Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody

A mouse/human chimeric antibody (ch14.18) was developed that reacts with the disialoganglioside GD2 on the surface of tumor cells of neuroectodermal origin. ch14.18 has the constant regions of a human IgG1 antibody and was expressed in a murine hybridoma. This antibody was produced in tissue culture at concentrations up to 180 mg/liter of spent culture fluid. ch14.18 was characterized and compared to 14.G2a, a murine mAb against GD2 of IgG2a isotype derived from the same parental hybridoma as ch14.18. Scatchard plot analysis of data from saturation binding studies on M21 melanoma cells showed identical binding for ch14.18 and 14.G2a. Indirect immunofluorescence revealed the same staining pattern for ch14.18 and 14.G2a on different melanoma cell lines. Both antibodies were equally capable of targeting M21 xenografts in athymic nude mice. ch14.18- and 14.G2a-activated human C-mediated cytolysis of melanoma cell; however, ch14.18-mediated antibody-dependent cytotoxicity of human effector cells against melanoma cells 50- to 100-fold more efficiently than 14.G2a.     

Specific inhibition of in vitro lymphocyte transformation by an anti-pan T cell (gp67) ricin A chain immunotoxin

The toxin A chain of ricin has been conjugated by a disulfide bond to a murine monoclonal antibody that recognizes the gp67kD antigen present on 95% of peripheral T lymphocytes. The immunotoxin retains both functions of its component parts: it binds to human peripheral blood lymphocytes, and it inhibits protein synthesis in a cellfree reticulocyte system. The immunotoxin has been evaluated for its ability to inhibit in vitro T lymphocyte transformation. In the presence of 20 mM NH4Cl, the immunotoxin decreases lymphocyte proliferation in response to phytohemagglutinin to less than 8% of untreated controls. The proliferative response in mixed lymphocyte culture and the development of allocytotoxic T cells is also dramatically inhibited by this immunotoxin. Monoclonal antibody alone does not inhibit these responses. Specificity of the immunotoxin has been established: the effect of the immunotoxin can be blocked by unconjugated monoclonal antibody, but not by a control monoclonal antibody that recognizes another T lymphocyte differentiation antigen or by a control monoclonal antibody that does not recognize human peripheral blood leukocytes. Treatment of human bone marrow cells with the immunotoxin preserves hematopoietic progenitor cells, as measured by granulocyte-macrophage, erythroid, and multipotential hematopoietic progenitor cell assays. These results indicate that an anti-pan T lymphocyte-ricin A chain immunotoxin is an effective agent against immunocompetent T lymphocytes in vitro, and may be an effective agent for use in clinical bone marrow transplantation.     

Engineered antibody approaches for Alzheimer's disease immunotherapy

The accumulation of amyloid-β-peptide (Aβ or A-beta) in the brain is considered to be a key event in the pathogenesis of Alzheimer's disease (AD). Over the last decade, antibody strategies aimed at reducing high levels of Aβ in the brain and or neutralizing its toxic effects have emerged as one of the most promising treatments for AD. Early approaches using conventional antibody formats demonstrated the potential of immunotherapy, but also caused a range of undesirable side effects such meningoencephalitis, vasogenic edema or cerebral microhemorrhages in both murine and humans. This prompted the exploration of alternative approaches using engineered antibodies to avoid adverse immunological responses and provide a safer and more effective therapy. Encouraging results have been obtained using a range of recombinant antibody formats including, single chain antibodies, antibody domains, intrabodies, bispecific antibodies as well as Fc-engineered antibodies in transgenic AD mouse and primate models. This review will address recent progress using these recombinant antibodies against Aβ, highlighting their advantages over conventional monoclonal antibodies and delivery methods.     

An immunotoxin for the treatment of T-acute lymphoblastic leukemic meningitis: studies in rhesus monkeys

Summary Monoclonal antibody WT1 (anti-CD7), conjugated to ricin A chain, was administered intrathecally to rhesus monkeys to test its suitability for use in the therapy of leukemic meningitis. The WT1-SMPT-dgRTA conjugate was cytotoxic to CEM (T-lymphoblastic leukemia) cells in vitro with an ID₅₀ of 53 pM. Immunoperoxidase testing showed no binding of WT1 to normal human tissues other than lymph nodes. Thirteen animals received one or more intrathecal 60-g doses of WT1-SMPT-dgRTA. All monkeys receiving repeated doses developed a cerebrospinal fluid (CSF) pleocytosis (primarily eosinophils), which was generally resolving by 3–4 weeks after therapy. Pharmacokinetic studies showed a half-life of 99 min, consistent with CSF clearance by bulk flow. Peak CSF immunotoxin concentrations exceeded the ID₅₀ for CEM cells by more than 2 log units and a concentration exceeding the ID₅₀ was maintained for as long as 24 h. All eight monkeys receiving repeated doses of immunotoxin developed serum antibodies against both WT1 and ricin A chain. In six of these monkeys antibodies were also present in the CSF. Both anti-WT1 and anti-(ricin A chain) antibodies were able to inhibit in vitro cytotoxicity of the immunotoxin for CEM cells; however, only anti-WT1 antibodies could block immunotoxin binding to the cell surface. No monkey developed anti-immunotoxin antibodies fewer than 7 days after the initiation of therapy, suggesting that repeated doses could be administered for up to 1 week without inhibition of clinical activity.     

Induction of multiple anti-c-erbB-2 specificities accompanies a classical idiotypic cascade following 2B1 bispecific monoclonal antibody treatment

 The bispecific monoclonal antibody (bsmAb) 2B1, targeting the extracellular domain of c-erbB-2, the protein product of the HER-2/neu proto-oncogene, and FcγRIII (CD16), expressed by human natural killer cells, neutrophils and differentiated monocytes, mediates the specific cytotoxic activity of these effector cells to tumor cells. A group of 24 patients with c-erbB-2-overexpressing tumors were treated with intravenously administered 2B1 in a phase I clinical trial and followed after treatment to evaluate the diversity and extent of the 2B1-induced humoral immune responses. As expected, 17 of 24 patients developed human anti-(murine Ig) antibodies (HAMA) to whole 2B1 IgG in a range from 100 ng/ml to more than 50 000 ng/ml; 10 of these patients (42%) had strong (at least 1000 ng/ml) HAMA responses, some of which were still detectable at day 191. These responses were usually associated with similar reactivity to the F(ab′)₂ fragments of the parental antibodies 520C9 (anti-c-erbB-2) and 3G8 (anti-CD16). We sought evidence of an idiotypic cascade induction, indicating a prolonged specific treatment-induced effect on at least one selected target of 2B1. Using competition-based enzyme-linked immunosorbent assays, specific anti-idiotypic antibodies (Ab2) were detectable against 520C9 in 11 patients and against 3G8 in 13 patients. Peak anti-idiotypic antibodies generally occurred 3–5 weeks from treatment initiation, with a downward trend thereafter. There was a statistically significant correlation among the induction of significant HAMA responses, anti-idiotypic antibody production and the development of antibodies to c-erbB-2. The anti-c-erbB-2 responses, which were distinct from anti-anti-idiotypic (Ab3) antibodies, were detected in the post-treatment sera of 6/16 patients examined. No obvious correlation could be made between the development of humoral immune responses, the dose received, and the clinical response. Future investigations involving 2B1 therapy will concentrate on investigating an association of these humoral responses to any c-erbB-2-specific cellular responses. Manipulations of 2B1 therapy effects that augment immunity to c-erbB-2 could provide additional avenues for immunotherapy with this and other bispecific antibodies. Received: 1 August 1996 / Accepted: 28 March 1997     

Evaluation and clinical relevance of patient immune responses to intravenous therapy with murine monoclonal antibodies conjugated to adriamycin

A retrospective study was performed in order to examine the clinical relevance of human anti-murine antibodies (HAMA) to concurrent clinical events in 21 patients receiving intravenous therapy with cocktails of murine monoclonal antibodies conjugated to Adriamycin. In vivo tumor localization of the murine antibodies was also evaluated. Serum levels of HAMA, human-murine immune complexes (HMIC), and murine antibodies were measured using an automated fluorescence immunoassay. Immunohistochemical staining was performed on frozen sections of tumor biopsies from eight of the patients to examine the in vivo binding of the murine antibodies. The patients were divided into low, intermediate, and high antibody dose groups. The incidence of allergic symptoms (80%) and HAMA correlation (75%) were highest in the low dose group. Specific IgM HAMA was the most highly correlated with allergic reactions, being present in 61.5% of the allergic patients. Thirteen of the 21 patients studied (61.9%) developed allergic symptoms after one or more doses of the murine monoclonal antibody conjugates. The percentages of total antibody doses in the patients' sera at varying intervals post-infusion varied widely from patient to patient for any given time point and dose, suggesting complex factors in the distribution and clearance of the murine antibodies. All eight of the patients biopsied during or post-therapy exhibited tumor localization of the murine monoclonal antibodies. Six of the eight had concurrent HAMA in their sera. Thus, the presence of HAMA did not prevent in vivo localization of the murine antibodies in the target tumors.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the clinical usefulness of a few sugar antigens and a galactosyl-transferase

Using human cultured cell lines or lymphocytes, two kinds of murine- and one human-monoclonal antibodies were produced, respectively and their clinical usefulness were investigated, and the possibility of galactosyl-transferase as a new tumor maker was also discussed. (1) A murine monoclonal antibody MSN-1, which was raised against human endometrial cancer cell line and recognized blood type sugar chain Leb, reacted with about 85% of endometrial cancer tissues, indicating that useful clinical information may be obtained by applying MSN-1 to immunohistochemistry and flow cytometry. (2) A new assay system using two murine monoclonal antibodies MA54 and MA61, which were raised against human lung cancer cell line and reacted with mucin sugar residues, revealed 76% positive rate in ovarian cancer patients, especially 82% in mucinous cystadenocarcinoma, indicating the clinical effectiveness as a new tumor maker compensating for the drawbacks of CA-125. (3) Galactosyl-transferase isozyme GT-2 was analyzed by the assay system using a newly produced monoclonal antibody. GT-2 was positive in 74% of ovarian cancers, especially in 89% of meso-nephroid cancer, indicating that GT-2 could be a useful tumor maker in ovarian tumors. (4) Human monoclonal antibody, which recognized "type 1 sugar chain" or iso-paragloboside, reacted about one half of endometrial cancer tissues. The production of human monoclonal antibody may contribute to the cancer imaging and the missile therapy.     

The effects of cyclophosphamide on the toxicity and immunogenicity of ricin A chain immunotoxin in rats

We conducted a study to determine if treatment with cyclophosphamide (CY) could suppress the formation of anti-murine and anti-ricin A chain antibodies in rats treated with a murine monoclonal antibody-ricin A chain immunotoxin (IT). Female Sprague-Dawley rats received intravenous doses of IT at a dose of 5 mg/kg body weight alone or in combination with CY at a dose level of either 10 or 20 mg/kg body weight. The IT was given as one or two courses consisting of five consecutive daily intravenous injections (days 0 to 4, or days 0 to 4 and days 21 to 25 of the study). Cyclophosphamide was given on days 2, 4, 6, 13, and 17 of the study to the group receiving a single course of IT; additional doses of CY were administered on days 23, 25, and 27 to the group receiving two courses of IT. On days 4, 14, 21, 28, and 35, animals from each group were evaluated for antibodies to murine IgG and ricin A chain, and for clinical laboratory parameters and histopathology. Animals receiving IT alone developed significant titers of both anti-murine and anti-ricin A chain antibodies. Compared with the response in the animals receiving single-course IT, the response to both of the components of the IT was significantly increased on days 28 and 35 in the animals receiving a second course of IT. The groups receiving a combination of either one or two courses of CY and IT demonstrated a significantly decreased antibody response to both the murine IgG and the ricin A chain compared with the group receiving IT alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneous antibody response to polyvalent melanoma vaccines in syngeneic mice

In this study, a human melanoma vaccine induced antibody responses in mice that varied significantly from animal to animal. BALB/c mice were immunized to a xenogenic human polyvalent melanoma vaccine that has been used in phase II clinical trials in over 600 patients. Mice were bled biweekly for up to 6 weeks to measure antibody responses. IgG antibody responses to the melanoma vaccine components were detectable within 2 weeks but were much stronger at 4 and 6 weeks. When the pooled sera were further analyzed by Western blot, a complex pattern of antigens was detected. When individual sera from identically immunized mice were assayed by Western blot, a consistent, reproducible pattern of antigen recognition was not seen. Rather, we found significantly different antibody responses among the mice. Both the intensity of antibody responses and the pattern of antigens recognized varied from animal to animal. Although there appeared to be immunodominant antigens that produced antibody responses in most mice, no single antigen induced antibody responses in all mice. These results demonstrate that polyvalent vaccines induce heterogeneous antibody responses in mice treated identically. Analysis of the response of selected melanoma patients immunized to the same vaccine revealed similar antibody responses to the antigens in the melanoma vaccine. Heterogeneity may hamper interpretation of vaccine immunogenicity and relevant tumor antigens in humans.     

Anti-idiotypic antibody identifies the cellular receptor of reovirus type 3

The binding and subsequent infectivity of reovirus to target cells are mediated by interaction with specific cell surface viral receptors. To gain a more detailed understanding of the biochemistry of the reovirus receptor and the cellular consequences of viral attachment, we have studied the binding of type 3 reovirus (Dearing strain) in a quantitative manner utilizing an antiidiotypic antibody probe. A syngeneic monoclonal antiidiotypic antibody (87.92.6) was prepared by immunization with hybridoma cells which secrete an antireovirus hemagglutinin-specific antibody. This antiidiotypic antibody was previously shown to specifically recognize the cell surface receptor for reovirus type 3. In this report, we demonstrate that antiidiotype mimicked reovirus tropism in binding to murine thymomas; antiidiotype inhibited the binding of reovirus to specific targets, but not the binding of anti-H-2; and cross linking of receptor-bound antiidiotype by antiimmunoglobulin induced patching, but not capping of reovirus receptors. Utilizing radiolabeled antiidiotype, we next quantitate the number of reovirus receptors on R1.1 and YAC thymoma cells and, finally, report on the preliminary identification of the reovirus receptor as a 67,000-Da membrane glycoprotein.     

Macrophage activation factor from EL-4, a murine T-cell line: antigenic characterization by hamster monoclonal antibodies to murine interferon-gamma

A cloned variant of the EL-4 murine T-cell line treated with phorbol myristate acetate (PMA) releases a factor that activates macrophages for nonspecific tumor cytotoxicity. This macrophage activation factor (MAF) is both physicochemically (Mr 25,000; pH 2 stable) and biologically different from interferon-gamma (IFN-gamma). However, EL-4 MAF may represent a breakdown product or otherwise altered fragment of IFN-gamma. We examined this possibility with a unique pair of hamster monoclonal antibodies against different epitopes of murine IFN-gamma. Both antibodies inhibited IFN-gamma-induced fibroblast antiviral activity; H21 but not H1 antibody also inhibited lymphokine (LK)-induced macrophage-mediated tumor cytotoxicity. Neither antibody, however, had any effect on the EL-4 MAF throughout a broad dose response. Moreover, passage through a H21 immunoaffinity chromatography column or addition of staphylococcal protein A and antibody completely inhibited LK-induced macrophage tumoricidal activity but did not affect the activity in EL-4 MAF. Identical effects in both fluid and solid phase were observed with polyclonal rabbit antisera to murine IFN-gamma. Results with all of these antibodies strongly suggest that the EL-4 MAF and murine IFN-gamma are antigenically distinct.     

High-dose,unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Summary Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to blockade nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 g of ¹³¹I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of ¹²⁵I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before ¹³¹I tumor-specific monoclonal antibody, will not enhance tumor imaging. Present address: Hybritech, San Diego, CA, USA     

Both VH and VL regions contribute to the antigenicity of anti-idiotypic antibody that mimics melanoma associated ganglioside GM3

Previously we developed a murine monoclonal anti-idiotype (antiid) antibody (4C10) that mimics the melanoma-associated ganglioside antigen GM₃, that is, it carries the internal image of GM₃. 4C10 was made against the human monoclonal antibody (HuMAb) L612, which reacts with several types of human cancer cells, including melanoma and breast cancer. To reduce mouse components of 4C10, the constant region was replaced by a human constant domain to form the murine/human chimeric anti-id antibody TVE-1. In the present study, we sought to determine which chain (V_H or V_L) of the anti-id is responsible for the antigenicity of GM₃. The TVE-1 V_H and V_L expression vectors were simultaneously transfected with either the V_H or V_L expression vector of a murine-human chimeric IgG antidansyl haptenic antibody, resulting in the construction of three different combinations of V_H and V_L chimeric antibodies. These IgG molecules were produced from the transfectomas, and their reactivity to HuMAb L612 was tested. Neither of the IgG proteins that had cross-combined the V_H-V_L pair showed positive results, suggesting that both heavy and light chains are required to express the antigenicity. The in vivo antigenicity of this chimeric anti-id was confirmed by skin tests in melanoma patients receiving active specific immuntherapy.     

Production of monoclonal antibodies against the B700 murine melanoma antigen and their antimetastatic properties.

Two unique murine melanoma antigens, termed B700 and B50, have been identified and isolated from several different murine melanoma cell lines. Both antigens can be detected on the cell surface, are actively shed in culture, and are often found in close association intracellularly. In previous studies, the antigen B700, which is related to serum albumin by biochemical and immunological criteria, was shown to function as a melanoma-specific tumor rejection antigen. We have also shown that animals sensitized to irradiated JB/RH melanoma cells produce antibodies which recognize B700 and/or B50, with B700 evoking the stronger humoral response. Animals testing positive by ELISA for antibody production to B700 or B50 were used for preparation of hybridomas and four different murine monoclonal antibodies have been produced whose specificities should facilitate epitope mapping. Clones have been used to generate ascites fluid in nude mice; the antibodies specifically recognize B700 and intact murine melanoma cells, but not B50. Two of these monoclonal antibodies have been administered systemically to C57Bl/6 mice bearing 5 day pulmonary metastases of the JB/MS melanoma, and significant inhibition of metastatic growth was observed for both antibodies.     

Monoclonal antibody storage conditions, and concentration effects on immunohistochemical specificity

Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.     

In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein.

Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies.     

Quantitation and intracellular localization of the 85K heat shock protein by using monoclonal and polyclonal antibodies.

Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by one-dimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.     

Remission of established disease in diabetic NOD mice induced by anti-CD3 monoclonal antibody]

Anti-CD3 monoclonal antibodies are potent immunosuppressants widely used in clinical transplantation to prevent or treat acute allograft rejection. We have used a hamster monoclonal antibody (145 2C11) specific for the epsilon chain of the murine CD3 complex to treat autoimmune insulin-dependent diabetes appearing spontaneously by 15 to 30 weeks of age in female Non Obese Diabetic (NOD) mice. Mice showing overt disease (glycosuria and glycemia > or = 4 g/l) were randomized in two groups receiving either anti-CD3 (5 micrograms/day i.v. for 5 consecutive days) or an identical dose of hamster polyclonal immunoglobulins. Progressive remission of disease was observed, 3 to 6 weeks after the end of treatment, in 80% of anti-CD3 treated mice as compared to 6% of mice in the control group. This remission was maintained long term namely, during the 4 to 5 months (after the end of treatment) observation period. These results open interesting perspectives on the possibility to treat recently diagnosed diabetic patients with therapy showing long term efficacy and no chronic toxicity.     

Single-chain antibody against human lipocalin-type prostaglandin D synthase: Construction, expression, purification, and activity assay

An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.     

Generation of human antibody fragments against <Emphasis Type="Italic">Streptococcus mutans</Emphasis>using a phage display chain shuffling approach

Background  

Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials.