Abstract Pritumumab is a natural human IgG1 kappa antibody that has been derived from a B-cell isolated from a regional draining lymph node of a patient with cervical carcinoma. Specificity analysis of the antibody with human tissues showed the target antigen, altered tumor-associated vimentin, to be highly restricted to various epithelial cancers and not normal cells and tissues. Daunomycin-pritumumab conjugates were generated that increased the effectiveness of the antibody. In various phase II clinical trials in Japan 249 patients with brain cancer were treated with unconjugated pritumumab. The overall response rate was between 25-30%, a 9-fold increase over standard care, with several survivors beyond 5-years post-treatment. The patients were on a low dose regimen of 1mg given twice a week for a course of 24 weeks for a total dose of 48 mgs per course. Pritumumab appears to be a safe and effective therapy in patients with brain cancer. Citation Format: Mark C. Glassy, Rishab Gupta. Pritumumab, a natural human IgG antibody defines a tumor-associated biomarker useful in treating brain cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2553. doi:10.1158/1538-7445.AM2014-2553

Abstract Pritumumab, a natural human IgG1 kappa antibody isolated from a lymph node of a patient with cervical carcinoma, has been used in a Phase II setting with brain cancer patients. The original human-human hybridoma secreted approximately 1 picogram of antibody per cell per day. This level of the antibody production posed cost constrains for late stage therapeutic clinical trials. To generate a more commercially viable antibody, a pritumumab-secreting Chinese hamster ovary (CHO) cell line was created using the GPEx® technology. This technology involved construction and cloning of heavy and light chain DNA. Both DNA sequences were cloned into the expression vector and transduced into HEK 293 cells that constitutively produces the MLV gag, pro, and pol. The pritumumab cell line was made by performing multiple rounds of transduction (multiplicity of > 1000 retrovector particles/cell) of the GPEx® Chinese Hamster Ovary (GCHO) parental cell line with retrovector made from the gene construct developed to express the pritumumab antibody light chain (LC) and the pritumumab antibody heavy chain (HC). Five independent transductions were performed: two LC and three HC. Limiting dilution method was used to establish clonal cell lines. Top clones were selected based on pritumumab antibody titer. Pritumumab expression was determined by Protein A HPLC using a generic IgG standard. Clearly, single copies of genes were inserted efficiently in unique locations throughout the genome of the CHO cells to obtain genetically stable cell line that expressed high levels of the antibody, and represented more than a 40-fold increase in production of the antibody over the original hybridoma. Flow cytometry binding and immunohistological analyses demonstrated comparability between the recombinant form of pritumumab and the hybridoma form of the protein. Thus, high level of specific productivities and titers of the CHO clones developed through the GPEx® system will allow the therapeutic antibody program to move forward into large and extended clinical trials. Citation Format: Gregory T. Bleck, Rishab K. Gupta, Beatrix Kotlan, Dona York, Mark C. Glassy. GPEx® system to increase production of pritumumab in a CHO cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2658. doi:10.1158/1538-7445.AM2014-2658

In the broadest sense there are no longer any technical limitations to making human mAbs. Biological issues involving the type and nature of either a synthetic or a natural antibody, advantages of various B cell immunological compartments, and various assays needed to qualitate and quantitate mAbs have essentially been solved. If the target antigen is known then procedures to optimize antibody development can be readily planned out and implemented. When the antigen or target is unknown and specificity is the driving force in generating a human mAb then considerations about the nature and location of the B cell making the sought after antibody become important. And, therefore, the person the B cell is obtained from can be an ethical challenge and a limitation. For the sources of B cells special considerations must be taken to insure the anonymity and privacy of the patient. In many cases informed consent is adequate for antibody development as well as using discarded tissues. After the antibody has been generated then manufacturing technical issues become important that greatly depend upon the amounts of mAb required. For kilogram quantities then special considerations for manufacturing that include FDA guidelines will be necessary.

Meeting abstracts Pritumumab is a natural human IgG1 kappa antibody that has been derived from a B cell isolated from regional draining lymph node of a cervical carcinoma patient. Specificity analysis of the antibody with human tissues showed the target antigen, altered tumor-associated vimentin,

This paper is based on the problem of accurate fault diagnosis by incorporating a waveform matching technique. Fault isolation and detection of a double circuit high voltage power transmission line is of immense importance from point of view of Energy Management services. Power SystemFault types namely single line to ground faults, line to line faults, double line to ground faults etc. are responsible for transients in current and voltage waveforms in Power Systems.Waveform matching deals with the approximate superimposition of such waveforms in discretized versions obtained from recording devices and Software respectively.The analogy derived from these waveforms is obtained as an error function of voltage and current, from the considered metering devices.This assists in modelling the fault identification as an optimization problem of minimizing the error between these sets of waveforms.In other words, it utilizes the benefit of software discrepancies between these two waveforms.Analysis has been done using the Bare Bones Particle Swarm Optimizer on an IEEE 2 bus, 6 bus and 14 bus system.The performance of the algorithm has been compared with an analogous meta-heuristic algorithm called BAT optimization on a 2 bus level.The primary focus of this paper is to demonstrate the efficiency of such methods and state the common peculiarities in measurements, and the possible remedies for such distortions.

Purpose: There is no consensus regarding the optimal tumor markers for melanoma. We compared 3 tumor markers, TA90-immune complex (TA90IC), melanoma-inhibiting activity (MIA) protein, and S100B protein in Stage III melanoma patients undergoing adjuvant vaccine immunotherapy. Experimental design: The serum of 75 patients representing 3 prognostic cohorts was assayed for the tumor markers prior to initiating immunotherapy and at 6 follow-up time points. Upper limits of normal for TA90IC, MIA and S100B were set at OD 0.41, 8.5 ng/ml, and 2.5 mu g/l, respectively. Results: At least 1 marker became elevated prior to 41 ( 80 percent) of 51 recurrences. TA90IC was the earliest elevated marker in 29 ( 57 percent), MIA in 11 ( 22 percent), and S100B in 4 ( 8 percent). Multivariate regression analysis revealed that TA90IC was an independent predictor of survival when elevation occurred between 2 weeks and 3 months, whereas MIA was an independent predictor at 4-6 months. In the poor prognostic cohort, mean values for MIA and S100B increased progressively, whereas TA90IC exhibited a parabolic curve. Conclusion: In this patient population, TA90IC and MIA were complementary; elevation of the immune complex preceded elevation of the tumor antigen in patients who developed recurrence. Additional studies in populations not receiving vaccine will further clarify the clinical utility of these assays.

The dynamic interaction between malignant melanoma cells and the host’s immune system has a profound influence on the development and progression of disease. Numerous studies have documented the therapeutic potential of immunological manipulation in patients with melanoma, and many tumorassociated antigens have been studied as possible targets of melanoma-specific immunotherapy. This chapter outlines the most significant melanoma-associated antigens, highlighting those with the greatest potential for influencing future treatment modalities. Specific antigens defined by T- and B-cells are discussed, and how these antigens are recognized, processed, and manipulated by the host’s immune system is explained. The role of gangliosides in distinguishing different stages of melanocyte differentiation is described, and a 90-kDa tumor-associated glycoprotein antigen which has considerable promise as a marker of melanoma tumor burden is introduced. Finally, this chapter reviews technological advances in assays used to detect various melanoma-associated antigens.

In cancer cells, cyclic AMP-dependent protein kinase (PKA) is secreted into the conditioned medium. This PKA, designated as extracellular protein kinase A (ECPKA), is markedly up-regulated in the sera of patients with cancer. The currently available tumor markers are based on the antigen determination method and lack specificity and sensitivity. Here, we present an ECPKA autoantibody detection method for a universal biomarker that detects cancer of various cell types. We tested sera from 295 patients with cancers of various cell types, 155 normal controls, and 55 patients without cancer. The specificity and sensitivity of this autoantibody enzyme immunoassay method were compared with the conventional antigen determination method by receiver-operating characteristic plots. In the sera, the presence of autoantibody directed against ECPKA was highly correlated with cancer. High anti-ECPKA autoantibody titers (frequency, 90%; mean titer, 3.0) were found in the sera of patients with various cancers, whereas low or negative titers (frequency, 12%; mean titer, 1.0) were found in the control group. The receiver-operating characteristic plot showed that autoantibody enzyme immunoassay exhibited 90% sensitivity and 88% specificity, whereas the enzymatic assay exhibited 83% sensitivity and 80% specificity. These results show that the autoantibody method distinguished between patients with cancer and controls better than the antigen method could. Our results show that autoantibody ECPKA is a universal serum biomarker for cancers of various cell types.

Journal of Surgical OncologyVolume 90, Issue 2 p. 69-70 Commentary Commentary—Prognosis in melanoma: the truth still lies in the node Mark B. Faries, Mark B. Faries John Wayne Cancer Insititute, Santa Monica, CASearch for more papers by this authorRishab K. Gupta, Rishab K. Gupta John Wayne Cancer Insititute, Santa Monica, CASearch for more papers by this authorDonald L. Morton, Corresponding Author Donald L. Morton [email protected] John Wayne Cancer Insititute, Santa Monica, CAJohn Wayne Cancer Insititute, 2200 Santa Monica Blud., Santa Monica, CA 90404.Search for more papers by this author Mark B. Faries, Mark B. Faries John Wayne Cancer Insititute, Santa Monica, CASearch for more papers by this authorRishab K. Gupta, Rishab K. Gupta John Wayne Cancer Insititute, Santa Monica, CASearch for more papers by this authorDonald L. Morton, Corresponding Author Donald L. Morton [email protected] John Wayne Cancer Insititute, Santa Monica, CAJohn Wayne Cancer Insititute, 2200 Santa Monica Blud., Santa Monica, CA 90404.Search for more papers by this author First published: 20 April 2005 https://doi.org/10.1002/jso.20241AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES 1 Kelley M, Jones R, Gupta R, et al.: Tumor-associated antigen TA90 immune complex assay predicts subclinical metastasis and survival for patients with early stage melanoma. Cancer 1998; 83: 1355– 1361. 2 Faries M, Gupta R, Ye X, et al.: Melanoma-inhibiting activity assay predicts survival in patients receiving a therapeutic cancer vaccine after complete resection of American Joint Committee on Cancer Stage III Melanoma. Ann Surg Oncol 2004; 11: 85– 93. 3 Hoon D, Bostick P, Kuo C, et al.: Molecular markers in blood as surrogate prognostic indicators of melanoma recurrence. Cancer Res 2000; 60: 2253– 2257. 4 Wilson L, Tran L, Morton D, et al.: Detection of differentially expressed proteins in early-stage melanoma patients using SELDI-TOF mass spectrometry. Ann N Y Acad Sci 2004; 1022: 317– 322. 5 Cochran A, Roberts A, Wen D, et al.: Update on lymphatic mapping and sentinel node biopsy in the management of patients with melanocytic tumours. Pathology 2004; 36: 478– 484. 6 Takeuchi H, Morton D, Kuo C, et al.: Prognostic significance of molecular upstaging of paraffin-embedded sentinel lymph nodes in melanoma patients. J Clin Oncol 2004; 22: 2671– 2680. Volume90, Issue21 May 2005Pages 69-70 ReferencesRelatedInformation

2564 Background: Strong humoral and cellular immune responses in patients with melanoma receiving a specific active immunotherapeutic (Canvaxin; CancerVax Corp, Carlsbad, CA) have been correlated with improved survival. We examined the impact of several immunomodulators on the strength of immune response. Methods: Analysis of sequential phase II clinical trials using Canvaxin alone (n=446) or in combination with cyclophosphamide (n=92), cimetidine (n= 357), indomethacin (n=54) or IL-2/INFα (n=71) was performed with regard to maximal immune response within 16 weeks of treatment. A randomized clinical trial (n= 97) of Canvaxin with or without the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) was also analyzed. Delayed-type hypersensitivity (DTH) to vaccine was measured in all trials. IgM and IgG responses to the TA90 tumor-associated antigen were measured prospectively in the GM-CSF trial. Results: DTH responses were greater in patients with stage IV melanoma with concurrent cyclophosphamide (mean induration 20.6 mm with cyclophosphamide vs. 14.2 without, p=0.011). This was not seen in patients with stage III disease or in any stage with cimetidine, indomethacin or IL-2/IFNα treatment. The randomized GM-CSF trial did not show a significant difference (by logistic regression) in maximal DTH response (15.9± 9.7 mm with GM-CSF vs. 16.7 ± 9.9 mm without). There was a significant increase in maximal anti-TA90 IgM titers in the GM-CSF arm (797 ± 622 vs. 541 ± 533; p=0.0123), but no difference in anti-TA90 IgG (680 ± 557 with GM-CSF vs. 612 ± 510 without GM-CSF.) Maximal absolute lymphocyte counts during treatment were also elevated in the GM-CSF arm. Adverse event profiles were not markedly different, but GM-CSF patients reported more grade 1–2 fatigue. Conclusions: These data suggest that the addition of cyclophosphamide to adjuvant active specific immunotherapy in stage IV melanoma impacts DTH responses, and GM-CSF increases IgM response. Since this represents an impact on both the cellular (DTH) and humoral (IgM) arms of immunity, further study of modulator combinations is warranted. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration CancerVax CancerVax

We aimed to test the hypothesis that the inducible form of nitric oxide synthase (iNOS) contributes to the development of an early subnormal retinal oxygenation response in preclinical models of diabetic retinopathy. In urethane anesthetized Sprague Dawley rats or C57BL/6 mice, functional magnetic resonance imaging was used to noninvasively measure the change in retinal oxygen tension (Delta PO(2)) during a carbogen-inhalation challenge. In the rat experiments, the retinal Delta PO(2) of the following groups were compared: control rats (n = 9), 3-month diabetic rats (n = 5), and 3-month diabetic rats treated orally with L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide, a prodrug of an inhibitor of iNOS (n = 6). In addition, the retinal Delta PO(2) of the following mouse groups were compared: C57BL/6 mice (n = 20), C57BL/6-Nos2(tm1 Lau) mice (n = 10), 4-month diabetic mice (n = 13), and 4-month diabetic knockout mice (n = 6). Only the Delta PO(2) of the superior hemiretina of the diabetic rat and mice groups were significantly subnormal (P < 0.05). The superior Delta PO(2) of the diabetic rats treated with the prodrug was not significantly (P > 0.05) different from their respective normal controls. In the mice experiments, the superior retinal Delta PO(2) of the iNOS null mice was not statistically different (P > 0.05) from that of normal control mice. iNOS is required for the development of an early subnormal Delta PO(2) in experimental diabetic retinopathy.

BACKGROUND:More than half of melanoma patients rendered disease free by lymph node dissection will experience disease recurrence. We hypothesized that serum levels of melanoma-inhibiting activity (MIA) protein might be useful to stratify risk and identify subclinical recurrence in patients undergoing adjuvant immunotherapy. We examined MIA levels in the serum of stage III patients treated after surgery with a therapeutic cancer vaccine. METHODS:Three cohorts of 25 patients were randomly selected from our melanoma database on the basis of time to death (group 1, <1 year; group 2, 1-5 years; group 3, >5 years.) Prospectively collected serum samples were assayed in a blinded fashion for MIA by enzyme-linked immunosorbent assay. RESULTS:MIA was increased at any time in 19 (76%) of 25, 4 (16%) of 25, and 1 (4%) of 25 patients in groups 1, 2, and 3, respectively. The median survival was 11 months for the 25 patients with increased MIA and >75 months for the 50 patients with normal MIA. MIA increased above normal a median of 1 month (mean, 75 days) before clinical recurrence. All patients with increased MIA after 2 months of treatment subsequently died of melanoma. One patient in whom initially increased levels decreased to normal within 2 months is disease free. CONCLUSIONS:Serum MIA levels provide important prognostic information early in the course of stage III melanoma and often detect melanoma recurrences before clinical evidence of disease.

This review summarizes the development and applicability of an assay to detect an immune complex (IC) of a 90kD glycoprotein tumor-associated antigen (TA90). The antigen is immunogenic in the cancer host and expressed by a variety of solid tumors. In circulation, it induces formation of endogenous antigen-antibody complexes. The presence of TA90-IC has been detected in significantly greater proportions of serum samples from cancer patients compared to normal controls. The postoperative presence of TA90-IC in circulation strongly correlated with subsequent development of clinically detectable recurrence within an average interval of 3 to 19 months. The TA90-IC status after surgery but before initiation of adjuvant therapy consistently correlated with disease-free survival and overall survival. Because of its ability to accurately detect subclinical metastasis and predict survival 1 to 3 months after surgical resection of early-stage cancer, TA90-IC represents a novel and feasible prognostic marker to select candidates for adjuvant therapies and to stratify patients in clinical trials. Expression of TA90 by melanoma, colon cancer, lung cancer, breast cancer and other solid neoplasms makes TA90-IC an ideal marker in the management of early-stage cancer. Its specificity can be significantly enhanced if positive samples are subsequently analyzed by a panel of tumor markers characteristic for a particular type of cancer, such as CA15-3 for breast cancer and carcinoembryonic antigen (CEA) for colon cancer. TA90-IC could be used in conjunction with these site-specific markers as part of a cancer screening program to identify high-risk individuals for closer surveillance.

2543 Background: The treatment of AJCC stage IV melanoma is inadequate; in a recent meta-analysis, median and 5-year survivals were 8.1 months and 2.3%. In phase II trials, we have evaluated the activity of active specific immunotherapy with a polyvalent therapeutic cancer vaccine (Canvaxin) and other immune adjuvants against stage IV melanoma. We compared the survival of vaccine-treated patients with the survival of comparable surgical and nonsurgical patients receiving nonvaccine therapies. Methods: Our study groups included all AJCC stage IV patients (N=1856) entered into our prospective database of 11,000 patients between 1/1/86 and 1/1/2001. Survival curves were estimated by Kaplan-Meier method, and statistical difference analyzed by log rank test and Cox regression according to the new AJCC staging system of Mla, Mlb and Mlc. Results: Although complete regression of evaluable disease was rare among vaccine patients, disease stabilization was common. Survival from initial stage IV diagnosis was prolonged almost twofold for the entire group of vaccine patients and for all vaccine-treated M1 subgroups (p = 0.001 - 0.05) except Mla (Table); 1-, 2-, 3- and 5-year survival rates were also significantly enhanced (p = 0.001). Toxicity of vaccine therapy was minimal: transient grade 1 or 2 fever, headaches, malaise and ulcerations at the injection sites. Skin-test and antibody responses to vaccine antigens were associated with enhanced survival. Conclusions: When compared with other postsurgical adjuvants and nonsurgical therapies, polyvalent vaccine immunotherapy significantly enhanced the survival of patients with resectable and nonresectable stage IV melanoma. This is the largest vaccine study yet reported, and the survivals are the longest yet observed in stage IV melanoma. These findings deserve further study in a multicenter randomized trial, which is currently underway. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration CancerVax Corp. CancerVax Corp. CancerVax Corp.

PURPOSE:To test the hypotheses that, in mice, breathing carbogen (95% O(2)-5% CO(2)) oxygenates the retina better than breathing 100% oxygen, the superior hemiretinal oxygenation response to carbogen inhalation is subnormal early in diabetes, and diabetes-induced elevation of retinal protein kinase C (PKC)-beta contributes to this pathophysiology. METHODS:Retinal oxygenation response (DeltaPO(2)) was measured using functional magnetic resonance imaging (MRI) and either carbogen or 100% oxygen inhalation challenge in C57BL/6J control (C) mice. Retinal DeltaPO(2) during carbogen breathing was also measured in PKCbeta knockout (C57BL6-Prkcb1; [KO]), 4 month C57BL/6J diabetic (D), and 4-month diabetic PKCbeta KO (D+KO) mice. Retinal PKCbeta protein expression was assessed by Western analysis. RESULTS:In C mice, retinal DeltaPO(2) during carbogen breathing was significantly greater (P < 0.05) than during oxygen breathing. In D mice, DeltaPO(2) during carbogen breathing was significantly lower than normal in the superior, but not the inferior, hemiretina and was normal (P > 0.0 5) in the KO group. In the D+KO mice DeltaPO(2) was normal (P > 0.05) only at distances less than 1.5 mm from the optic nerve head. PKCbeta expression was elevated in the retina in diabetes (P < 0.05), but was significantly decreased (P < 0.05) in D+KO mice. CONCLUSIONS:The present study confirms that, in the mouse, retinal DeltaPO(2) patterns during different inhalation challenges or in the presence of diabetes are similar to what has been reported in rats. Diabetes-induced elevation of PKC appears to contribute only focally to subnormal retinal DeltaPO(2). This raises the possibility that PKC inhibition therapy may be only regionally effective in treating retinal pathophysiology associated with diabetic retinopathy.

PURPOSE:A therapeutic polyvalent cancer vaccine (Canvaxin vaccine; CancerVax Corp, Carlsbad, CA) induces antibodies to a glycoprotein tumor-associated antigen (TA90). However, endogenous immune responses to TA90 have also been reported. This study examined anti-TA90 antibody responses with respect to the survival of patients who received adjuvant vaccine immunotherapy after resection of thick (> or = 4 mm) primary cutaneous melanoma.PATIENTS AND METHODS:Serum specimens were obtained from 54 patients immediately before and then 1, 2, 4, and 6 months after wide local excision of thick primary cutaneous melanoma and sentinel lymphadenectomy. All patients were offered adjuvant therapies with the vaccine, high-dose interferon, or other agents. An enzyme-linked immunosorbent assay was used to determine serial serum titers of immunoglobulin G (IgG) and IgM antibodies against TA90. These titers were correlated with clinical course.RESULTS:Forty-three patients chose vaccine therapy, and 11 patients chose postoperative observation. Preoperative anti-TA90 IgG and IgM titers were similar for vaccine and observation groups (P =.184). At a median follow-up of 26 months, univariate analysis of Cox regression showed that disease-free survival and overall survival of vaccine patients were significantly correlated with maximal IgM response (P =.0006 and.006, respectively) but not with maximal IgG response (P =.73 and.95, respectively). Neither response predicted survival in the observation group.CONCLUSION:Postoperative vaccine therapy may enhance IgG and IgM immune responses to TA90 after surgical resection, but only the IgM response is correlated with improved survival. These findings may become useful to guide selection of patients for postoperative adjuvant therapy of high-risk melanoma.