High-throughput cytotoxicity and antigen-binding assay for screening small bispecific antibodies without purification.

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies.

Influence of ovarian cancer type I and type II microenvironment on the phenotype and function of monocyte-derived dendritic cells

PURPOSE. The aim of this study was to evaluate the influence of ovarian cancer cell lysates isolated from type I or type II ovarian cancer (OC) on the phenotype of monocyte-derived dendritic cells (Mo-DCs) and the cytokine profile. We also determined whether the Mo-DCs and tumor microenvironment, reflected by peritoneal fluid (PF) from type I or II ovarian cancer, could promote regulatory T cell (Tregs) differentiation from naive CD4+ lymphocytes in vitro. RESULTS. Our results show a significant role of the ovarian cancer microenvironment reflected by PF from type I or II OC in the inhibition of the DC differentiation process. Interestingly, the percentage of cells co-expressing CD45 and CD14 antigens in the cultures stimulated with PF from both type I and type II OC was higher than in the control. Furthermore, the percentage of cells expressing CD1a, i.e., a marker of immature DCs, was significantly reduced in the cultures stimulated with PF from type I and type II OC. The results obtained show that ovarian cancer type II lysates induce differentiation of monocytes into macrophage-like cells with a CD1a+/HLA-DR+/CD83− phenotype and significantly higher CD86/HLA-DR expression. We show that ovarian cancer type II Mo-DCs are able to prevent an immune response by release of IL-10, whereas OC type I Mo-DCs can promote the generation of Tregs. CONCLUSIONS. We demonstrate that each type of ovarian cancer can induce a unique phenotype of DCs and differentiation of Tregs, both associated with immune-suppressive function, which may be an obstacle while developing effective anticancer dendritic cell vaccination (AU)

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Development and validation of an HPLC/UV assay for separation and quantification of peptide antigens from a liposomal vaccine delivery platform.

The development and validation of an HPLC method for the quantification of eight peptide antigens from the therapeutic cancer vaccine DPX-0907 is described. The antigens were formulated in DepoVax™, a patented liposomal vaccine delivery platform used in a phase 1 study for breast, ovarian, and prostate cancers. A gradient reversed-phase method with UV detection was optimized for separating and quantifying the peptide mixture. Several extraction methods investigated to extract the peptides from the lipids led to poor recovery of one or more of the peptides. A simple, reproducible, and high-recovery extraction procedure for the simultaneous quantification of hydrophilic and hydrophobic peptides was discovered using a liquid-liquid extraction with water-saturated n-butanol and sodium bicarbonate (0.1 M). The method was found to be specific, linear, accurate, precise, and reliable within the range of 50-150% of the nominal concentration for DPX-0907. The validated method was successfully applied to the assay of peptide content in pre-clinical and clinical batches of DPX-0907.

Regular dose of gemcitabine induces an increase in CD14+ monocytes and CD11c+ dendritic cells in patients with advanced pancreatic cancer.

OBJECTIVE: Chemotherapy and immunotherapy often seem to contradict each other. However, recent reports suggested that the anticancer effects in some chemotherapeutic agents were concerned with immune response. This study was designed to evaluate the immunological reaction by gemcitabine for future clinical trial of combination therapy with gemcitabine and cancer vaccines. METHODS: We evaluated several immunological parameters in patients with advanced pancreatic cancer who received a conventional dose of gemcitabine for 2 months. Twenty-eight patients with metastasis or locally advanced tumor, including 18 gemcitabine-naïve and 10 with a history of preceding gemcitabine treatment, were enrolled in this study. The patients received gemcitabine 1000 mg/m(2) for 3 weeks, followed by 1 week of rest. We monitored the kinetics of lymphocytes, natural killer cells, monocytes, dendritic cells (DC), human leukocyte antigen (HLA)-multimer conjugated with CMV or WT1 peptide, and intracellular cytokine production of interferon-gamma and interleukin-4 by flow cytometry. The T cell receptor (TCR) repertoire was also analyzed. RESULTS: The absolute number and percentage of CD14(+) monocytes and CD11c(+) (myeloid) DC increased with gemcitabine treatment (P = 0.033 and P = 0.021). The percentage of CD123(+) (plasmacytoid) DC also increased (P = 0.034), whereas no significant change was observed in other immune parameters, including multimer, intracellular cytokine production and TCR repertoire. CONCLUSIONS: Our finding that gemcitabine treatment induced the proliferation of CD14(+) monocytes and CD11c(+) DC could support combination therapy with gemcitabine and specific immunotherapy such as peptide vaccination against pancreatic cancers.

Cluster intradermal DNA vaccination rapidly induces E7-specific CD8+ T-cell immune responses leading to therapeutic antitumor effects.

Intradermal administration of DNA vaccines via a gene gun represents a feasible strategy to deliver DNA directly into the professional antigen-presenting cells (APCs) in the skin. This helps to facilitate the enhancement of DNA vaccine potency via strategies that modify the properties of APCs. We have previously demonstrated that DNA vaccines encoding human papillomavirus type 16 (HPV-16) E7 antigen linked to calreticulin (CRT) are capable of enhancing the E7-specific CD+ T-cell immune responses and antitumor effects against E7-expressing tumors. It has also been shown that cluster (short-interval) DNA vaccination regimen generates potent immune responses in a minimal time frame. Thus, in the current study we hypothesize that the cluster intradermal CRT/E7 DNA vaccination will generate significant antigen-specific CD8+ T-cell infiltrates in E7-expressing tumors in tumor-bearing mice, leading to an increase in apoptotic tumor cell death. We found that cluster intradermal CRT/E7 DNA vaccination is capable of rapidly generating a significant number of E7-specific CD8+ T cells, resulting in significant therapeutic antitumor effects in vaccinated mice. We also observed that cluster intradermal CRT/E7 DNA vaccination in the presence of tumor generates significantly higher E7-specific CD8+ T-cell immune responses in the systemic circulation as well as in the tumors. In addition, this vaccination regimen also led to significantly lower levels of CD4+Foxp3+ T-regulatory cells and myeloid suppressor cells compared to vaccination with CRT DNA in peripheral blood and in tumor-infiltrating lymphocytes, resulting in an increase in apoptotic tumor cell death. Thus, our study has significant potential for future clinical translation.

Immunologic and clinical effects of injecting mature peptide-loaded dendritic cells by intralymphatic and intranodal routes in metastatic melanoma patients.

PURPOSE: A phase I/II trial was conducted to evaluate clinical and immunologic responses after intralymphatic and intranodal injections of mature dendritic cells. EXPERIMENTAL DESIGN: Fourteen patients with a metastatic melanoma received matured dendritic cells, loaded with Melan-A/MART-1 and/or NA17-A peptides and keyhole limpet hemocyanin. The cells were matured overnight with Ribomunyl, a toll-like receptor ligand, and IFN-gamma, which ensured the production of high levels of interleukin-12p70. Dendritic cells were injected at monthly intervals, first into an afferent lymphatic and then twice intranodally. Immunologic responses were monitored by tetramer staining of circulating CD8(+) lymphocytes and delayed-type hypersensitivity tests. RESULTS: Dendritic cell vaccination induced delayed-type hypersensitivity reactivity toward NA17-A-pulsed, keyhole limpet hemocyanin-pulsed, and Melan-A-pulsed dendritic cells in 6 of 10, 4 of 11, and 3 of 9 patients, respectively. Four of the 12 patients analyzed by tetramer staining showed a significantly increased frequency of Melan-A-specific T cells, including one patient vaccinated only with NA17-A-pulsed dendritic cells. Furthermore, 2 of the 12 analyzed patients had a significant increase of NA17-A-specific T cells, including one immunized after an optional additional treatment course. No objective clinical response was observed. Two patients were stabilized at 4 and 10 months and three patients are still alive at 30, 39, and 48 months. CONCLUSIONS: Injections into the lymphatic system of mature peptide-loaded dendritic cells with potential TH1 polarization capacities did not result in marked clinical results, despite immunologic responses in some patients. This highlights the need to improve our understanding of dendritic cell physiology.

Study on biological characters of SGC7901 gastric cancer cell-dendritic cell fusion vaccines.

AIM: To detect the biological characters of the SGC7901 gastric cancer cell-dendritic cell fusion vaccines. METHODS: The suspending living SGC7901 gastric cancer cells and dendritic cells were induced to be fusioned by polyethylene glycol. Pure fusion cells were obtained by selective culture with the HAT/HT culture systems. The fusion cells were counted at different time points of culture and their growth curves were drawn to reflect their proliferative activities. The fusion cells were also cultured in culture medium to investigate whether they could grow into cell clones. MTT method was used to test the stimulating abilities of the fusion cells on T lymphocytes' proliferations. Moreover, the fusion cells were planted into nude mice to observe whether they could grow into new planted tumors in this kind of immunodeficiency animals. RESULTS: The fusion cells had weaker proliferative activity and clone abilities than their parental cells. When they were cultured, the counts of cells did not increase remarkably, nor could they grow into cell clones in culture medium. The fusion cells could not grow into new planted tumors after planted into nude mice. The stimulating abilities of the fusion cells on T lymphocytes' proliferations were remarkably increased than their parental dendritic cells. CONCLUSION: The SGC7901 gastric cancer cell-dendritic cell fusion vaccines have much weaker proliferative abilities than their parental cells, but they keep strong abilities to irritate the T lymphocytes and have no abilities to grow into new planted tumors in immunodeficiency animals. These are the biological basis for their anti-tumor biotherapies.

Immunotherapy and cancer vaccines in the management of breast cancer.

Besides the traditional therapeutic options, treatment with antibodies specific for the receptor tyrosine kinase HER-2/neu has been established as a standard therapy in the clinical management of advanced breast cancer. Ongoing clinical studies focus on the improvement of application protocols in order to minimize side effects and evaluate the potential therapeutic benefit of anti-HER-2/neu antibodies in combination with conventional chemotherapy. Various similar strategies to target other tumour-associated antigens or proangiogenic factors with inhibitory antibodies are currently investigated in promising preclinical and clinical trials. In addition, research efforts are made to develop procedures to generate tumour-specific cellular immune responses in breast cancer patients. Therapeutic vaccination is, however, still at an early stage of development, despite encouraging results of animal studies. We summarise and discuss vaccination strategies with tumour-specific proteins or peptides, pulsed dendritic cells, and modified tumour cells as well as antibody-based therapeutic concepts to target HER-2/neu, EGF receptor, MUC-1, uPA/uPAR, and VEGF.

Efficient and scalable method for scaling up cell free protein synthesis in batch mode.

A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 microL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 microL reaction volume, the PANOx SP system produced 560 +/- 36 microg/mL of active CAT and 99 +/- 10 microg/mL of active GLH protein using the new thin film approach whereas 500 microL test tube reactions produced 250 +/- 42 microg/mL and 72 +/- 7 microg/mL of active CAT and GLH respectively. Similarly, 500 microL cell-free synthesis reactions with the Cytomim system produced 481 +/- 38 microg/mL of active CAT and 109 +/- 15 microg/mL active GLH respectively in thin films compared to 29 +/- 7 microg/mL of active CAT and 5 +/- 2 microg/mL of active GLH protein in 500 microL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume.

Whole cell ELISA for detection of tumor antigen expression in tumor samples.

We have developed a whole cell enzyme-linked immunosorbent assay (ELISA) that detects the melanoma antigens gp100 and S-100 in tumor samples from patients with metastatic melanoma and the antigen CA 125 in tumor samples from patients with ovarian carcinoma. The assay is relatively simple to perform and interpret without specialized expertise in pathology. It is both sensitive and selective and is amenable to being automated. The results correlate very well with those obtained by flow cytometry and are in good accord with published values obtained by immunohistochemistry. We believe that this assay should be very helpful for characterizing autologous cell cancer vaccines and may also represent a useful alternative to immunohistochemistry for cancer diagnosis. It should be adaptable to other types of cancer where tumor antigens have been identified and good antibodies are available.