Ellen S. Vitetta

Immunotoxins: magic bullets or misguided missiles?

Thirteen years have passed since specific in vitro and in vivo killing of tumour cells by immunotoxins was first described. Why, then, has it taken so long to determine whether these drugs will have a major impact on the treatment of cancer, AIDS and autoimmune disease? The answer is that the transfer of basic discoveries to the clinic is a slow, multistep, interdisciplinary process. Thus, immunotoxin molecules must be designed and redesigned by the basic scientist depending on the efficacy and toxicity shown in vitro and in relevant experimental models. Next, each version must be evaluated by clinicians in humans through a lengthy process (1-3 years) in which the dose regimen is optimized and in which new problems and issues frequently emerge. These problems must again be modelled and studied in animals before additional clinical trials are initiated. In this article, Ellen Vitetta and colleagues discuss both basic and clinical aspects of the development of immunotoxin therapy.

Cellular Interactions in the Humoral Immune Response

Publisher Summary This chapter discusses the interactions between T and B cells and the role of accessory cells and cytokines in the generation of specific antibody responses. The first part of the chapter is a historic perspective followed by a summary of the present-day concepts. The final part of the chapter speculates on how the different components of the immune system might function in vivo. A highly complex system of communication has developed among the various cell types in the immune sytem. One important mechanism of communication is the requirement for interactions among cells for the activation and differentiation of resting B lymphocytes into antibody-secreting cells. These cellular interactions involve both cell-cell contact and the release of mediators (cytokines) that can act in either an autocrine or paracrine fashion on cells both within and outside the immune system. The chapter describes two types of functionally different Th cells and the role of lymphokines in the regulation of T and B cell function. Although Th1 versus Th2 cells have different effects on B cells and other cells in vitro, their role in vivo is less well understood. Nonetheless, studies in vivo have confirmed the requirement for IL-4 and IFN-У in IgE and IgG2, responses, respectively.

The protein products of the murine 17th chromosome. Genetics and structure.

Publisher Summary This chapter focuses on the analysis of the protein products of the murine 17th chromosome. Many of the gene complexes that map between the centromere and TLa play an essential role in cell–cell interactions and the control of immune responsiveness, embryogenesis, and differentiation. All the products of these genes are glycoproteins, most of which are expressed on cell surfaces presumably as receptors and often in association with β-microglobulin. The chapter discusses the present state of biochemical knowledge about these glycoproteins, focusses on their common features and interrelationships, and explores the implications of these qualities for the evolutionary origins of the associated genes. This segment of chromosome 17 acts in some ways as a “super gene” in which the genes and gene complexes coding for a number of functionally interrelated molecules are closely linked, presumably as a result of selective pressures. Significant data concerning the genetics, function, and structure of the I region and its products have been obtained. There is great heterogeneity in the I region. I -region genes and products are involved in a fundamental way with the regulation of the immune response.

The differential expression of homing and adhesion molecules on virgin and memory T cells in the mouse

We have used an anti-CD45R mAb to identify virgin and memory CD4+ T cells. In this report, we demonstrate that in mice, as in humans, virgin and memory T cells express different levels of adhesion molecules. Splenic CD45Rlo memory T cells express higher levels of PgP-1, Lyt-1, and LFA-1 than do CD45Rhi virgin cells. In contrast, CD45Rlo memory cells express lower levels of antigens identified by the mAbs. Mel-14 (leukocyte homing receptor) and SM3G11. These observations are in agreement with work in humans and rats which suggests that differences in the levels of adhesion molecules and homing receptors might be related to the efficiency of activation and patterns of recirculation among virgin and memory T cells.

IFN-γ enhances secretion of IgG2a from IgG2a-committed LPS-stimulated murine B cells: Implications for the role of IFN-γ in class switching☆

Abstract IFN-γ is a pleiotropic lymphokine that influences the isotypes of immunoglobulin secreted by B cells. IFN-γ inhibits the secretion of IgG3, IgG2b, IgG1, and IgE, and enhances the secretion of IgG2a. We have examined the mechanism of IFN-γ-mediated enhancement of IgG2a secretion in sorted populations of B cells and find that IFN-γ reproducibly stimulates a twofold increase in the precursor frequency of IgG2a-secreting cells in the sIgG2a+ population. Additionally, we find that IFN-γ does not induce an increase in the clone size of IgG2a-secreting cells. IFN-γ stimulates a twofold increase in the precursor frequency of IgG2a-secreting cells from sIgG− and unsorted B cells which can be attributed to an increase in IgG2a secretion from IgG2a-committed cells in these populations. Hence, under the culture conditions utilized in these studies, IFN-γ enhances IgG2a secretion from IgG2a-committed cells and does not induce a class switch.

Immunotoxins : Magic bullets or misguided missiles?

Thirteen years have passed since specific in vitro and in vivo killing of tumor cells by immunotoxins was first described. Why, then, has it taken so long to determine whether these pharmaceuticals will have a major impact on the treatment of cancer, AIDS and autoimmune disease? The answer is that the transfer of basic discoveries to the clinic is a slow, multistep, interdisciplinary process. Thus, immunotoxin molecules must be designed and redesigned by the basic scientist depending on the efficacy and toxicity shown in vitro and in relevant experimental models. Next, each version must be evaluated by clinicians in humans through a lengthy process (1-3 years) in which the dose regimen is optimized and in which new problems and issues frequently emerge. These problems must again be modeled and studied in animals before additional clinical trials are initiated. In this article, Ellen Vitetta and colleagues discuss both basic and clinical aspects of the development of immunotoxin therapy.

Cytotoxicity of a recombinant ricin-A-chain fusion protein containing a proteolytically-cleavable spacer sequence

Chimeric proteins composed of ricin toxin A chain (RTA) and staphylococcal protein A (PA) have been produced in E. coli. Constructs consisting of N‐terminal RTA and C‐terminal PA (RTA‐PA) or N‐terminal PA and C‐terminal (PA‐RTA) were capable of binding to immunoglobulin G (via PA) and of specifically depurinating 28 S ribosomal RNA (via RTA). However, neither fusion protein was cytotoxic to antigen‐bearing target cells in the presence of an appropriate monoclonal antibody presumably because the RTA could not be released from the PA within the cytosol where the ribosomal substrate of RTA is located. The overcome this, a short amino acid sequence from diphtheria toxin was engineered between the RTA and PA to produce a disulfide‐linked loop containing a trypsin sensitive cleavage site. Cleavage of this fusion protein with trypsin converted the RTA‐DT‐PA to the two chain form consisting of RTA linked by a disulfide bond to PA. The cleaved fusion protein was highly toxic to Daudi cells coated with anti‐immunoglobulin antibody suggesting that the RTA could be released from the PA by reduction within the cytosol.

The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and a hindered disulfide bond

The large scale preparation of two second generation immunotoxins containing murine monoclonal antibodies and deglycosylated ricin A chain is described. The procedure for the preparation of immunotoxins consists of the derivatization of antibody with SMPT and reduction of dgA with DTT followed by their reaction to establish a hindered interchain disulfide bond. The purification of the immunotoxin includes affinity chromatography on Blue-Sepharose to remove the free antibody and gel filtration on Sephacryl S-200HR to remove any high molecular weight material and free dgA. The two immunotoxins were prepared by GLP procedures and tested for yield, composition, purity, sterility and biological activity.

The toxicity of chemically deglycosylated ricin A-chain in mice

Tumor-reactive antibodies coupled to ricin or its A-chain (immunotoxins) have been used in rodents and humans to treat a variety of neoplastic diseases. Side-effects of such treatment include hepatotoxicity, vascular leak syndrome, myalgia and low grade fever. At high doses, severe toxicities include liver damage, pulmonary edema, aphasia, rhabdomyolysis and kidney failure. There have been a limited number of toxicologic studies on uncoupled ricin or its A-chain and none on deglycosylated A-chain. Since the latter has been utilized in second generation immunotoxins, the current studies were carried out to evaluate the toxicities induced by deglycosylated ricin A-chain (dgA) in mice. The administration of dgA to normal BALB/c mice causes early (24 h) weight loss and late (10 day) accumulation of ascites. These effects could be partially altered by changing the route of injection of dgA from i.v. to i.p. Thus, i.p. administration caused weight loss but not ascites, whereas i.v. administration caused both. Weight loss was associated with reduced fluid intake by the treated mice, and was not associated with increased levels of serum TNF-alpha. SCID mice injected with the same dose of dgA as normal BALB/c mice developed ascites, but it was of lesser severity, suggesting that a functional immune system, differences in microbial flora, or strain differences may be involved in the development of ascites.

Intracellular routing rather than cross-linking or rate of internalization determines the potency of immunotoxins directed against different epitopes of sIgD on murine B cells

Several variables influence the potency of an immunotoxin (IT) prepared with a monoclonal antibody (mAb) and ricin A chain (IT-A). These include the affinity of the mAb, the nature and density of the target antigen (Ag), the epitope on the target Ag bound by the mAb, the type of cell target, and the rate of endocytosis and route of internalization of the bound IT-A. In a previous report, we demonstrated that anti-delta mAbs directed against epitopes which are putatively more proximal to the plasma membrane make more effective IT-As than those directed against epitopes that are putatively more distal from the plasma membrane. It is known that the latter mAbs cross-link sIgD less effectively than the former. Therefore, in the present study, we determined whether the differential cytotoxicity of IT-As directed against these epitopes is related to their ability to cross-link their specific surface antigen (sIgD). We further determined whether they were internalized at different rates by normal B cells. Our results show that neither cross-linking nor rate of internalization account for the different potencies of anti-Fc vs anti-Fd IT-As. However, when these IT-As were used in the presence of the lysosomotropic agent chloroquine, the less potent IT-A became 100-fold more potent and was as cytotoxic as the effective anti-Fc IT-A. Taken together with the results of other studies, these findings further support the hypothesis that the epitope specificity of a given mAb may be an important factor in determining the intracellular routing of an IT-A after internalization.