor. Jurkat cells transfected with the cFLIP gene were used to evaluate the role of cFLIP in IRX-2-mediated protection. Effects of CHX on IRX-2-mediated protection and activation of NF-κB upon the TMV/IRX-2 treatment were also measured. Results IRX-2 protected T cells from apoptosis by preventing Fas overexpression induced by TMV and blocking caspase 8 activation by up-regulating cFLIP. Jurkat cells overexpressing cFLIP were more resistant to TMV-induced apoptosis than the mock-transfected cells ( p < 0.02). Signaling via the PI3K/Akt pathway, IRX-2 corrected the imbalance of pro- versus anti-apoptotic proteins induced by TMV and promoted NF-κB translocation to the nucleus. CHX abolished IRX-2-mediated protection in T cells, suggesting that IRX-2 induces de novo synthesis of one or more proteins that are required for protection. Conclusions This biologic may be therapeutically useful for protection of activated T cells from tumor-induced immune suppression and death.

Objectives:Head and neck squamous cell carcinoma (HNSCC) is associated with profound defects in cellular immunity. IRX-2, a primary cell-derived biologic containing multiple cytokines, has enhanced immune responses and induced tumor rejection in preclinical studies. This phase 1 open label study aimed to determine the clinical and laboratory safety of an IRX-2 regimen in patients with HNSCC. Methods:Patients with HNSCC who had failed surgery and/or radiation therapy were enrolled. IRX-2 was injected subcutaneously at 115 units per dose, 2 doses/d over 10 days, starting on day 4. Patients received low-dose cyclophosphamide infusion on day 1 and took oral indomethacin and zinc daily from day 1 through day 21. Safety and laboratory assessments were undertaken throughout the treatment and 4 weeks after completion of the regimen. Results:A total of 13 patients with advanced disease were enrolled in the safety/intent-to-treat population; all experienced treatment-emergent adverse events (AEs). The most frequent AEs were blood and lymphatic disorders, followed by gastrointestinal disorders. Most AEs were mild to moderate in severity. Three patients discontinued the study due to an AE, including 2 deaths. Two patients died after the study period due to tumor progression. No death or discontinuation was considered related to the study drugs. Antitumor responses were noted by radiographic assessment. In the 8 patients who had antitumor data at day 21, 1 patient had complete response, 5 had stable disease, and 2 had progressive disease. Conclusions:The IRX-2 regimen was tolerated in patients with advanced HNSCC who failed surgery and/or radiation therapy. The safety and antitumor activity observed warrants further studies.

Therapeutic cancer vaccines are attractive due to the prospect of specificity and their lack of toxicity; however, their clinical development has been hampered by several biologic and clinical challenges. One of the most important biologic challenges is the relative lack of effective cellular immune adjuvants. Effective physiologic immune responses are characterized by the local generation of a complex cytokine environment that activates and regulates multiple immune cell types. IRX-2 is a primary cell-derived biologic with physiological levels of multiple active cytokine components, produced under pharmaceutical standards. The hypothesis that IRX-2 amplifies the T cell response to defined antigens was assessed in mice by measuring the T cell-specific peptide response to a dominant mouse peptide (NFT) derived from human prostate-specific membrane antigen (PSMA). IRX-2 enhances the T cell response to NFT when antigens were delivered either via irradiated cells expressing human PSMA, NFT peptide in Incomplete Freund's adjuvant (IFA) or NFT peptide conjugated to KLH. The T cell-specific activity was measured in spleen or lymph nodes cells by IFN-γ ELISpot and/or IFN-γ secretion over 6 days or in vivo by peptide-specific delayed-type hypersensitivity reaction (DTH). Further more, a single administration of IRX-2 with the antigen was not active as compared to 4 or 9 additional administrations which were sufficient to enhance the T cell response to antigens. The influence of IRX-2 on the B cell response to ovalbumin when it was used as a carrier protein was measured by ELISA. IRX-2 was compared to a commercially available combination adjuvant (MPL+TDM in squalene/Tween 80) which based on the literature is a potent adjuvant in murine systems. In the T cell assay IRX-2 was superior to the commercially available combination adjuvant and while IRX-2 also increased antibody titer, it was not as potent as the combination adjuvant. Mice immunized with IRX-2 and antigen also exhibited delayed tumor progression following challenge with PSMA-expressing tumor cells. These studies demonstrate that IRX-2 is an immunomodulator with adjuvant activity which preferentially enhances the T cell-specific responses to tumor associated antigens. Based on these studies, IRX-2 is a candidate for evaluation as a T cell adjuvant in a variety of preclinical vaccine delivery systems as well as in human clinical trials with cancer vaccine candidates.

IRX-2 is a cytokine-based biologic agent that has the potential to enhance antitumor immune responses. We investigated whether IRX-2 can protect T cells from tumor-induced apoptosis. Tumor-derived microvesicles (MV) expressing FasL were purified from supernatants of tumor cells and incubated with activated CD8 + T cells. MV induced significant CD8 + T-cell apoptosis, as evidenced by Annexin binding (64.4±6.4%), caspase activation (58.1±7.6%), a loss of mitochondrial membrane potential (82.9±3.9%) and DNA fragmentation. T-cell pretreatment with IRX-2 prevented apoptosis. IRX-2-mediated cytoprotection was dose and time dependent and was comparable to effects of IL-2, IL-7 or IL-15. IRX-2 prevented MV-induced downregulation of JAK3 and TCR ζ chain and induced STAT5 activation in T cells. IRX-2 prevented MV-induced Bax and Bim upregulation ( P <0.005–0.05), prevented cytochrome c release and Bid cleavage, and concurrently restored the expression of Bcl-2, Bcl-xL, FLIP and Mcl-1 ( P <0.005–0.01) in T cells. In addition, IRX-2 reversed MV-induced inhibition of the PI3K/Akt pathway. An Akt inhibitor (Akti-1/2) abrogated protective effects of IRX-2, suggesting that Akt is a downstream target of IRX-2 signaling. Thus, ex vivo pretreatment of CD8 + T cells with IRX-2 provided potent protection from tumor-induced apoptosis. IRX-2 application to future cancer biotherapies could improve their effectiveness by bolstering T-cell resistance to tumor-induced immunosuppression.

A method of increasing immunological effect in a patient by administering an effective amount of a primary cell derived biologic to the patient, inducing immune production, blocking immune destruction, and increasing immunological effect in the patient. Methods of treating an immune target, treating a tumor, immune prophylaxis, and preventing tumor escape.

IRX-2 is a uniform, well-defined set of natural cytokines currently in Phase II clinical trials for squamous cell carcinoma of the head and neck (HNSCC). In preliminary clinical studies of HNSCC patients, IRX-2 therapy has shown promising results, increasing overall survival of patients from 32% to 61% at 48 months. Although it is known that specific cytokines in IRX-2 enhance T cell activity [e.g., interleukin-2 (IL-2), interferon-gamma, IL-1beta], we chose to investigate the influence of IRX-2 on monocyte-derived dendritic cells (Mo-DCs) isolated from human peripheral blood in an effort to further understand the clinical findings. We show here that IRX-2 treatment of human monocyte-derived DC resulted in morphologic, phenotypic, and functional changes consistent with the development of mature activated DC. Specifically, IRX-2-treated DC increased expression of CD83 and CCR7, markers for DC maturation and migration, respectively, and increased the expression of HLA-DR, CD54, and the costimulatory molecules CD86 and CD40, which are critical mediators of T cell activation. Functional changes in DC induced by IRX-2 included a reduced endocytic capacity, increased ability to stimulate T cells and increased IL-12 cytokine production. These results provide a plausible mechanistic explanation for the in vivo clinical activity of IRX-2 and an additional rationale for the use of IRX-2-based immunotherapy in patients.

Hematology analyzers designed to count platelets in samples of whole blood are used to enumerate the total number of platelets in components prepared for transfusion. This report addresses the issue of variability in platelet counts obtained with different models of hematology analyzers. The influence of a common calibration procedure, involving one level of porcine platelets, on the extent of variability was also evaluated. Identical sets of samples of simulated and apheresis-derived human platelets were counted by multiple laboratories in 3 separate studies. In the first 2 exercises, 7 samples of both porcine platelets and modified goat erythrocytes with targeted platelets counts from 0.2 to 4.0 x 10(12)/L were counted without prior dilution. In both exercises, the samples were counted multiple times after routine calibration using instructions provided by the manufacturers of the various hematology analyzers used. In the second exercise, the samples were recounted after the hematology analyzers were recalibrated with a common calibrant consisting of porcine platelets at a targeted concentration of 0.5 x 10(12)/L. In the first and second exercises, 20 and 18 hematology analyzers were used, respectively. In the third exercise, 6 samples prepared from a single unit of apheresis platelets with targeted counts from 0.2 to 1.64 x 10(12)/L were shipped by an overnight courier and counted in triplicate on the day of arrival. Eleven hematology analyzers were used. The influence of recalibration was evaluated statistically by using the 95% prediction interval for the mean of a future set of observations. The platelet counts measured with a specific type of hematology analyzer provided the data to calculate the 95% prediction interval. With routine calibration, a wide variability in platelet counts was observed with all levels of both simulated and apheresis-derived human platelets. For example, with porcine platelets at a targeted level of 0.4 x 10 (12)/L, the platelet counts ranged from 0.31 to 0.47 x 10(12)/L. Recalibration reduced the extent of variability observed with all levels of simulated and apheresis-derived human platelets by increasing the observed platelet counts determined with a subset of hematology analyzers that produced platelet counts in the lower portion of the range. With recalibration, the mean platelet counts obtained with most hematology analyzers, especially with samples having targeted platelet levels no greater than 1.0 x 10(12)/L, were within or near the 95% prediction interval determined with the instruments that provided the highest platelet counts with routine calibration. With recalibration, the reproducibility of the platelet counts was considered to be good for all hematology analyzers with all levels of simulated and apheresis-derived human platelets for most of the instruments. The coefficient of variance did not exceed 6%, with most of the values ranging from 1% to 3%. This study therefore found that the platelet counts of platelet concentrates can be markedly influenced by the type of hematology analyzer used. A common calibration procedure designed specifically for the range of platelet counts in platelet products may be beneficial considering that many different hematology analyzers are being used to count platelets.

The results of scanning electron microscopic (SEM) and fatty acid methyl ester (FAME) characterization of the bacterial species shown to penetrate conventional 0.2/0.22 micron rated "sterilizing grade" filters are presented. SEM data suggest that retention of bacteria by these filters appears to be strongly influenced by the morphology, and especially the width of bacteria and less so by length. When the bacterial cell width is small, less than 0.3 micron or so, the cell length does not appear to limit the ability to penetrate 0.2/0.22 micron rated filters. As the bacterial width increases, there is also a strong, almost exponential, decrease in the allowable length for penetration, with most penetrative cells tending to be coccoid beyond a width of 0.5 micron. Significant percentages of the bacteria (40-50%) that were observed downstream of these filters were larger than B. diminuta, the standard organism used to qualify 0.2/0.22 micron rated filters. The average sizes of natural waterborne bacteria that penetrated the filters tested were 20-40% larger in width, and 40-70% larger in length, compared to B. diminuta. These results indicate that size exclusion is not the sole mechanism governing bacterial retention. All isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. Most of the bacteria recovered downstream of 0.2/0.22 micron rated filters were gram negative, oxidase positive, motile, nonfermentors.

The results of bacterial challenge tests conducted on several 0.2 and 0.22 micron rated "sterilizing grade" filter cartridge types with bacteria from a natural water source are presented. Eight different 0.2/0.22 micron rated "sterilizing grade" filter types from four different filter manufacturers, claimed to be capable of retaining Brevundimonas diminuta at a challenge level of 10(7) CFU/cm2, were tested. The filters tested included nylon 6.6 and polyamide filters from two manufacturers, modified or hydrophilic PVDF filters from two manufacturers, modified or asymmetric PES filters from three manufacturers, and cellulose acetate filters from a single manufacturer. Consistent bacterial penetration was observed, over the 18-24 h challenge period, for all twenty-five integral 0.2 and 0.22 micron rated filter cartridges tested, at challenge levels of about 10(1)-10(4) CFU/cm2, indicating that natural waterborne bacteria were more penetrative than B. diminuta. The observed penetration was thus qualitatively independent of filter media type or manufacturer. These results add to the growing body of evidence that shows 0.2 and 0.22 micron rated filters may not remove all microorganisms under all conditions. These results further establish that bacterial penetration of 0.2/0.22 micron rated filters is not limited just to (1) specific membrane types, or (2) extended duration challenges (>> 24 h), or (3) extremely high challenge levels, or (4) bacteria that can only exist in a penetrative state in an artificial laboratory setting.

The results of bacterial challenge tests conducted on several 0.2 and 0.22 mum rated "sterilizing grade" filter cartridge types with bacteria from a natural water source are presented. Eight different 0.2/0.22 mum rated "sterilizing grade" filter types from four different filter manufacturers, claimed to be capable of retaining Brevundimonas diminuta at a challenge level of 10(7) CFU/cm(2), were tested. The filters tested included nylon 6,6 and polyamide filters from two manufacturers, modified or hydrophilic PVDF filters from two manufacturers, modified or asymmetric PES filters from three manufacturers, and cellulose acetate filters from a single manufacturer. Consistent bacterial penetration was observed, over the 18-24 h challenge period, for all twenty-five integral 0.2 and 0.22 mum rated filter cartridges tested, at challenge levels of about 10(1)-10(4) CFU/cm(2), indicating that natural waterborne bacteria were more penetrative than B. diminuta. The observed penetration was thus qualitatively independent of filter media type or manufacturer. These results add to the growing body of evidence that shows 0.2 and 0.22 mum rated filters may not remove all microorganisms under all conditions. These results further establish that bacterial penetration of 0.2/0.22 mum rated filters is not limited just to (1) specific membrane types, or (2) extended duration challenges (>> 24 h), or (3) extremely high challenge levels, or (4) bacteria that can only exist in a penetrative state in an artificial laboratory setting.

We have previously reported on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. This article reports on the retention efficiencies of a large panel of 0.2/0.22 microm and 0.1 microm rated filter cartridges for H. pseudoflava (ATCC 700892) versus the retention capabilities of the same filters for Brevundimonas diminuta (ATCC 19146). A total of thirty-two 0.2/0.22 microm rated filter cartridges, spanning nine different "sterilizing grade" filter types from four different filter manufacturers, were challenged with H. pseudoflava at challenge levels exceeding 10(7) cfu/cm2. H. pseudoflava was shown to penetrate every 0.2/0.22 microm rated filter tested, with log titer reduction (LTR) values ranging from 3.5 to 7.7 logs. H. pseudoflava was shown to be more penetrative than B. diminuta under the same challenge conditions. B. diminuta was fully retained by nineteen of the twenty 0.2/0.22 microm rated filters that were challenged with both organisms. In the case of 0.1 microm rated filters, eighteen filter cartridges, spanning five different filter types from three manufacturers were tested. H. pseudoflava was consistently retained by four out of the five filter types tested, with LTR values in excess of 11.5 to 12.2 logs. The 0.1 microm rated filter type that was penetrated by H. pseudoflava has been previously demonstrated to be not fully retentive for naturally occurring bacteria. The data show that H. pseudoflava penetrates 0.2/0.22 microm rated filters just as readily as B. diminuta penetrates 0.45 microm rated filters. In addition, titer reductions provided by 0.2/0.22 microm rated filters for H. pseudoflava are comparable to those reported for A. laidlawii mycoplasma, albeit under different conditions. This study demonstrates that H. pseudoflava meets all criteria for use as a standard organism for qualifying the microbial removal performance of 0.1 microm rated filters for enhanced sterility assurance.

Although 0.1 microm rated filters intended for pharmaceutical sterilization applications have been commercially available for at least 15 years, there is no industry-wide standard for qualifying the microbial removal performance of these filters. In this article, we report on the bacterial challenge methodology used to screen four bacterial species for potential utility as a standard challenge organism to qualify 0.1 microm rated filters. These isolates were, in their natural state, demonstrated to penetrate 0.2/0.22 microm rated filters in prior studies. In the screening challenges described in this study, three out of these four candidates tested demonstrated consistent penetration of one 0.22 microm rated filter type tested (when cultured in a low nutrient medium under standard laboratory conditions). These included 6204-22 (FAME ID Acidovorax avenae citrulli), 6266-15 (FAME ID Comamonas acidovorans), and 6266-34 (FAME ID Hydrogenophaga pseudoflava). Of these, H. pseudoflava (6266-34) was chosen for additional experiments with other 0.2 microm rated filter membranes. In total, seventeen 0.2 and 0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from three different filter manufacturers were tested. H. pseudoflava penetration was observed for every filter tested. Under the same challenge conditions, H. pseudoflava was consistently retained by a 0.1 microm rated hydrophilic PVDF (polyvinylidenefluoride) filter with a specified high titer reduction claim for Acholeplasma laidlawii. In order to ensure selection of the most stable penetrative phenotype (i.e., select for nonrevertants), H. pseudoflava was subjected to three rounds of "filter cloning," and these results are described herein. The advantages of using H. pseudoflava for qualifying the microbial removal performance of 0.1 microm rated filters are also discussed.

In this article, we report on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. Filter-cloned H. pseudoflava (ATCC 700892) was easily cultured in a low nutrient broth (R2A broth) under standard laboratory conditions, reaching high titers of 10(8)-10(9) cfu/mL within 48-65 hours of incubation at 25+/-5 degrees C. Under these conditions, H. pseudoflava is a rod-shaped bacterium, averaging 0.25+/-0.03 microm by 1.65+/-0.35 microm, and appears to be smaller than Brevundimonas diminuta in width (0.31+/-0.03 microm), but somewhat longer in length (0.88+/-0.19 microm), which may partly explain the observed penetration. In total, thirty-five 0.2/0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from two different filter manufacturers were challenged with H. pseudoflava. In all cases, H. pseudoflava was shown to consistently penetrate every 0.2/0.22 microm rated filter disc tested. These tests also spanned three different challenge durations, including short-term challenges (30-40 minutes), and two different challenge fluids. The use of serial (double) 0.22 mm rated filters, which is a common industry practice to reduce the prefiltration bioburden to the final "sterilizing" filter, was also shown to be inadequate to fully retain H. pseudoflava under the challenge condition used. In contrast, two different 0.1 microm rated filter types functionally qualified with a specified high titer reduction claim for Acholeplasma laidlawii, were shown to consistently and fully retain H. pseudoflava, and retention by these two filter types was shown to be robust and independent of the challenge duration.

The term bubble point test refers to a colloection of many different test methods. In this Part I of a two-part article, the authors report on the effect of test methodology on bubble point measurements, including those using automated instruments. Results indicate that bubble points measured for the same filter cartridge can vary by ∼5-6 psi, depending on the exact bubble point type test used and that all tests dectected bubble points for the filter cartridges at pressures well above the intrinsic transition point marking the onset of bulk flow. The results clearly show that the use of bubble point specification as a correlated (critical) test on a process filter is most valid when the test is performed using the same specific protocol, equipment, and filter area used to establish the primary correlation in the first place.

Part II presents results of bubble point type tests on filter disks removed from 10-in, filter cartridges. Results show a 10-20% variation in the bubble points measured for the same filter disk by the different bubble point type tests used. Irrespective of the test used, water-wet bubble points measured on 47-mm disks were mostly higher than those measured on the corresponding 10-in, filter cartridge from which they were removed, even when the same test method was used for both determinations. Results also indicate that the use of a bubble point specification as a correlated (critical) test on a process filter is most valid when the filter user performs the test using the same protocol, equipment, and filter area used to establish the correlation in the first place.