Anti-drug Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities.

Use of In Vitro Assays to Assess Immunogenicity Risk of Antibody-Based Biotherapeutics.

An In Vitro Comparative Immunogenicity Assessment (IVCIA) assay was evaluated as a tool for predicting the potential relative immunogenicity of biotherapeutic attributes. Peripheral blood mononuclear cells from up to 50 healthy naïve human donors were monitored up to 8 days for T-cell proliferation, the number of IL-2 or IFN-γ secreting cells, and the concentration of a panel of secreted cytokines. The response in the assay to 10 monoclonal antibodies was found to be in agreement with the clinical immunogenicity, suggesting that the assay might be applied to immunogenicity risk assessment of antibody biotherapeutic attributes.

Preclinical development of AMG 139, a human antibody specifically targeting IL-23.

Background And Purpose: AMG 139 is a human anti-IL-23 antibody currently in a phase II trial for treating Crohn's disease. To support its clinical development in humans, in vitro assays and in vivo studies were conducted in cynomolgus monkeys to determine the pharmacology, preclinical characteristics and safety of this monoclonal antibody.

Experimental Approach: The in vitro pharmacology, pharmacokinetics (PK), pharmacodynamics and toxicology of AMG 139, after single or weekly i.

Development of a human antibody tolerant mouse model to assess the immunogenicity risk due to aggregated biotherapeutics.

We describe a novel human immunoglobulin G2 (IgG2 )-tolerant and immune-competent heterozygous mouse model (Xeno-het) developed by crossbreeding a human Ig-tolerized XenoMouse® with a C57BL/6J wild-type mouse. The Xeno-het mouse expresses both mouse and human immunoglobulin G (IgG) genes, resulting in B-cells expressing human and mouse IgG, and secretion of human and mouse Ig into serum. This model was utilized to evaluate the immunogenicity risk of aggregated and chemically modified human antibodies.

A detailed examination of the antibody prevalence and characteristics of anti-ESA antibodies.

Background: The antibody characteristics in erythropoiesis-stimulating agent (ESA)-treated patients who develop antibody-mediated pure red cell aplasia (PRCA; amPRCA) can be described as high-affinity, neutralizing anti-ESA antibodies with a mixed immunoglobulin G (IgG) subclass. The characteristics of an early-onset anti-ESA antibody response are not well documented, especially in the months prior to the development of amPRCA. Therefore, a detailed characterization of anti-ESA antibodies was performed in patients in both clinical studies and in a post-market setting.

The future of protein particle characterization and understanding its potential to diminish the immunogenicity of biopharmaceuticals: a shared perspective.

Biopharmaceuticals represent an important and growing class of medicines. Immunogenic responses to biopharmaceuticals in patients can sometimes result in reduced safety and efficacy. Although multiple factors are known to influence immunogenicity, our understanding of the complex underlying mechanisms remains imperfect.

Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses.

Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K.

Considerations for optimization and validation of an in vitro PBMC derived T cell assay for immunogenicity prediction of biotherapeutics.

An immune response to a biotherapeutic can be induced when the therapeutic is processed and presented by antigen presenting cell to T helper cells. This study evaluates the performance of an in vitro assay that can elicit antigen specific effector T cell responses. Two biotherapeutics with known clinical immunogenicity [FPX1 and FPX2] were assessed for their ability to induce antigen-specific IFN-γ secreting T cells in peripheral blood mononuclear cells (PBMC).

Molecular alterations in pediatric sarcomas: potential targets for immunotherapy.

Purpose/results/discussion. Recurrent chromosomal translocations are common features of many human malignancies. While such translocations often serve as diagnostic markers, molecular analysis of these breakpoint regions and the characterization of the affected genes is leading to a greater understanding of the causal role such translocations play in malignant transformation.

Intradermal vaccination of MUC1 transgenic mice with MUC1/IL-18 plasmid DNA suppresses experimental pulmonary metastases.

MUC1 (mucin 1) is a transmembrane glycoprotein normally expressed on epithelia of the pancreas, breast, prostate, colon, and lung. However, this self-antigen is over-expressed and aberrantly glycosylated in adenocarcinomas, thereby making it a potential target for immunotherapy. Toward this goal, DNA plasmids encoding human MUC1 (pMUC1) and mouse interleukin-18 (pmuIL-18) were developed, and previous work demonstrated pMUC1/pmuIL18 vaccination protected MUC1 transgenic mice (MUC1.

Engineering human IL-18 with increased bioactivity and bioavailability.

Cytokines in plasmid form can act as potent adjuvants when co-administered with DNA vaccines, resulting in an enhanced immune response to the DNA-encoded antigen. This is true of interleukin-18 (IL-18), which has been shown to serve as an adjuvant in conjunction with certain DNA vaccines. To determine if the properties of IL-18 could be optimized for use as a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to identify variants of human IL-18 that were predicted to improve receptor interactions and potentially bioactivity.

A MUC1/IL-18 DNA vaccine induces anti-tumor immunity and increased survival in MUC1 transgenic mice.

MUC1 (mucin 1) is a tumor-associated antigen that is overexpressed in many adenocarcinomas. Active immunotherapy targeting tumors expressing MUC1 could have great treatment value. MUC1 DNA vaccines were evaluated in MUC1 transgenic (MUC1.

Induction of dendritic cell maturation by IL-18.

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18.

Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy.

Background: There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical scale method to prepare autologous DCs for cancer clinical trials.

Methods: PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF.

Pilot trial of tumor-specific peptide vaccination and continuous infusion interleukin-2 in patients with recurrent Ewing sarcoma and alveolar rhabdomyosarcoma: an inter-institute NIH study.

Background: Patients with recurrent Ewing sarcoma and alveolar rhabdomyosarcoma have poor prognoses and limited therapeutic options. We have investigated the use of peptide pulsed vaccination in an attempt to immunologically target the breakpoint region of tumor specific fusion proteins expressed in these tumors.

Procedure: Sixteen patients with recurrent, translocation positive, Ewing sarcoma, and alveolar rhabdomyosarcoma underwent apheresis for collection of peripheral blood mononuclear cells.

Antigenicity of fusion proteins from sarcoma-associated chromosomal translocations.

Synovial sarcoma (SS), clear cell sarcoma (CCS), and desmoplastic small round cell tumor (DSRCT) are soft-tissue malignancies occurring primarily in adolescents and young adults. These tumors contain specific chromosomal translocations that fuse the 5' region of one gene with the 3' region of another, resulting in the formation of characteristic fusion proteins. These translocations are unique to tumor cells and may be required for persistence, thereby serving as targets for immunotherapy.

Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer.

Immunosuppression may contribute to the progression of cancer. In this study we assessed the structural and functional status of T cells from tumor specimens obtained from patients with early stage non-small cell lung cancer and late-stage ovarian cancer. Although some groups have described structural alterations in the TCR in patients with other malignancies, we did not observe decreased expression of the CD3zeta subunit in the tumor-associated T cells.

Differential effects of CD40 ligand/trimer stimulation on the ability of dendritic cells to replicate and transmit HIV infection: evidence for CC-chemokine-dependent and -independent mechanisms.

The role of exogenous stimulation of CD40 by CD40 ligand (CD40L) in dendritic cell (DC) maturation, CC-chemokine production, and CCR5 receptor expression was examined using a soluble trimeric CD40L agonist protein (CD40LT). Stimulation of monocyte-derived DCs with CD40LT enhanced the production of the CC-chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and RANTES and diminished surface expression of CCR5. Based on these findings, the functional role of CD40LT stimulation on the ability of DCs to replicate and transmit HIV viral infection was studied.

gp100/pmel 17 is a murine tumor rejection antigen: induction of "self"-reactive, tumoricidal T cells using high-affinity, altered peptide ligand.

Many tumor-associated antigens are nonmutated, poorly immunogenic tissue differentiation antigens. Their weak immunogenicity may be due to "self"-tolerance. To induce autoreactive T cells, we studied immune responses to gp100/pmel 17, an antigen naturally expressed by both normal melanocytes and melanoma cells.

CD40 ligand/CD40 stimulation regulates the production of IFN-gamma from human peripheral blood mononuclear cells in an IL-12- and/or CD28-dependent manner.

CD40 ligand (CD40L)/CD40 costimulation is an important regulator of Th1 responses. Two mechanisms by which CD40L/CD40 stimulation may enhance IFN-gamma are via direct induction of IL-12 and augmentation of the expression of costimulatory molecules such as B7 from APCs. We examined the ability of CD40L/CD40 stimulation to regulate the production of IFN-gamma through IL-12 and/or CD28 costimulation from human PBMCs stimulated with T cell-specific stimuli.

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein.

A challenge for subunit vaccines whose goal is to elicit CD8(+) cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin.

Delivery of antigens to the MHC class I pathway using bacterial toxins.

Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules. Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts. Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway.

Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine.

Recombinant poxviruses encoding tumor-associated antigens (TAA) are attractive as candidate cancer vaccines. Their effectiveness, however, will depend upon expression of the TAA in appropriate antigen-presenting cells. We have used a murine model in which the TAA is beta-galactosidase (beta-gal) and a panel of recombinant vaccinia viruses (rVV) in which beta-gal was expressed under early or late promoters at levels that varied over 500-fold during productive infections in tissue culture cells.

Genetic analysis of indefinite division in human cells: evidence for a common immortalizing mechanism in T and B lymphoid cell lines.

Somatic cell hybrids are useful for gaining insight into the process(es) by which normal human cells undergo senescence. In previous studies, we found that hybrids generated by fusing normal human diploid fibroblasts, T lymphocytes, and endothelial cells with various immortal human cell lines exhibited limited division potential. This leads to the conclusion that the phenotype of cellular senescence is dominant and that immortal cells arise due to recessive changes in normal growth control mechanisms.