Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

Phenotypic analysis of lymphoproliferative disorders is now considered mandatory for accurate classification which is the basis for optimum patient management. This is presently carried out in most cases using a range of antibodies recognizing B and T-cell antigens effective in paraffin sections, and an antibody to CD3 is currently a key member of such panels, indicating T-cell phenotype. Current antibodies to CD3 are polyclonal with the inherent disadvantages of this type of reagent compared to monoclonal antibodies. In this study, we have used a recombinant fusion protein representing part of the epsilon subunit of the CD3 molecule to generate a novel monoclonal antibody (NCL-CD3-PS1) effective in paraffin sections. The antibody has been characterized biochemically and by immunohistochemistry using a wide range of normal and pathological tissues. Lineage and phenotype specificity have been supported in our study and results from other laboratories are awaited with interest.     

Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

Phenotypic analysis of lymphoproliferative disorders is now considered mandatory for accurate classification which is the basis for optimum patient management. This is presently carried out in most cases using a range of antibodies recognizing B and T-cell antigens effective in paraffin sections, and an antibody to CD3 is currently a key member of such panels, indicating T-cell phenotype. Current antibodies to CD3 are polyclonal with the inherent disadvantages of this type of reagent compared to monoclonal antibodies. In this study, we have used a recombinant fusion protein representing part of the epsilon subunit of the CD3 molecule to generate a novel monoclonal antibody (NCL-CD3-PS1) effective in paraffin sections. The antibody has been characterized biochemically and by immunohistochemistry using a wide range of normal and pathological tissues. Lineage and phenotype specificity have been supported in our study and results from other laboratories are awaited with interest.     

Anti‐CD40 ligand monoclonal antibody delays the progression of murine autoimmune cholangitis

While there have been significant advances in our understanding of the autoimmune responses and the molecular nature of the target autoantigens in primary biliary cirrhosis (PBC), unfortunately these data have yet to be translated into new therapeutic agents. We have taken advantage of a unique murine model of autoimmune cholangitis in which mice expressing a dominant negative form of transforming growth factor β receptor II (dnTGFβRII), under the control of the CD4 promoter, develop an intense autoimmune cholangitis associated with serological features similar to human PBC. CD40‐CD40 ligand (CD40L) is a major receptor–ligand pair that provides key signals between cells of the adaptive immune system, prompting us to determine the therapeutic potential of treating autoimmune cholangitis with anti‐CD40L antibody (anti‐CD40L; MR‐1). Four‐week‐old dnTGFβRII mice were injected intraperitoneally with either anti‐CD40L or control immunoglobulin (Ig)G at days 0, 2, 4 and 7 and then weekly until 12 or 24 weeks of age and monitored for the progress of serological and histological features of PBC, including rigorous definition of liver cellular infiltrates and cytokine production. Administration of anti‐CD40L reduced liver inflammation significantly to 12 weeks of age. In addition, anti‐CD40L initially lowered the levels of anti‐mitochondrial autoantibodies (AMA), but these reductions were not sustained. These data indicate that anti‐CD40L delays autoimmune cholangitis, but the effect wanes over time. Further dissection of the mechanisms involved, and defining the events that lead to the reduction in therapeutic effectiveness will be critical to determining whether such efforts can be applied to PBC.     

Analysis of the tumour-associated antigen TAG-12 by monoclonal antibody 7A9 in normal,benign and malignant mammary tissues

Summary A monoclonal antibody 7A9 was raised against the tumour-associated glycoprotein TAG-12 purified from T47-D breast carcinoma cells. In immunoblots from cytosol of T47-D cells and from sera of breast cancer patients, antibody 7A9 detects the high molecular weight mucin-like TAG-12 antigen. A series of paraffin sections of normal, benign and malignant mammary tissues have been studied with monoclonal antibody 7A9 and the immunoalkaline phosphatase method. In resting gland, proliferating gland and fibroadenoma ducts, reactivity of 7A9 was mainly restricted to luminal membranes of epithelial cells and secretions. 77/79 primary breast carcinomas including ductal, lobular and various other carcinoma types showed cytoplasmic and/or membrane-associated staining with 7A9 in most tumour cells. Metastases (31/31) from different sites were also positive. Strong immunoreactivity with single tumour cells was noted in cytological preparations from freshly resected breast cancer tissue. Thus, monoclonal antibody 7A9 seems to be very useful for the targeting of breast carcinoma cells.     

Immunohistochemical staining of non-Hodgkin's lymphoma in paraffin sections using the MB1 and MT1 monoclonal antibodies

We have performed a single blind trial to assess the value of the monoclonal antibodies MB1 and MT1 in lymphoma classification. Sixty cases of non-Hodgkin's lymphoma (NHL) were stained with MB1 and MT1 using an indirect immunoperoxidase technique in paraffin sections. The majority of B tumours (27/33) stained with MB1, and most of the T tumours (24/27) stained with MT1. The MB1 antibody often produced rather weak staining but it was apparently highly specific for B cells, with only three (3/27) of the T tumours (two cases of 'malignant histiocytosis' of the intestine (MHI) and one pleomorphic T-cell lymphoma) displaying 'false' positivity. The MT1 antibody generally produced very strong staining, but it was not very selective, with 14/33 of the B lymphomas displaying 'false' positivity. the cross-reactivity observed in 17 cases led to only three misdiagnoses, two B tumours being designated as T lymphomas and one T tumour being designated as a B lymphoma. In a few cases (7/17), dual staining with both antibodies precluded firm diagnosis. In other cases (6/17), classification was possible despite some of the tumour cells showing dual staining. The seventeenth case was a plasmacytoma displaying MT1 positivity only. While the monoclonal antibodies MB1 and MT1 are of use in classifying lymphomas in paraffin section, they are not entirely lineage-specific, and the uncritical use of these two reagents alone may give rise to misdiagnosis; the use of a panel of monoclonal antibodies may yield more accurate results. As with any immunohistochemical marker, their limitations should be recognized; interpretation must be judicious and always in the context of the histological appearances.     

Application of monoclonal antibody 44-3A6 in the cytodiagnosis and classification of pulmonary carcinomas

Thirty-five pulmonary carcinomas were studied retrospectively with monoclonal antibody (MCA) 44-3A6 raised against a human adenocarcinoma cell line. The antibody was applied to cytologic smears of bronchial brushings originally stained with the Papanicolau method, and to conventional tissue sections. Ten of 12 adenocarcinomas (ADC) immunostained strongly in sections and smears, as did five of seven large-cell "undifferentiated" carcinomas (LCUC). Eight neuroendocrine carcinomas (NEC) and eight squamous-cell carcinomas (SCC) were negative, except for rare weakly positive foci. We conclude that MCA 44-3A6 can be effectively applied on cytologic smears, and that it could be valuable in the precise classification of pulmonary carcinomas. The immunoreactivity of the ADC and SCC was predictable. Positive immunostaining in some LCUC confirms that these constitute a heterogeneus tumor class that includes cases that are phenotypically ADC despite the absence of obvious glands. Occasional immunostaining in NEC suggests focal exocrine differentiation as previously noted by electron microscopy.     

Ligation of B7 with CD28/CTLA-4 on T cells results in CD40 ligand expression,interleukin-4 secretion and efficient help for antibody production by B cells

It has been extensively shown that when T cells are co-stimulated with B7-CD28 interaction, a strong proliferative as well as cytolytic T cell response can be induced. In contrast, there exists only indirect evidence that the B7-CD28 interaction is of importance for the induction of T cell helper functions in B cell responses. Here we have used mouse fibroblasts transfected with the human Fcγ receptor type II and human B7 to address this issue. We found that T cells, when activated through the T cell receptor (TcR)/CD3 complex with monoclonal antibodies and co-stimulated by B7-CD28 interaction, can provide efficient help for the induction of both IgM and IgG production by resting B cells. This helper activity is, at least in part, mediated by the interaction between the CD40 ligand on the T cells and CD40 on the B cells. We also demonstrate that more than one signal to the T cell is required for the induction of the CD40 ligand, one being delivered through the TcR/CD3 complex and the second by ligation of CD28 with the B7 molecule. In addition to the induction of cognate T helper function, we provide evidence that co-stimulation of T cells with B7-CD28 interaction can result in the secretion of both Th1- and Th2-type lymphokines.     

Characterization of a monoclonal antibody to a novel glycan-dependent epitope in the V1/V2 domain of the HIV-1 envelope protein,gp120

Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of additional antibodies to GDEs.     

Prevention of adjuvant arthritis by the W3/25 anti-CD4 monoclonal antibody is associated with a decrease of blood CD4(+)CD45RC(high) T cells

Imbalance between Th1 and Th2 functions is considered to play a key role in the induction and development of several autoimmune diseases, and the correction of that imbalance has led to effective therapies of some experimental pathologies. To examine whether CD4(+)CD45RC(high) (Th1-like) and CD4(+)CD45RC(low) (Th2-like) lymphocytes play a role in the pathogenesis of adjuvant arthritis (AA) and in its prevention by anti-CD4 antibody, CD45RC expression on CD4(+) T cells was determined in arthritic rats and in animals treated with an anti-CD4 MoAb (W3/25) during the latency period of AA. The phenotype of regional lymph node lymphocytes from arthritic rats in the active phase of the disease was determined by flow cytometry. Peripheral blood lymphocytes from rats treated with W3/25 MoAb were also analysed for 2 weeks after immunotherapy finished. IgG2a and IgG1 isotypes of sera antibodies against the AA-inducing mycobacteria, considered to be associated with Th1 and Th2 responses, respectively, were also determined by ELISA techniques. Fourteen days after arthritis induction, regional lymph nodes presented an increase in CD4+CD45RC(high) T cell proportion. Preventive immunotherapy with W3/25 MoAb inhibited the external signs of arthritis and produced a specific decrease in blood CD4(+)CD45RC(high) T cells and a diminution of antibodies against mycobacteria, more marked for IgG2a than for IgG1 isotype. These results indicate a possible role of CD4(+)CD45RC(high) T lymphocytes in the pathogenesis of AA, and suggest that the success of anti-CD4 treatment is due to a specific effect on CD4(+)CD45RC(high) T subset that could be associated with a decrease in Th1 activity.     

Formation of subepithelial dense deposits in rats induced by a monoclonal antibody against the glomerular cell surface antigen

We developed a monoclonal antibody, H5H3, of IgG1 subclass by hybridization technique using spleen cells of mice immunized with plasma membrane fraction of isolated rat glomeruli. H5H3 recognized main bands at about 220 kD by immuno-overlay technique and bound to the glomerulus as well as brush border of proximal tubules by indirect immunofluorescence (IF) microscopy on normal rat kidney frozen sections. By immunoelectron microscopy (IEM) it bound to the surface of mainly glomerular epithelial cell and weakly to the endothelial cell. After injection to Wistar rats it remained granularly in the glomerulus for more than 2 weeks seen by IF. When rats were preimmunized with murine IgG 4 days before the injection of H5H3, mouse IgG, rat IgG and C3 were strongly visible granularly in the glomerulus in 14 days by IF. Numerous dense deposits were formed at subepithelial area seen by transmission electron microscopy. Perfusion experiment of H5H3 into rat left kidney showed granular distribution of mouse IgG in 48 h, indicating that the reaction occurred in situ. H5H3 bound diffusely in fine granular pattern on the surface of cultured glomerular epithelial cells (GEC) studied by IF and IEM. Antigenic redistribution occurred on GEC after incubation of H5H3 at 37 C. These results suggested the required conditions to form subepithelial immune dense deposits, namely that H5H3 after reaction with antigen could stay for long time in the glomerulus; that H5H3 became an antigen in autologous phase to induce large immune complexes; and H5H3 could induce antigenic modulation.     

Divalency of the monoclonal antibody 5-1-6 is required for induction of proteinuria in rats.

Leucocyte-endothelial adhesion molecules have been implicated in the pathogenesis of inflammatory diseases. To evaluate their role as markers of disease activity in tuberculosis, we have used an antigen capture ELISA to measure the serum concentrations of circulating intercellular adhesion molecule-1 (cICAM-1), E-selectin (cE-selectin) and vascular cell adhesion molecule-1 (cVCAM-1) in 34 patients with active tuberculosis (27 with pulmonary disease and seven with lymph node disease) before the commencement of standard chemotherapy, 15 subjects who had previously completed treatment for pulmonary tuberculosis, and 27 healthy volunteers. Circulating ICAM-1 and E-selectin levels were significantly elevated in patients with active tuberculosis when compared to those with treated disease (P < or = 0.01), and healthy controls (P < 0.02). Circulating VCAM-1 was raised in patients with active or old pulmonary tuberculosis (P < 0.02 versus healthy controls) but not in those with tuberculous lymphadenitis. Significant correlations were observed between the levels of cICAM-1 and cE-selectin (p = 0.63, P = 0.0001), and between cICAM-1 and cVCAM-1 (p = 0.28, P = 0.016). Taking the mean +2 s.d. of the serum level in healthy controls as the upper limit of normal range, circulating ICAM-1 had the best discriminative power in identifying active tuberculosis, being elevated in about 80% of patients but was raised in only 6.7% of subjects with treated disease and in 3.7% of normal subjects. Our data support the possibility that three adhesion molecules may be involved in the pathogenesis of tuberculosis and cICAM-1 may be a useful marker of disease activity.     

A novel monoclonal antibody (FUN-1) identifies an activation antigen in cells of the B-cell lineage and Reed—Sternberg cells

The characterization of a new monoclonal antibody (MoAb) recognizing a human B-cell activation antigen, designated FUN-1, is described in this paper. Immunoprecipitation revealed that FUN-1 recognizes an antigen with a molecular weight (MW) of 75 kD. FUN-1 reacts with pokeweed mitogen-activated B lymphocytes and monocytes of peripheral blood, but not with unstimulated lymphocytes or granulocytes. It also reacts with large lymphoid cells in germinal centres, Epstein–Barr virus-transformed B cell lines, large B-cell lymphomas, Ki-1-positive anaplastic largecell lymphomas, and Reed–Sternberg cells of Hodgkin's disease, but not with Concanavalin A-activated T cells, acute lymphoblastic leukaemias, T-cell lymphomas, or low-grade B-cell leukaemias. These findings indicate that FUN-1 recognizes a previously unreported B-cell activation antigen. This MoAb appears to be useful for the study of maturation and differentiation in the B-cell lineage as well as for the immunohistochemical diagnosis of B-cell lymphomas and Hodgkin's disease.     

Identification of structural variations in the carboxyl terminus of Alzheimer's disease-associated beta A4[1-42] amyloid using a monoclonal antibody.

The accumulation of amyloid plaques and amyloid congophilic angiopathy (ACA) in the brains of affected individuals is one of the main pathological features of Alzheimer's disease. Within these deposits, the beta A4 (Ass) polypeptide represents a major component with the C-terminal 39-43 amino acid variants being most abundant. Using a mouse IgG1 MoAb produced by hybridoma beta A4[35-43]-95.2 3B9, which reacts with the epitope is defined by the amino acid residues beta A438[GVV]40, this study has identified a unique conformation within the carboxyl terminus of human beta A4[1-42]. Although the beta A438[GVV]40 sequence is present within the C-termini of human beta A4[1-40] and beta A4[1-43] and the beta A4-containing region of human APP, the beta A4[35-43]-95.2 3B9 MoAb (designated MoAb 3B9) does not bind these polypeptides, demonstrating a high degree of specificity for the beta A438[GVV]40 epitope as presented within the beta A4[1-42] sequence. The beta A4[1-42] epitope bound by MoAb 3B9 is sensitive to heating (100 degrees C for 5 min) and is denatured by SDS but not by oxidative radio-iodination of beta A4 or by adsorption to plastic surfaces or nitrocellulose. The recognition of beta A4 plaque deposits and ACA by MoAb 3B9 within formalin-fixed sections of human AD brain demonstrates the potential of these antibodies for investigating the role of the unique beta A4[1-42] conformation in the development of Alzheimer's disease.     

Use of the direct linear plot to estimate, in an immunoglobulin solution, the proportion which is specific monoclonal antibody against an unknown cell surface antigen

A Cornish-Bowden direct linear plot was used to assess the level of specific monoclonal antibody in a protein A preparation from mouse ascitic fluid. With this method, experimental observations are plotted directly as lines in parameter space and estimates of kinetic parameters are read directly from the plot without need for further calculation. This method is particularly well suited for the analysis of systems, such as the one outlined here, where conventional kinetic analysis is not possible because the preparation contains both specific and non-specific antibody. Results obtained with the direct linear plot showed that estimates of the level of specific immunoglobulin present in a given protein A preparation are consistent, not only within individual experiments, but also throughout a series of experiments using more than one labelled antibody preparation.     

Use of a monoclonal, anti Plasmodium berghei antibody, cross reacting with P. falciparum, for the detection of P. falciparum in in vitro infected blood

This report describes an immunoradiometric assay for Plasmodium falciparum in infected blood, based on a cross-reacting monoclonal antibody (mAb) raised against P. berghei. In this assay, binding of the mAb to intact P. berghei parasites coated on microtiter plates is inhibited by solubilized P. falciparum infected red blood cells. The use of P. berghei parasites in conjunction with monoclonal antibodies should facilitate the development of an inexpensive and reproducible test for the immunodiagnosis of malaria.     

Distinction of naive and memory BoCD4 lymphocytes in calves with a monoclonal antibody, CC76, to a restricted determinant of the bovine leukocyte-common antigen, CD45.

A murine IgG1 monoclonal antibody (mAb), CC76, has been produced that, based on findings of the relative molecular mass of polypeptides that it recognized, staining of leukocytes in blood and tissues, and the biological properties of the T lymphocyte subpopulations with which it reacts, is considered to identify an isoform of the leukocyte common antigen (LCA) family of molecules in cattle. The mAb is more similar to human CD45R which detect products requiring the presence of the B exon within the LCA gene and to the anti-rat mAb MRC-OX22, than to CD45RA or CD45R0. mAb CC76 reacts with an antigen expressed by subpopulations of cells in bovine blood that express BoCD2 and either the BoCD4 or BoCD8 antigens. T cells that express the gamma/delta T cell receptor identified with mAb to BoWC1 antigen did not react with CC76. The molecule detected is expressed on B cells but not on monocytes or granulocytes. Only 2% of cells in thymic suspensions stained with mAb CC76. Immature cortical thymocytes that were BoCD1+ did not react with CC76 and 90% of the cells in thymic suspensions that were CC76+ had the phenotype of mature thymocytes. These cells were primarily in the medulla. The LCA isoform detected thus appears to be acquired by mature cells shortly before emigration from the thymic medulla into the periphery. Expression of the molecule detected by mAb CC76 on cells from lymph nodes was similar to that in blood, but expression on cells from the gut mucosa was quite different. Almost all, 95% and 93% respectively, of the BoCD4+ cells in the gut mucosa or discrete Peyer's patches were CC76-. A greater proportion of BoCD8+ cells from these sites, 35% and 26%, expressed the antigen. Lymphocytes from animals that had been immunized with Trypanosoma brucei were sorted into BoCD4+, CC76+ and BoCD4+, CC76- populations and cultured in vitro with the variable surface glycoprotein antigen from the parasite. Lymphocyte transformation responses were entirely within the CC76- population indicating that the mAb distinguished naive from memory BoCD4+ T cells in cattle. Major histocompatibility complex (MHC) class I-restricted cytotoxic precursor cells that expressed the BoCD8 antigen sorted from cattle that were immune to Theileria parva were both CC76+ and CC76- indicating that different isoforms of the LCA may be expressed on MHC class I- and class II-restricted memory cells and that BoCD8 memory cells are heterogeneous with respect to the LCA isoform that they express.     

A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--I. Identification of a novel epitope on a killer cell surface bimolecular complex important in the cytotoxic response

This paper presents the initial characterization of a mouse monoclonal antibody (RH1-38) which blocks, in the absence of complement, three different systems of cell-mediated cytotoxicity. This monoclonal antibody markedly inhibits cytotoxicity mediated by human natural killer cells, a monocyte-like cell [phorbol myristate acetate (PMA) stimulated HL-60], and cytotoxic T-lymphocytes generated in a mixed leukocyte reaction. RH1-38 is not nonspecifically toxic to cells since antibody-dependent cellular cytotoxicity was not inhibited and viability as assessed by trypan blue exclusion was not affected. Inhibition is specific since control hybridoma culture supernatants, parent (NS-1) ascites supernatant, monoclonal anti-HLA and normal mouse IgG were not significantly inhibitory. In the NK system, the inhibitory effect appears to be due to binding of monoclonal antibody to effector cell surface since exposure of targets to antibody followed by washing yielded no inhibition of killing. Inhibition requires the antigen-binding portion of the antibody molecule and thus appears to be related to steric hindrance of an effector cell surface molecule which is important in the expression of cell-mediated cytotoxicity. Immunoprecipitation of surface-radioiodinated membranes from PMA-stimulated HL-60 cells and analysis on sodium dodecyl sulfate-polyacrylamide gels revealed a bimolecular complex (195,000 and 125,000 daltons) without significant change under reducing conditions. Control immunoprecipitates yielded no peaks of activity. This monoclonal antibody should serve as a useful probe of the function and biochemistry of a killer cell surface antigen important in the expression of cell-mediated cytotoxicity. Since RH1-38 inhibits cytotoxicity mediated by at least three apparently unrelated effector cells, the relevant antigen may be part of a common mechanistic step. As the companion paper demonstrates, this monoclonal antibody does not affect the conjugation step, but appears to block a late step in the NK cytolytic mechanism. Thus, RH1-38 recognizes either an epitope district from previously-described anti-LFA-1 antibodies or alternatively recognizes a distinct functional killer cell surface molecule.     

Partial and transient modulation of the CD3–T‐cell receptor complex,elicited by low‐dose regimens of monoclonal anti‐CD3, is sufficient to induce disease remission in non‐obese diabetic mice

It has been established that a total of 250 μg of monoclonal anti‐mouse CD3 F(ab′)₂ fragments, administered daily (50 μg per dose), induces remission of diabetes in the non‐obese diabetic (NOD) mouse model of autoimmune diabetes by preventing β cells from undergoing further autoimmune attack. We evaluated lower‐dose regimens of monoclonal anti‐CD3 F(ab′)₂ in diabetic NOD mice for their efficacy and associated pharmacodynamic (PD) effects, including CD3–T‐cell receptor (TCR) complex modulation, complete blood counts and proportions of circulating CD4⁺, CD8⁺ and CD4⁺ FoxP3⁺ T cells. Four doses of 2 μg (total dose 8 μg) induced 53% remission of diabetes, similarly to the 250 μg dose regimen, whereas four doses of 1 μg induced only 16% remission. While the 250 μg dose regimen produced nearly complete and sustained modulation of the CD3 –TCR complex, lower doses, spaced 3 days apart, which induced similar remission rates, elicited patterns of transient and partial modulation. In treated mice, the proportions of circulating CD4⁺ and CD8⁺ T cells decreased, whereas the proportions of CD4⁺ FoxP3⁺ T cells increased; these effects were transient. Mice with greater residual β‐cell function, estimated using blood glucose and C‐peptide levels at the initiation of treatment, were more likely to enter remission than mice with more advanced disease. Thus, lower doses of monoclonal anti‐CD3 that produced only partial and transient modulation of the CD3–TCR complex induced remission rates comparable to higher doses of monoclonal anti‐CD3. Accordingly, in a clinical setting, lower‐dose regimens may be efficacious and may also improve the safety profile of therapy with monoclonal anti‐CD3, potentially including reductions in cytokine release‐related syndromes and maintenance of pathogen‐specific immunosurveillance during treatment.     

Evaluation of safety and clinical activity of multiple doses of the anti-CD80 monoclonal antibody, galiximab, in patients with moderate to severe plaque psoriasis

Background: Reduction in lesional, activated T cells induces improvement in psoriatic plaques. Galiximab (IDEC-114), an IgG(1) anti-CD80 antibody, binds to CD80, a costimulatory molecule involved in T-cell activation. Objective: A Phase I/II, multidose, multischedule, dose-finding study of galiximab to evaluate safety, pharmacokinetics, and clinical activity was conducted in 35 patients with moderate to severe plaque psoriasis. Methods: Seven cohorts of five patients received galiximab intravenously on three different schedules at different dose levels. Results: Adverse events (AEs) commonly occurred as mild and self-limiting. Improvements were observed in most cohorts without evidence of a dose response in Psoriasis Area and Severity Index (50% or greater reduction in PASI score in 40% of patients), Physician's Global Psoriasis Assessment (PGA rating of Good or above in 57% of patients), and Psoriasis Severity Scale (PSS, baseline mean of 7.6 decreased by Study Day 127 to 5.0). An association was observed between reduction in CD3(+) cell count in histologic studies and reduction in PASI score. No antibodies to galiximab were detected. Conclusion: Galiximab appears to be safe and well tolerated with preliminary evidence of clinical and histologic response.     

A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--II. Evidence that the epitope recognized is involved in a late step in the cytolytic mechanism

A monoclonal antibody (RH1-38) which blocks multiple systems of cell-mediated cytotoxicity was functionally characterized. RH1-38 specifically blocks, in the absence of complement, natural killer (NK) activity (K562 targets) without any effect on NK-K562 conjugate formation. Kinetic studies suggested that the antibody blocks a step that occurs 30-120 min after effector populations are mixed with target cells. Single-cell cytotoxicity assays in agarose, combined with standard 51Cr release assays and Michaelis-Menten analysis revealed that RH1-38 markedly decreases Vmax and the number of active NK cells, again without any effect on the number of target-binding cells. The maximum recycling capacity was usually decreased, but in some experiments unchanged, in the presence of the monoclonal antibody. RH1-38 inhibited equally well whole peripheral blood mononuclear leukocytes (PBML), Percoll-fractionated lymphocytes enriched for NK activity, and interferon (IFN)-boosted NK activity. PBML exposed to RH1-38 and then washed mediated depressed NK activity which was partially reversed by subsequent treatment with IFN. These studies are most consistent with the hypothesis that RH1-38 inhibits a step late in the NK cytolytic mechanism rather than through an effect on conjugate formation. The primary effect is probably not on the IFN-generating or boosting mechanism, but a secondary effect on IFN-related mechanisms cannot be ruled out. Inhibition through an effect on a small lymphocyte modulator of NK activity is also unlikely but not rigorously excluded. Thus, RH1-38 appears to inhibit NK activity through a direct effect on NK effector cells, probably by interfering with a cell-surface molecule which is important in the expression of NK activity. The companion paper demonstrates that this monoclonal antibody immunoprecipitates a molecule which is very similar or identical to the LFA-1 antigen. Thus, RH1-38 recognizes either a novel epitope on the LFA-1 molecule or alternatively a distinct, functional killer cell surface molecule. The epitope appears to be involved in a late step in the cytolytic mechanism, possibly part of the effector cell lytic machinery.