Downstream processing of proteins: recent advances.

This review on the downstream processing of proteins describes innovations that have occurred in the field since 1983. Several areas have seen particularly high levels of achievement, and are accorded expanded coverage relative to our previous review [1]. As an example, the increasing integration of downstream operations with upstream technologies, such as molecular biology and fermentation, has led to the development of some very powerful processes. The degree to which organizations understand that there needs to be one unified process, rather than the independent steps of cloning, fermentation and recovery, seems directly related to the ultimate speed and success of the development effort. In 1983 one of the most active development areas was chromatography, especially affinity chromatography. This is still true today, and this topic has been expanded to include biospecific adsorptions that would not traditionally be classified as chromatography. With more proteins being developed for human administration, there has been an increased emphasis on all aspects of process hygiene. In addition, there has been much discussion about the impact of regulatory demands on the design and development of the manufacturing processes. Therefore, a section has been added which covers several of the regulatory issues that have been raised for products of the new biotechnology. Finally, as some of the early process development achievements are now beginning to bear fruit in the form of patents, we have increased our citation of this area of the literature.

Calcium dependency of antigen-specific (T3-Ti) and alternative (T11) pathways of human T-cell activation.

Human T lymphocytes are activated by two lineage-specific surface components: the antigen/major histocompatibility complex receptor (T3-Ti) and the unrelated T11 molecule. Interaction of either of these with their respective ligands leads to T-cell proliferation via an interleukin 2(IL-2) dependent autocrine mechanism. To begin to characterize the molecular details of the activation process, the role of Ca2+ was examined using human T-cell clones and monoclonal antibodies directed against their surface components. Here, we show that within minutes of triggering either the T3-Ti or T11 molecule, there is a large increase in intracellular Ca2+ concentration, as measured by quin-2 fluorescence. This is essential for induction of T-cell proliferation in inducer, suppressor, and cytotoxic clones and therefore presumably is required at an early step in the autocrine growth pathway. Thus, chelating exogenous Ca2+ with EGTA specifically inhibits proliferation triggered by anti-T3-Ti or anti-T11 monoclonal antibodies, but it does not affect triggering by exogenous IL-2. In addition, the Ca2+ ionophore A23187 can, by itself, initiate clonal proliferation.

An alternative pathway of T-cell activation: a functional role for the 50 kd T11 sheep erythrocyte receptor protein.

A series of seven monoclonal antibodies was produced against the T-lineage-specific 50 kd T11 sheep erythrocyte rosette (SRBC) receptor protein in order to define the function of the molecule. Three distinct epitopes were detected: T11(1), the SRBC binding site expressed on all T lymphocytes and thymocytes; T11(2), an epitope unrelated to the SRBC binding site but with a similar distribution; and T11(3), a neo-epitope expressed only upon T-cell activation. Simultaneous triggering of T11(2) and T11(3) epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen and/or antigen-presenting cells. This antigen-independent mode of triggering is distinct from that involving the T3-Ti antigen receptor complex and represents an alternate pathway of T-cell activation. Given that T11 is the earliest T-lineage surface glycoprotein to appear in thymic ontogeny and is thus expressed before T3-Ti, the former may be involved in clonal expansion and/or differentiation during early development.

Triggering of the T3-Ti antigen-receptor complex results in clonal T-cell proliferation through an interleukin 2-dependent autocrine pathway.

Human T-cell clones and anti-T-cell-receptor antibodies (clonotypic) directed at surface receptors for antigen (T3-Ti molecular complex) as well as anti-interleukin 2 (IL-2) and anti-IL-2-receptor antibodies were utilized to investigate the mechanism by which alloantigens or antigen plus self-major histocompatibility complex (MHC) (i.e., physiologic ligand) trigger specific clonal proliferation. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand, enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either the IL-2 receptor or IL-2 itself. These results suggest that induction of IL-2 receptor expression but not IL-2 release occurs in the absence of T3-Ti receptor cross-linking. Perhaps more importantly, the findings demonstrate that antigen-induced proliferation is mediated through an autocrine pathway involving endogenous IL-2 production, release, and subsequent binding to IL-2 receptors.

Identification of the receptor for antigen and major histocompatibility complex on human inducer T lymphocytes.

Human T cell clones and monoclonal antibodies directed at their surface structures were used to define the receptor for the antigen and major histocompatibility complex on inducer T lymphocytes. The results indicated that the receptor is a single complex consisting of the monomorphic T3 molecule with a molecular weight of 20,000 to 25,000 and a clonotypic disulfide linked heterodimer Ti with a molecular weight of 90,000. Sepharose-bound monoclonal antibodies (anti-Ti4 or anti-T3) to the receptor could activate clonal proliferation and inducer function for B cell immunoglobulin secretion and thus substitute for the appropriate combination of major histocompatibility complex gene product and specific antigen.

Peptide variability exists within alpha and beta subunits of the T cell receptor for antigen.

The T cell receptor for antigen (Ti) has recently been identified as a 90-kdalton T3-associated clonotypic structure composed of one 49-51-kdalton alpha and one 43-kdalton beta subunit, which are disulfide linked. Here, Ti molecules from two alloreactive CTL clones derived from the same donor but of differing specificities (CT8III and CT4II) are directly compared following isolation with anticlonotypic monoclonal antibodies. Isoelectric focusing shows that the alpha subunits (pI 4.4-4.7) are more acidic than the beta subunits (pI 6.0-6.2) but that each glycoprotein species is distinctive. More importantly, two-dimensional peptide maps of 125I-labeled surface receptors indicate that the beta chains of Ti1 and Ti2 appear unique and share only two peptides in common. In contrast, peptide maps of Ti1 and Ti2 alpha chains are more related although not identical. These results suggest that the human T cell receptor is composed of constant as well as variable regions and that at least one of the latter is located within the beta subunit.

Antigen-like effects of monoclonal antibodies directed at receptors on human T cell clones.

Recent studies suggested that the clonally unique Ti epitopes defined by non-cross-reactive monoclonal antibodies might represent the variable regions of the antigen receptor. Here we determine whether such anti-Ti antibodies could trigger clonal T cell activation. Anticlonotypic monoclonal antibodies to the 49/43-kdalton heterodimer of a given clone or antibodies to the 20/25-kdalton membrane associated monomorphic T3 molecule selectively induce proliferation and IL-2 secretion when linked to a solid support. In contrast, anti-T4 and anti-T8 antibodies under the same conditions have no effect. In conclusion, these results imply that anticlonotypic antibody functions in a fashion analogous to antigen and further support the notion that the T3-Ti molecular complex represents the antigen receptor on human T lymphocytes.

Immunoregulatory human T lymphocytes triggered as a consequence of viral infection: clonal analysis of helper, suppressor inducer and suppressor effector cell populations.

The regulatory functions of a series of human T cell clones specific for an autologous Epstein-Barr virus transformed B lymphoblastoid cell line were examined. Two T4+ T cell clones, termed AT4II and AT4IV, and one T8+ clone, AT8III, were maintained in culture for greater than or equal to 9 months and were characterized in detail. Both T4+ clones provided helper function for autologous B cell immunoglobulin production when added to unstimulated peripheral blood mononuclear cells. In addition, these same clones produced soluble inducer factors after specific antigenic stimulation. However, when AT4II, AT4IV and their subclones were tested on pokeweed mitogen stimulated peripheral blood mononuclear cells, it was found that AT4IV provided help for immunoglobulin production whereas AT4II cells were strongly suppressive. This suppression by AT4II was indirect and required the presence of fresh, autologous, unirradiated T8+ cells. In contrast, the T8+ AT8III clone markedly inhibited Ig production by autologous B cells in the absence of any additional T8+ cells from peripheral blood and produced a soluble suppressor factor upon specific antigenic triggering. Thus, after stimulation with autologous Epstein-Barr virus transformed cells, at least three discrete regulatory human T cell populations can be defined at the clonal level: helper, inducer of suppression and suppressor effector clones.

Comparison of T3-associated 49- and 43-kilodalton cell surface molecules on individual human T-cell clones: evidence for peptide variability in T-cell receptor structures.

Two monoclonal antibodies, anti-Ti1A and anti-Ti1B, were shown to define the clonally unique surface receptor on CT8III, a human cytolytic T lymphocyte specific for a class I major histocompatibility gene product. In the present report, this surface structure was characterized and related to the 20-kilodalton (kDa) T3 glycoprotein present on all mature human T lymphocytes. The results demonstrated that the anti-clonotypic antibodies react with an epitope on a disulfide-linked heterodimer of 49- and 43-kDa subunits exclusively expressed by CT8III. This structure is associated with T3 in the cell membrane. Similar T3-associated 49/43-kDa molecules were detected on eight additional clones, although these did not express the determinant defined by anti-Ti1A or anti-Ti1B. By probing clones of differing specificities derived from the same donor with anti-T3, it was possible to compare these T3-associated heterodimers. Biochemical analysis indicated that the 49/43-kDa structures, but not the T3 molecules themselves, had isoelectric point variability and unique peptide maps after digestion with chymotrypsin or staphylococcal protease V8. These findings support the idea that the 49/43-kDa heterodimer contains the variable region of the T cell's antigen receptor structure.

Human cytotoxic T cell clones directed at autologous virus-transformed targets: further evidence for linkage of genetic restriction to T4 and T8 surface glycoproteins.

Human cytotoxic T cell clones were generated against autologous EBV-transformed B lymphocytes. Whereas the majority of the clones expressed the T8 surface glycoproteins and showed a specificity for class I MHC gene products on the target cell, a minority expressed the T4 surface glycoprotein and demonstrated a class II specificity. Monoclonal antibodies to T4 and T8 inhibited cytotoxic effector function of reactive clones in a fashion analogous to their effect on alloreactive CTL clones. Each autoreactive T cell clone was cytotoxic for EBV-transformed B lymphocytes but not pokeweed mitogen-activated or resting autologous lymphocytes, suggesting a dual specificity for an MHC gene product as well as an antigen induced and/or encoded by virus. Taken together, the present findings provide further support for the notion that T4 and T8 serve as associative recognition elements on T lymphocytes for MHC gene products.

Evidence for the T3-associated 90K heterodimer as the T-cell antigen receptor.

Several surface molecules appear to be involved in antigen recognition by human T lymphocytes including the monomorphic 20/25K T3 structure present on all mature T lymphocytes and the subset-specific associative recognition elements, T4 and T8 (refs 1-8). More recently, Ti1, a clonally unique antigen recognition structure comprised of a 49,000 molecular weight (49K) alpha-chain and a 43K beta-chain, linked to T3 was identified on a major histocompatibility complex (MHC) class I specific T8+ T-cell clone, CT8III (ref. 9). To determine whether analogous receptor molecules could be found on other T-cell clones of differing specificity, we produced monoclonal antibodies against a clonal structure (Ti2) on an MHC class II specific T4+ lymphocyte, CT4II, derived from the same donor as CT8III. The Ti2 structure on CT4II is shown here to be a disulphide-linked heterodimer like Ti1 on CT8III and is composed of subunits of similar molecular weight. Monoclonal antibodies against Ti2 or Ti1 block antigen specific functions of the respective clone without showing any cross-reactivity. These findings suggest that each T lymphocyte, regardless of subset derivation or specificity, uses an analogous Ti heterodimer for antigen specific function. The latter is linked to T3 and expressed on the cell surface at an identical density (30,000-40,000 sites per cell).

Clonotypic structures involved in antigen-specific human T cell function. Relationship to the T3 molecular complex.

Monoclonal antibodies were produced against a human cytotoxic T cell clone, CT8III (specificity: HLA-A3), with the view of defining clonally restricted (clonotypic) surface molecules involved in its antigen recognition function. Two individual antibodies, termed anti-Ti1A and anti-Ti1B, reacted exclusively with the CT8III clone when tested on a panel of 80 additional clones from the same donor, resting or activated T cells, B cells, macrophages, thymocytes, or other hematopoietic cells. More importantly, the two antibodies inhibited cell-mediated killing and antigen-specific proliferation of the CT8III clone but did not affect the functions of any other clone tested. This inhibition was not secondary to generalized abrogation of the CT8III clone's function, because interleukin 2 responsiveness was enhanced. To examine the relationship of the structures defined by anti-clonotypic antibodies with known T cell surface molecules, antibody-induced modulation studies and competitive binding assays were performed. The results indicated that the clonotypic structures were associated with, but distinct from, the 20,000-mol wt T3 molecule expressed on all mature T lymphocytes. Moreover, in contrast to anti-T3, anti-Ti1A and anti-Ti1B each immunoprecipitated two molecules of 49,000 and 43,000-mol wt from 131I-labeled CT8III cells under reducing conditions. The development of monoclonal antibodies to such polymorphic T cell surface structures should provide important probes to further define the surface receptor for antigen.

Effects of leucine5 - and methionine5 - beta h - endorphin on behavior and electroencephalogram in cats.

Intracerebroventricular administration of leu5 - and met5 - beta h - endorphin produces sequential behavioral changes characterized by restlessness, eye fixation, head tremor, and exaggerated orienting reaction to external auditory stimuli, accompanied by EEG alterations. These changes take place during three stages. Met5- produces an increase in amplitude and reactivity of the theta rhythm in the hippocampus. Leu5- produces a masking of the theta rhythm by the appearance of spiking activity. Leu5- produces a loss of reactivity to visual stimuli not observed with met5-. The exaggerated reaction to auditory stimuli accompanied by an increased response of the reticular formation indicates a sensitivity of some mesencephalic structures (concerned with the modulation of sensory input) to both endorphins. Naloxone blockade of the subcortical (limbic) activity prominent in Stage II reveals the persistence of the first seen Stage I characteristics. These reticular-neocortical effects persist also into Stage III where they are seen intermixed with the limbic effects most prominent in Stage II. This suggests the presence of two endorphin-sensitive neural systems, only one of which is Naloxone reversible, and is that which in the cat covaries with the excited, hyperattentive state rather than the sedation and immobility phase seen in the rat. This dual system is compared to that described by Jacquet (13).

Surface structures involved in target recognition by human cytotoxic T lymphocytes.

Cloned human cytotoxic T lymphocytes and monoclonal antibodies inhibiting their function (anti-T3A, anti-T4A, and anti-T8A) were used to elucidate the role of T cell surface glycoproteins in cell-mediated lympholysis involving individual classes of gene products of the major histocompatibility complex on target cells. The results indicate that several surface molecules are required for specific target recognition: T3 and T4 on T4+ cytotoxic T lymphocytes and T3 and T8 on T8+ cytotoxic T lymphocytes.