Biological molecules are increasingly becoming a part of the therapeutics portfolio that has been either recently approved for marketing or those that are in the pipeline of several biotech and pharmaceutical companies. This is largely based on their ability to be highly specific relative to small molecules. However, by virtue of being a large protein, and having a complex structure with structural variability arising from production using recombinant gene technology in cell lines, such therapeutics run the risk of being recognized as foreign by a host immune system. In the context of immune-mediated adverse effects that have been documented to biological drugs thus far, including infusion reactions, and the evolving therapeutic platforms in the pipeline that engineer different functional modules in a biotherapeutic, it is critical to understand the interplay of the adaptive and innate immune responses, the pathophysiology of immunogenicity to biological drugs in instances where there have been immune-mediated adverse clinical sequelae and address technical approaches for their laboratory evaluation. The current paradigm in immunogenicity evaluation has a tiered approach to the detection and characterization of anti-drug antibodies (ADAs) elicited in vivo to a biotherapeutic; alongside with the structural, biophysical, and molecular information of the therapeutic, these analytical assessments form the core of the immunogenicity risk assessment. However, many of the immune-mediated adverse effects attributed to ADAs require the formation of a drug/ADA immune complex (IC) intermediate that can have a variety of downstream effects. This review will focus on the activation of potential immunopathological pathways arising as a consequence of circulating as well as cell surface bound drug bearing ICs, risk factors that are intrinsic either to the therapeutic molecule or to the host that might predispose to IC-mediated effects, and review the recent literature on prevalence and intensity of established examples of type II and III hypersensitivity reactions that follow the administration of a biotherapeutic. Additionally, we propose methods for the study of immune parameters specific to the biology of ICs that could be of use in conjunction with the detection of ADAs in circulation.

The process of nuclear protein transport requires the interaction of several different proteins, either directly or indirectly with nuclear localization or targeting sequences (NLS). Recently, a number of karyopherins ␣, or NLS-binding proteins, have been identified. We have found that the karyopherins hSRP1 and hSRP1␣ are differentially expressed in various leukocyte cell lines and could be induced in normal human peripheral blood lymphocytes. We show that the two karyopherins bind with varied specificities in a sequence specific manner to different NLSs and that the sequence specificity is modulated by other cytosolic proteins. There was a correlation between binding of karyopherins ␣ to different NLSs and their ability to be imported into the nucleus. Taken together, these data provide evidence for multiple levels of control of the nuclear import process.Active nuclear transport of proteins with molecular weights greater than 40 -60 kDa requires at least four different proteins, which act in a sequential manner with karyophilic proteins containing nuclear localization targeting sequences (NLS) 1 (1-4). There appear to be several discrete steps in the import process which involves: 1) binding of the NLS-binding protein, karyopherin ␣, to an NLS; 2) interaction of this complex with karyopherin ␤; 3) targeting to nuclear pore proteins; and 4) the ATP/GTP-dependent translocation through the nuclear pore mediated by ran (1,5,6).Recently, the proteins involved in NLS binding and transport have been identified. Those proteins that interact directly with the NLS have been termed karyopherins ␣ (7-11). The Xenopus protein importin 60 was the first karyopherin ␣ to be cloned, sequenced, and shown to be involved in nuclear protein import (7). Subsequently, a number of other karyopherins ␣ have been identified, which suggests that there is a family of these NLS-binding proteins. The two major groups of karyopherins ␣ include 1) the yeast protein SRP1 (12) and the human proteins hSRP1 and NPI-1 (8, 9), and 2) importin 60 (7) and the human proteins hSRP1␣ (11) and Rch1 (10). In this report we have termed hSRP1 and hSRP1␣, K1 and K2, respectively. Each of these karyopherins ␣ are capable of binding to NLSs and facilitating nuclear import. Recently it was shown that there was tissue-specific expression of the mouse K1 (mSRP1) and K2 (mPendulin). The levels of K1 RNA appear higher in the brain and cerebellum, whereas K2 RNA was found mostly in the thymus and spleen (13).Similar to karyopherin ␣, there are several homologs of karyopherin ␤, (also called importin 90 or p97) (11,14). The function of karyopherin ␤ appears to be the targeting of the karyophile-karyopherin ␣ complex to the nuclear pore (11,16). The interaction of karyopherin ␤ with karyopherin ␣ has been shown to enhance the latter protein's affinity for the NLS containing protein (1). Although the protein factors described above are sufficient to support nuclear protein transport, there are accessory factors which are also important for regulating nuclear transp...

The CD28/B7 costimulatory pathway is generally considered dispensable for memory T cell responses, largely based on in vitro studies demonstrating memory T cell activation in the absence of CD28 engagement by B7 ligands. However, the susceptibility of memory CD4 T cells, including central (CD62Lhigh) and effector memory (TEM; CD62Llow) subsets, to inhibition of CD28-derived costimulation has not been closely examined. In this study, we demonstrate that inhibition of CD28/B7 costimulation with the B7-binding fusion molecule CTLA4Ig has profound and specific effects on secondary responses mediated by memory CD4 T cells generated by priming with Ag or infection with influenza virus. In vitro, CTLA4Ig substantially inhibits IL-2, but not IFN-γ production from heterogeneous memory CD4 T cells specific for influenza hemagglutinin or OVA in response to peptide challenge. Moreover, IL-2 production from polyclonal influenza-specific memory CD4 T cells in response to virus challenge was completely abrogated by CTLA4Ig with IFN-γ production partially inhibited. When administered in vivo, CTLA4Ig significantly blocks Ag-driven memory CD4 T cell proliferation and expansion, without affecting early recall and activation. Importantly, CTLA4Ig treatment in vivo induced a striking shift in the phenotype of the responding population from predominantly TEM in control-treated mice to predominantly central memory T cells in CTLA4Ig-treated mice, suggesting biased effects of CTLA4Ig on TEM responses. Our results identify a novel role for CD28/B7 as a regulator of memory T cell responses, and have important clinical implications for using CTLA4Ig to abrogate the pathologic consequences of TEM cells in autoimmunity and chronic disease.

This volume covers many topics in the field of T-cell costimulation. The need for such a volume is testament to the growth of the field. From its beginning as a concept in the 1980s, we have now progressed to the point where many molecules now have functionally defined roles in T-cell costimulation. In addition, the field has progressed 'from bench to bedside'. Abatacept [cytotoxic T-lymphocyte antigen-4 (CTLA-4)-immunoglobulin (Ig) (CTLA-4-Ig)], an inhibitor of CD28-mediated T-cell costimulation, was approved for the treatment of moderate-to-severe rheumatoid arthritis in 2006 by the Food and Drug Administration and in 2007 by the European Medicines Agency. This chapter first presents a personal historical perspective on the early basic studies on the elucidation of the CD28/B7 T-cell costimulatory pathway and the discovery of CTLA-4-Ig. We next present an overview of studies of CTLA-4-Ig in preclinical animal studies. The material discussed in these first two sections is selective rather than exhaustive; their purpose is to provide context for the final section, a summary of human clinical studies performed with abatacept.

Deoxyspergualin (DSG) is a potent immunosuppressant whose mechanism of action remains unknown. To elucidate its mechanism of action, an intracellular DSG binding protein was identified. DSG has now been shown to bind specifically to Hsc70, the constitutive or cognate member of the heat shock protein 70 (Hsp70) protein family. The members of the Hsp70 family of heat shock proteins are important for many cellular processes, including immune responses, and this finding suggests that heat shock proteins may represent a class of immunosuppressant binding proteins, or immunophilins, distinct from the previously identified cis-trans proline isomerases. DSG may provide a tool for understanding the function of heat shock proteins in immunological processes.