Junming Le

Construction and initial characterization of a mouse-human chimeric anti-TNF antibody

Tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of a variety of human diseases including septic shock, cachexia, graft-versus-host disease and several autoimmune diseases. Monoclonal antibodies directed against TNF provide an attractive mode of therapeutic intervention in these diseases. We have generated a murine monoclonal antibody (A2) with high affinity and specificity for recombinant and natural human TNF. To increase its therapeutic usefulness, we used genetic engineering techniques to replace the murine constant regions with human counterparts while retaining the murine antigen binding regions. The resulting mouse-human chimeric antibody should have reduced immunogenicity and improved pharmacokinetics in humans. Molecular analysis of light chain genomic clones derived from the murine hybridoma suggests that two different alleles of the same variable region gene have rearranged independently and coexist in the same hybridoma cell. The chimeric A2 antibody (cA2) exhibits better binding and neutralizing characteristics than the murine A2 which was shown to contain a mixture of two kappa light chains. The properties of cA2 suggest that it will have advantages over existing murine anti-TNF antibodies for clinical use.

Interleukin 6: a multifunctional cytokine regulating immune reactions and the acute phase protein response.

Cytokines are protein mediators of cell-to-cell communication important in a variety of physiologic and pathophysiologic processes. Only a decade ago information about cytokines was limited to the description of a multitude of ill-defined “factors” present in crude super- natants of cultures of mononuclear cells. Modern techniques of protein chemistry and molecular biology have led to the isolation and precise functional definition of many cytokines, including the interferons, colony-stimulating factors, and a heterogeneous group of agents termed interleukins. The name “interleukins” was originally conceived to designate proteins produced by lymphocytes or monocytes, affecting the growth and/or differentiation of lymphoid, monocytic or myeloid cells. With time it has become apparent that many interleukins are not produced exclusively by white blood cells, nor are their actions restricted to leukocytes. The terms “interleukin” or “lymphokine” are now being used for cytokines that can be produced by a variety of cell types and that can affect the functions of many nonhematopoietic cells.

Monoclonal antibodies to human immune interferon and their application for affinity chromatography

Two IgG1/kappa class monoclonal antibodies specific for human immune interferon (IFN-gamma), designated B1 and B3, were developed. Specific binding of both monoclonal antibodies to natural or Escherichia coli-derived recombinant human IFN-gamma was demonstrated in a solid-phase radioimmunoassay or by immunoprecipitation. Antibody B3 showed potent neutralizing activity against both natural and recombinant IFN-gamma. Antibody B1, which showed neutralizing activity only when very high concentrations were employed, was used for preparing immunosorbents for affinity chromatography of IFN-gamma. When a highly purified preparation of 125I-labeled natural IFN-gamma was loaded onto the affinity column, all of the biological activity was retained on the column. The bulk of 125I-labeled IFN-gamma bound to the affinity column be eluted in biologically active form, suggesting that antibody B1 could be used for the purification of human IFN-gamma. Analysis of IFN-gamma eluted from the column by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE) indicated that both of the known molecular weight subspecies of IFN-gamma (25,000 and 20,000 MW), as well as the presumed dimer of 45,000 MW, were retained by the B1 antibody affinity column.

Synthesis of alpha and gamma interferons by a human cutaneous lymphoma with helper T-cell phenotype

Abstract A cloned human cutaneous lymphoma Hut102-B2 with helper T-cell phenotype (Leu1 + , Leu2a − , Leu3a + ) was found to produce substantial quantities of interferon (IFN) on induction with the phorbol ester, 12- O -tetradecanoylphorbol-13-acetate (TPA). Whereas only trace amounts of IFN were secreted by Hut102-B2 cells spontaneously, up to 8000 laboratory units/ ml of IFN were synthesized under the optimal conditions of TPA induction. Characterization studies including neutralization by specific antisera to IFNs and determination of the activities in human and bovine cells disclosed that the IFN produced by Hut102-B2 cells exposed to TPA was a mixture of immune IFN (IFN-γ) and leukocyte IFN (IFN-α) made in approximately equal amounts in terms of antiviral activity.

HIV-1 infection and modulation of cytokine and growth factor expression in Kaposi's sarcoma-derived cells in vitro.

ObjectivesHIV-1 transcripts have been detected in AIDS-related Kaposis sarcoma (KS) tissues within the factor Xllla + dermal dendrocytes present in the tumor. Various cytokines and growth factors have been shown to influence the growth of KS-derived cells in vitro. HIV-1 preferentially infects CD4 + cells and has also been found to infect some CD4− cells in vitro. The susceptibility of cultured KS cells in vitro to infection with HIV-1 and the expression of interleukin (IL)-1β, IL-6 and basic fibroblast growth factor (bFGF) after exposure to HIV-1 was examined. MethodsThe susceptibility of two different KS-derived cell cultures to HIV-1 infection was examined by the expression of p24 antigen, detection of proviral sequence and electron microscopy. The expression of IL-1β, IL-6 and bFGF was detected by enzyme-linked immunosorbent assay and reverse transcriptase polymerase chain reaction. ResultsKS-derived cells can be infected by HIV-1 in vitro. Both KS-derived cells were found to express CD4 mRNA. The expression of IL-1β and IL-6 was increased, whereas the expression of bFGF was not stimulated after exposure of KS cells to HIV-1. ConclusionThese experiments describe the in vitro infection of KS-derived cells by HIV-1 and the expression of various cytokines and growth factor following infection. The increased production of cytokines observed following such infection may be involved in the pathogenesis of AIDS-related KS.

Determination of human T cell activity in response to allogeneic cells and mitogens: An immunochemical assay for γ-interferon is more sensitive and specific than a proliferation assay

T lymphocytes proliferate and secrete lymphokines in response to allogeneic cells, mitogens and other stimuli. Cell proliferation as measured by [3H]thymidine ([3H]Tdr) incorporation into DNA has been routinely used to determine T cell responses in research and clinical laboratories. We have compared the sensitivity of an immunoradiometric assay (IRMA) for human gamma-interferon (IFN-gamma) (Chang et al., 1984), with that of the conventional [3H]Tdr incorporation assay in the measurement of T cell responses to antigens and mitogens in culture. Peripheral blood mononuclear cells (PBMs) were incubated in the presence and absence of phytohemagglutinin (PHA) or mononuclear cells from another individual for various periods of time. The culture fluids were collected for determining IFN-gamma and the cells were assayed for [3H]Tdr incorporation. Results of measurements were expressed in terms of stimulation indices. Both IFN-gamma secretion and thymidine incorporation were measurable in mixed lymphocyte cultures after incubation for 3 days, and in PHA stimulated culture after 24 h of incubation. The stimulation indices reflecting increased gamma-interferon were found to be more pronounced and more consistent than those of [3H]Tdr incorporation.

Activation of Thymocytes and T Cells by Interleukin-6a

Interleukin-6 (IL-6), also termed B cell-stimulatory factor 2 (BSF-2),’.* 26-kDa protein,’ or so-called interferon p,,“.’ is a pleiotropic cytokine with multiple functions on a wide range of target c e k 6 IL-6 induces the final maturation of B cells into antibody-forming cells, thereby enhancing immunoglobulin production in activated B cells as well as in Epstein-Barr virus (EBV)-transformed B lymphoblastoid cells.’ IL6 has been shown to function as a potent growth factor for murine B cell hybridomas and plasmacytomas,8 thus suggesting an essential role of IL-6 in the pathogenesis of multiple myeloma^.^ IL-6 also has been found to be an hepatocyte-stimulating factor regulating the major acute-phase protein response in liver cells.’o In addition, IL-6 appears to play an important part in stimulation of hemopoietic progenitors.’’ In this paper, we demonstrate that recombinant human IL-6 stimulates the proliferation of activated murine thymocytes and T cells via both interleukin-2 (IL-2)-dependent and IL-2-independent pathways. The activation of thymocytes can be down-regulated by cyclosporin A.

Interferon-γ enhances target cell sensitivity to monocyte killing

Abstract The mechanism of human peripheral blood monocyte-mediated cytotoxicity was investigated using the HT-29 human colon adenocarcinoma line, A673 human rhabdomyosarcoma line, and A375 human melanoma line as target cells. Pretreatment of these target cells with 100 U/ml of recombinant human interferon (IFN)-γ for 48 hr increased their susceptibility to monocyte killing. Increased susceptibility to the lytic action was particularly pronounced at low effector/target cell ratios. Unlike IFN-γ human IFN-α did not potentiate monocyte cytotoxicity, and pretreatment of HT-29 with IFN-α also had virtually no effect on their susceptibility to monocyte killing. However, IFN-γ appeared to prime either monocytes or target cells to become responsive to IFN-α. Our data suggest that IFN-γ can promote the killing of tumor cells by monocytes through two separate actions, one on the monocyte and one on the target cell.

Activation of human CD8-positive T cells via the CD8/HLA class I complex

Cross-linking of CD8 and HLA class I molecules with appropriate monoclonal antibodies (mAb) and goat anti-mouse Ig (GaMIg) antibody resulted in a marked proliferation of resting human CD8 cells in the presence of interleukin-2 (IL-2). These cells also expressed IL-2 receptor (IL-2R), transferrin receptor, HLA-DR and -DQ antigens. Activation of the cross-linked CD8 cells is apparently independent of accessory monocytes. Various anti-CD8 and anti-HLA class I mAb recognizing nonpolymorphic antigenic determinants were examined for the efficacy of activating CD8 cells. Among mAb specific for HLA class I molecules, PA2.6, MB40.5, BB7.7, A1.4, and W6/32 mAb markedly stimulated the proliferation of cross-linked CD8 cells, whereas BBM.1, Q1/28, and HC10 mAb were found inactive. Footprinting analysis of HLA class I molecules suggested that the activity of these anti-HLA class I mAb appeared to be related to the corresponding peptides they protect from enzymatic digestion. In contrast to the anti-HLA class I mAb, all anti-CD8 mAb examined (C8, OKT8A, and anti-Leu-2a) induced the proliferation of CD8-HLA class I cross-linked cells with similar efficacy. These results suggest that physical interaction between CD8 and at least one specific region of HLA class I molecules can trigger the activation of resting human CD8 cells.

[6] Induction of human interferon gamma with phorbol esters and phytohemagglutinin

Publisher Summary This chapter discusses the induction of human interferon gamma with phorbol esters and phytohemagglutinin (PHA). It is well known that immune interferon (IFN-γ) is produced by lymphocytes in response to specific antigens or nonspecific mitogens. The T cell mitogen, PHA has been widely employed as an inducer of IFN-γ in cultures of human mononuclear cells from peripheral blood. 12- O -tetradecanoylphorbol-13-acetate (TPA) (also termed phorbol myristate acetate (PMA)) is a potent tumor-promoting agent exerting hormone-like pleiotropic effects on growth, differentiation, and many other cell functions. Recently, it is shown that the cellular receptor for TPA and related phorbol esters is likely to be protein kinase C. Buffy coats can be employed as a source of cells instead of plateletpheresis residues. However, the average IFN-γ yields obtained from buffy coat-derived cultures are only about half of the yields obtained with cells from plateletpheresis residues. The reason for this difference is not known.