Idiotypic Antibodies Research Articles

Isolation, production, and characterization of a new single chain anti-idiotypic antibody against benzo[a]pyrene

ABSTRACTPolycyclic aromatic hydrocarbons are chemical carcinogens which could induce the development of human cancers. Anti-idiotypic antibodies against benzo[a]pyrene (BP) are perspective for human cancer immunoprophylaxis and tumor immunodiagnostic techniques. The purpose of this study was to isolate anti-idiotypic antibodies against BP from human lymphocytes naïve phage library. The anti-idiotypic antibody, named B5, was selected. Analysis of the nucleotide and amino acid sequences B5 showed no similarity to known protein databases antibodies. B5 bound idiotypic antibodies against BP in direct and competitive ELISA. It was suggested that the B5 carried an immunological image of BP and bound the idiotypic antibodies against BP.Abbreviations: scFv: single-chain variable fragment; Ab1: idiotypic antibodies; Ab2: anti-idiotypic antibodies; CBD: cellulose binding domain; BSA: bovine serum albumin; PBS: phosphate buffer; BP-BSA: benzo[a]pyrene-BSA conjugate; Cr-BSA: chrysene-BSA conjugate; Py-BSA: pyrene-BSA conjugate; Ac-BSA: anthracene-BSA conjugate; Ba-BSA: benz[a]anthracene-BSA conjugate; PAH: polycyclic aromatic hydrocarbons; pSh: mouse idiotypic single-chain variable fragment against benzo[a]pyrene; T72: human idiotypic single-chain variable fragment against benzo[a]pyrene.

New human single chain anti-idiotypic antibody against benzo[a]pyrene.

The nal¨ve library from the lymphocytes of healthy humans was screened by murine single-stranded idiotypic antibodies against benzo[a]pyrene (pSh). The phage clone which contained of anti-idiotypic antibody against benzo[a]pyrene, designated as A4, was chosen for further work because of highly specific to pSh. The available protein databases were searched. The A4 amino acid sequence was unique and 76% identical to a sequence in antibody against interferon g. The A4 protein was expressed in bacteria and purified by two different methods: His-tagged A4 and CBD-fusion A4. Both the A4 bound to pSh and also to the human single chain idiotypic antibody against the benzo[a]pyrene (T72) by ELISA. The Kd values of A4 for pSh and T72 were very close: 4.44 × 10-7 M and 5.71 × 10-7M, respectively. A4 was a competitor with benzo[a]pyrene for binding sites of both idiotypic pSh and T72 in competitive ELISA. Thus, A4 was a high affinity anti-idiotypic against benzo[a]pyrene which recognised pSh and T72 active sites.

Autoantibodies to Neuropeptides in the Different States of Opium Addiction

During the last years the addiction rate remains stable high. While the neurochemical drug effect remains unclear. To analyze the changes of the idiotypic (аАТ1) and anti-idiotypic (аАТ2) autoantibodies to the neuroproteins S100, MBP, GFAP, NGF on the different stages of opium addiction and to indicate prognosis criteria of their effect. 70 patients (only men, aged 22−38) with diagnosis opium addiction underwent examination. According to the results of testing, we detected the intoxication in 24 patients, withdrawal ― in 24, and 22 patients were at remission stage of 21−28 days. The control group included only healthy people (n=18). The survey was focused on the rate detection of the idiotypic and anti-idiotypic IgG class antibodies in relation to the rate of neural proteins (S100, MBP, GFAP, NGF) in the serum with the IEA. In patients with opium intoxication, we revealed statistical assurance in the rate of autoantibodies amount and their counterweights to the neural proteins rate between control and experimental groups. Only the rate of the аАТ2 protein significantly decreased relatively to the MBP. In patients with abstinence, the rate of аАТ1 to the MBP, GFAP (р≤0,05) increased. The rate of аАТ2 in relation to the GFAP and MBP also increased (р≤0,05), at the same time it decreased in relation to the S100 and NGF (р≤0,05). The autoantibodies amount at the remission stage corresponded to the amount at the intoxication stage. The comparative analysis of the patient groups with the different stages of opium addiction detected the identity criteria both in the intoxication and remission. We revealed statistical assurance in the rates of аАТ1 to MBP and аАТ2 to NGF in patients with intoxication and abstinence, and in the rates of аАТ1 to GFAP, MBP, and аАТ2 to GFAP (decreased in the remission) and to S100, MBP (increased in the remission) in patients with abstinence and at remission. Levels of idiotypic and anti-idiotypic antibodies to the neural proteins S100, MBP, GFAP, NGF (especially аАТ2 to MPB) could be used as diagnostic factor and for accessing different states of opium addiction.

A non-toxic enzyme-linked immunosorbent assay for aflatoxin B1 using anti-idiotypic antibodies as substitutes.

Immunoassays are widely employed techniques to detect aflatoxins since they are rapid, selective and sensitive. One common disadvantage of them is using aflatoxins as standard substances, which may trigger exposure risks to operators and environmental contamination without proper handling. Anti-idiotypic antibodies (anti-Ids or Ab2s), also named as internal-image anti-Ids, are able to mimic and function as antigens, so a non-toxic enzyme-linked immunosorbent assay (ELISA) for aflatoxin B1 (AFB1 ) is developed and validated using anti-Ids as substitutes. Mouse monoclonal anti-idiotypic antibody (McAb2) to AFB1 was generated by the hybridoma technique using Fab fragments of rabbit anti-AFB1 idiotype antibody (Ab1) as immunogen. As indicated by indirect competitive ELISA, McAb2, represented an internal-image of antigen AFB1 , was able to bind Fab with competition to AFB1 . Then, analysis of AFB1 in spiked samples by non-toxic ELISA using anti-Ids as substitutes was developed, and it showed no significant differences with comparison to AFB1 as competitive antigens. Our work demonstrated that anti-Ids could be used as internal-image mimicry of AFB1 , and it had potential applications in immunoassays for antigen substitution to reduce operational risk for operators and environmental contamination. © 2016 Society of Chemical Industry.

Antibody idiotype plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity (ADCC) against influenza A virus

Abstract The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. Recently, elicitation of broadly-neutralizing antibodies which target the conserved hemagglutinin stalk domain have emerged as a promising “universal” influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions, such as antibody-dependent cell-mediated cytotoxicity, has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza-binding antibody idiotypes in vivo has never been defined. Here, we demonstrate that complex interactions among viral glycoprotein-binding antibody idiotypes regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We propose that antibody idiotype serves as a critical determinant in the activation of ADCC. This is the first demonstration that cross-talk among antibody idiotypes is important for the regulation of Fc-dependent effector functions. This phenomenon will have major implications for not only the development of universal influenza virus vaccines, but also in any scenario wherein Fc-dependent effector functions are engaged in the context of polyclonal responses containing multiple antibody idiotypes.

Anti-bevacizumab idiotype antibody vaccination is effective in inducing vascular endothelial growth factor-binding response, impairing tumor outgrowth.

Tumors require blood supply and, to overcome this restriction, induce angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in this process, which explains the great number of antiangiogenic therapies targeting VEGF. The research and development of targeted therapy has led to the approval of bevacizumab, a humanized anti‐VEGF monoclonal antibody (mAb), in clinical settings. However, side effects have been reported, usually as a consequence of bolus‐dose administration of the antibody. This limitation could be circumvented through the use of anti‐idiotype (Id) antibodies. In the present study, we evaluated the efficacy of an active VEGF‐binding immune response generated by an anti‐bevacizumab idiotype mAb, 10.D7. The 10.D7 anti‐Id mAb vaccination led to detectable levels of VEGF‐binding anti‐anti‐Id antibodies. In order to examine whether this humoral immune response could have implications for tumor development, 10.D7‐immunized mice were challenged with B16‐F10 tumor cells. Mice immunized with 10.D7 anti‐Id mAb revealed reduced tumor growth when compared to control groups. Histological analyses of tumor sections from 10.D7‐immunized mice showed increased necrotic areas, decreased CD31‐positive vascular density and reduced CD68‐positive cell infiltration. Our results encourage further therapeutic studies, particularly if one considers that the anti‐Id therapeutic vaccination maintains stable levels of VEGF‐binding antibodies, which might be useful in the control of tumor relapse.

Bioanalysis of bevacizumab and infliximab by high-temperature reversed-phase liquid chromatography with fluorescence detection after immunoaffinity magnetic purification

This study presents two simple and rapid methods for the quantification of therapeutic mAbs based on LC. Two mAbs (bevacizumab and infliximab) in plasma samples were purified using magnetic beads immobilized with a commercially-available idiotype antibody for each mAb. Purified mAbs were separated with HT-RPLC and detected with their native fluorescence. Using immunoaffinity beads, each mAb was selectively purified and detected as a single peak in the chromatogram. The HT-RPLC achieved good separation for the mAbs with sharp peaks within 20 min. The calibration curves of the two mAbs ranged from 1 to 20 μg mL−1 (bevacizumab) and 1–10 μg mL−1 (infliximab), and they had strong correlation coefficients (r2 > 0.998). The LOD of bevacizumab and infliximab was 0.07 and 0.15 μg mL−1, and the LLOQ of bevacizumab and infliximab was 0.12 and 0.25 μg mL−1, respectively. Thus, the sensitivities were sufficient for clinical analysis. Immunoaffinity purification with HT-RPLC produced a selective and accurate bioanalysis without an LC-MS/MS instrument. Both methods could become general-purpose analytical methods and complement the results obtained with conventional LBA.

A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5μg/mL) in the presence of 0.78μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function.

Anti-idiotypic nanobody as citrinin mimotope from a naive alpaca heavy chain single domain antibody library.

Compared with peptide-based mimotope, anti-idiotypic antibodies (AIds) are considered as promising biosynthetic surrogate antigen because these antibodies display stable protein conformation. Nevertheless, conventional AIds are generated by immunizing animals with heterologous idiotypic antibody in vivo; isolated AIds commonly exhibit a higher affinity to primary antibodies than target analytes because AIds undergo an affinity-matured process during immune responses, resulting in low sensitivity in competitive immunoassay. In the present study, an anti-citrinin monoclonal antibody (anti-CIT McAb) was designed as primary antibody; one β-type AI alpaca heavy chain single domain antibody (β-AI VHH) was selected as a citrinin (CIT) surrogate from a naive phage-displayed VHH library. The affinity constant (K D) of obtained β-AI VHH to anti-CIT McAb (160 nM) is 2.35 times lower than that of CIT and ovalbumin conjugates (CIT-OVA) to anti-CIT McAb (68 nM). The developed VHH-based enzyme-linked immunosorbent assay (V-ELISA) can be used to perform dynamic linear detection of CIT in 10% (v/v) methanol/PBS from 5.0 to 300.0 ng/mL, with a median inhibitory concentration (IC50) of 44.6 ng/mL (n = 3); this result was twice as good as that of indirect competitive ELISA (ic-ELISA, IC50 = 96.2 ng/mL) with CIT-OVA as a coating antigen. Moreover, the precision of V-ELISA was evaluated by analyzing average recoveries and coefficient of variations of CIT-spiked cereal sample; the reliability of V-ELISA was also validated with a conventional ic-ELISA. In summary, the proposed strategy has a great potential for panning other β-AI VHH toward small organic molecules from a naive VHH library.

Intracellular expression of an anti-idiotypic antibody single-chain variable fragment reduces porcine reproductive and respiratory syndrome virus infection in MARC-145 cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome; it is one of the most economically important viral diseases affecting the swine industry worldwide. At present, neither live-attenuated nor inactivated PRRSV vaccines can provide sustainable disease control. Our previous studies have demonstrated that PRRSV infection can produce the auto-anti-idiotypic antibodies (aAb2s) specific to the idiotypic antibodies against PRRSV GP5, which plays an important role in the host immune responses to PRRSV infection. In the present study, a single-chain variable antibody fragment (scFv) from the monoclonal anti-idiotypic antibody specific for the idiotypic antibody against GP5 was expressed in MARC-145 cells and its effect on virus infection in vitro was evaluated. An scFv was constructed from the anti-idiotypic antibody (Mab2-5G2) and was named 5G2scFv. The lentiviral vector system was used as a vehicle to deliver 5G2scFv into MARC-145 cells. The effect of 5G2scFv expression in MARC-145 was analysed by determining the PRRSV N protein level and the virus titre in the supernatant. Virus attachment and the level of type I interferon (IFN) were determined to elucidate the mechanism of the scFv effect. 5G2scFv was delivered in MARC-145 cells using the lentiviral vector system as confirmed by the western blot and indirect immunofluorescence assays. The PRRSV challenge experiments demonstrated that expressed 5G2scFv in MARC-145 strongly reduced PRRSV infection and replication by inhibiting protein synthesis and progeny virus production. This effect was not due to the change of viability or virus binding, but increased IFN-α at messenger RNA and protein levels. The expression of the anti-idiotypic antibody 5G2scFv in MARC-145 cells has the interferential effect on PRRSV infection in the cells by induction of IFN-α, which provides a novel therapeutic approach for PRRSV infection.

THE IMMUNOLOGICAL STRUCTURE OF POPULATION OF KARAKUL LAMBS BUCCINATED AGAINST CONDITIONALLY AND ACUTE PATHOGENOUS BACTERIA

Antibodies, developing in animal and human organisms against antigens of external and internal origin are not limited with opsonization functions, lying on the base of formation of humoral immunity. Being the product of somatic mutations of plasmatic cells, according to clonal selection theory of immunity, they themselves are also alien proteins for organisms. Thus, they themselves also begin to display antigen pressure on the immune competent system. So, they will start themselves the elaboration of antibodies against them, which are called anti–idiotypic ones. Anti–idiotypic antibodies, imitating the corresponding bacterial and virus antigens are able to initiate the immune response of animal organism against them, which play great immune modulating role. The immunological structure of populations of Karakul sheep lambs vaccinated against E. coli bacteria, salmonellosis and anthrax antigen from the age of 2–3 days till 2–3 weeks was studied. Experiments were carried out in the 1–2, 3–4, 5–6, 7–8, 9–10 and 11–12 weeks old Karakul lambs. Experimental lambs during appraisal were vaccinated once by associated hydroxide aluminum form vaccine against paratyphoid and coli bacteriosis of calves, pigs and lambs. In another experiment, the lambs were immunized by rare naturally occurring bacterial antigen. As such an immunogenic was selected sterile soluble anthrax antigen. Developed and tested in the application of Ascoli reaction the role of anti–idiotype antibodies in the formation of natural immunity against opportunistic pathogens identified the nature of normal antibodies was studied. The nature of wavelike character of the immune reaction of an organism of newborn animals of vaccination was revealed. Also the immunomodulation role of anti–idiotypic antibodies was determined.

Single-chain anti-idiotypic antibody retains its specificity to porcine reproductive and respiratory syndrome virus GP5

Monoclonal anti-idiotypic antibody (Mab2-5G2) raised against idiotypic antibodies to membrane glycoprotein GP5 of porcine reproductive and respiratory syndrome virus (PRRSV). The variable regions of the heavy chain (VH) and light chain (VL) of Mab2-5G2 were cloned and connected with a (Gly4Ser)3 linker. The recombinant scFv gene was cloned into the pEasy-E1 vector and expressed in E. coli as inclusion bodies. The expressed scFv-His proteins renatured in a pH and urea gradient buffer retained the same immunological properties as that of Mab2-5G2. Renatured scFv-His protein retained the same characteristics as that of Mab2-5G2 by recognizing and binding to Marc-145 cells. Furthermore, renatured scFv-His along with Mab2-5G2 were used to immunize rabbits to produce anti-anti-idiotypic antibodies (Ab3) that neutralized PRRSV infection of Marc-145 cells. These results demonstrated that the expressed scFv-His protein possessed the same characteristics of Mab2-5G2 and will be suitable for future investigations of Mab2-5G2 antibody structure and its ability to interact with potential PRRSV cellular receptor as well as immunological properties against PRRSV infection.

The idiotypic network in the regulation of autoimmunity: Theoretical and experimental studies

The regulation of autoimmunity is a key issue in fundamental immunology. Despite outstanding achievements on this front, we currently have more questions than answers. The idea of an immune network as a regulatory mechanism is quite attractive, since it enables us to explain the selectivity (specificity), and moreover the clonality, of the regulation. Nevertheless it remains unclear how this mysterious network of immune cells is organized, how it operates, and how it exerts control over autoimmunity. This article presents an attempt to understand how the immune network functions and how it controls autoreactivity. We present a mathematical model of the immune network that is based on principles of immune network organization and function that we arrived at from a survey of the available literature. To test the principles on which the mathematical model is based, we studied the model and compared the different responses to antigen that it generated with the results obtained from experimental studies of immune response. The modeled kinetics of idiotype and anti-idiotype in response to the administration of antigen are in good agreement with the experimental kinetics of idiotypic and anti-idiotypic antibodies. To obtain evidence of the existence of idiotypic mechanisms for regulating autoimmunity, we studied a mathematical model containing autoclones and compared the model results with data from experimental studies in a model of autoimmune hemolytic anemia in mice. Because the results from the theoretical and the experimental studies coincide, there is justification to conclude that autoreactive lymphocytes are normal components of the immune network within which they are regulated. We discuss a possible molecular/cellular mechanism for negative control of autoreactive cells as affected by anti-idiotypic antibodies.

A complex immunological idiotypic network for maintenance of tolerance.

A complex immunological idiotypic network for maintenance of tolerance.

Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

Elimination of Progressive Mammary Cancer by Repeated Administrations of Chimeric Antigen Receptor-Modified T Cells

Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

Rheumatoid factor in idiotypic regulation of autoimmunity

Rheumatoid factor (RF) is known to be heterogeneous, and RFs detected by various methods exhibit different characteristics. In addition to interacting with the Fc region of immunoglobulin G (IgG), certain RFs are able to recognize idiotypes of antibodies. Given the important role of idiotypic interactions in regulating autoimmunity, we hypothesize that RF is involved in regulation of lymphocyte activity against autoimmune disease-inducing antigens via idiotype-anti-idiotype interactions with these lymphocytes. RF level and the existence of idiotype-anti-idiotype interactions between RF and antibodies to autoimmunity-inducing antigens were studied in rats resistant and sensitive to collagen-induced arthritis, encephalomyelitis and atherosclerosis. RF was assayed by agglutination of tanned IgG-loaded erythrocytes. Rat resistance to autoimmune disease is associated with high RF production during the initiation of the immune response, and a low RF level during this period may be a preclinical marker of experimental autoimmune disease manifestation. RF-containing sera compete with an antigen if the RF-containing sera were obtained from rats immunized with that antigen, and they non-specifically inhibit binding of different antigen-antibody pairs. This suggests that RFs are anti-idiotypic antibodies that carry two kinds of paratopes: a particular paratope that recognizes the antigen-binding sites of antibodies, and a shared paratope that serves to recognize the recurrent idiotype on antibodies. Antigenic epitopes for the shared RF paratope can be created in the hinge region of Fc fragments of homologous IgG. RF detected by agglutination of tanned IgG-loaded erythrocytes is involved in negative idiotypic regulation of lymphocytes specific to autoimmunity-inducing antigens.

Role of disorders related to idiotypic and anti-idiotypic interactions in slowing down the achievement of negative serologic reactions in patients with early onset forms of syphilis after specific therapy

Goal. To study the humoral immunity state in patients with the slowed down achievement of negative serologic reactions after the treatment of early onset forms of syphilis by means of examining the blood serum level of idiotypic and anti-idiotypic antibodies to cardiolipin and p17 Treponema Pallidum antigenic protein. Materials and methods. The study involved 324 patients (39.5% male and 60.5% female) with the slowed down achievement of negative serologic reactions. Primary (idiotypic) antibodies to cardiolipin and p17 protein were obtained using immunochromatographic assays with the help of the Bio Logik LP system. Purified antibodies were concentrated using the ultrafiltration technique with the aid of the XM-100А membrane. To obtain the rabbit antiserum to p17 Treponema Pallidum protein, chinchilla rabbits were immunized using the commercial recombinant p17 protein. To determine anti-cardiolipin idiotypic antibodies in the blood serum, the ELISa method optimized for detecting anti-cardiolipin antibodies was applied. To determine anti-cardiolipin anti-idiotypic antibodies as well as idiotypic and anti-idiotypic antibodies to p17 Treponema Pallidum protein, the standard ELISA method was applied. The following antigens were used to process the pads: F(ab)2 fragments of anti-cardiolipin antibodies (5 μg/mL), recombinant р17 T. pallidum protein (5 μg/mL) and F(ab)2 fragments of antibodies to р17 T. pallidum protein (10 μg/mL). The level of antibodies was assessed based on the absorbancy and expressed in conventional activity units using the K coefficient being the absorbancy of the serum under examination to the mean absorbancy of control serums ratio. The K value exceeding 1.5 conventional units indicated the increased level of antibodies. Results. Patients with the slowed down achievement of negative serologic reactions demonstrated a selective increase in the level of anti-idiotypic antibodies (AIAB) relative to T. pallidum antigens, cardiolipin and p17 protein, vs. first-order antibodies, which points at abnormal mutual regulation between idiotypic antibodies (IAB) and AIAB; the discovered phenomenon lays the immunochemical basis for the formation of a self-sustaining “vicious circle” contributing to the induction of high levels of antibodies to treponema antigens even when the pathogen was destroyed.

PReS13-SPK-1283: Pathogenesis, diagnosis and management of neuropsychiatric SLE

Neuropsychiatric (NP) manifestations pose diagnostic and therapeutic challenges in systemic lupus erythematosus (SLE). Less than one-third of these events can be directly attributed to SLE; accordingly attribution to SLE remains a considerable challenge. Increased generalized SLE disease activity or damage, previous or concurrent major neuropsychiatric SLE (NPSLE) events, and persistently positive moderate-to-high antiphospholipid antibody titers are established risk factors. NPSLE patients have increased genetic burden and novel genomic approaches are expected to elucidate its pathogenesis. In animals with disturbed blood-brain barrier, autoantibodies against the NR2 subunits of the N-methyl-D-aspartate receptor and 16/6 idiotype antibodies, may cause diffuse NP manifestations through neuronal apoptosis or brain inflammation; data in humans are still circumstantial. In NPSLE, advanced neuroimaging uncovers structural and metabolic abnormalities in brain regions with normal appearance on conventional magnetic resonance imaging. The European League Against Rheumatism (EULAR) has published evidence and expert based opinion for the management of NPSLE(1).. According to them, diagnostic evaluation is guided by the presenting manifestation with MRI used to visualize brain or spinal pathologies. For neuropsychiatric events believed to reflect an immune or inflammatory process, or when these events occur in the context of active generalized disease, evidence (primarily from uncontrolled studies) supports the use of glucocorticoids alone or in combination with immunosuppressive therapy. In refractory cases, uncontrolled studies suggest a beneficial role of rituximab. Antiplatelet and/or anticoagulation therapy is recommended for NPSLE manifestations related to antiphospholipid antibodies, especially for thrombotic cerebrovascular disease. For the future, we anticipate that novel biomarkers and advanced neuroimaging tests will better define the underlying pathologic mechanisms of SLE-related neuropsychiatric disease, and help guide therapeutic decisions.

131I-Tositumomab Consolidation Radioimmunotherapy (RIT) In Patients With B-Cell Chronic Lymphocytic Leukemia (CLL) Or Small Lymphocytic Lymphoma (SLL) In First Remission

BackgroundDespite the initial response to combination chemotherapy in CLL/SLL patients, disease relapse and minimal residual disease (MRD) remain major issues in treatment of these diseases. Therefore, developing more effective treatments for CLL/SLL patients is a necessity. One strategy is to eliminate persistent disease using radioimmunotherapy (RIT), such as 131I-tositumomab (Bexxar®), as consolidation after an objective complete (CR) or partial response (PR) after an induction regimen. In this study we investigated the tolerability and efficacy of standard non-myeloablative doses of 131I-tositumomab following primary induction chemotherapy in CLL/SLL patients in first remission. Patients and MethodsPatients older than 18 with CLL/SLL with indication for treatment were included if they were in first CR or PR from prior treatments and had <25% bone marrow involvement and acceptable peripheral counts. 131I-tositumomab (75 cGy total-body dose) was delivered between days 90 and 180 from the first day of the last chemotherapy treatment. The dose was reduced to 65 cGy in cases of thrombocytopenia (between 100,000-150,000/µL). Three months after the treatment dose, efficacy and response criteria were specified per NCI working group guidelines and toxicity assessments were recorded based on the CTCAEv3.0. Rituximab levels were determined using ELISA with a monoclonal anti-rituximab idiotype antibody. Results16 patients (CLL11, SLL 5) received consolidative 131I-tositumomab in first remission between 2005-2012. The median age was 61 (38-78). Seven patients (43.7%) had high-risk disease based on cytogenetics or molecular profile. Two patients (12.5%) had 11q deletion and one had mutated TP53 (6.25%). Increased CD38 and ZAP-70 expression was present in 3 of 11 and 6 of 7 patients who were tested and unmutated IgVH was detected in 1 of 3 patients. Twelve patients (75%) were in PR when entered the study while 4 (25%) were in CR. Eight patients (50%) had minimal residual disease (MRD) assessed by multiparametric flow cytometry (MFC). Prior chemotherapy consisted of FR in 9 patients (56.2%), FCR in 4 patients (25%), BR in 2 patients (12.5%) and R-CHOP in 1 patient (6.2%). The median time from the first day of the last treatment cycle to the RIT dose was 15.4 weeks (10 - 29). 7 patients (43.7%) needed dose reduction. At 3 months, CR was achieved or sustained in 12 patients (80%). Conversion from PR to CR following RIT occurred in 4 of 8 patients (50%). Likewise, 131I-tositumomab eliminated MRD in 4 of 8 patients (50%) by negative MFC at 3 months. One patient (6.6%) had PR, one (6.6%) had nodular PR and one had disease progression (6.6%) after administration of 131I-tositumomab. Lymphadenopathy was improved in 83% (5 of 6) of the patients with measureable disease prior to 131I-tositumomab. Overall, the patients with CR at 3 months had significantly higher levels of pre-treatment Rituximab levels (11.0 vs. 2.32 µg/ml, p = 0.01. There was no difference in the pre-treatment levels in patients with pre-treatment MRD who had a negative MFC analysis at 3 months compared to the ones to had residual MRD (12.5 vs. 7.6 µg/ml, p = 0.50) or in patients with PR before receiving 131I-tositumomab who achieved CR at 3 months compared to the ones who did not (11.7 vs. 5.8 µg/ml, p = 0.19). These Results suggest that the levels of Rituximab may be blocking CD20 sites at the lymph node and/or marrow level. The median follow-up was 9.4 months (2.7-54.3). Hematologic toxicities at 3 months were grade 3 anemia in 1 patient (6.2%), grade 3 or 4 neutropenia in 13 (81%), and grade 3 or 4 thrombocytopenia in 8 (50%). Six patients (37.5%) required blood or platelet transfusions. Two patients (12.5%) needed myeloid growth factor support. One patient was hospitalized within 3 months of RIT for neutropenia-related sepsis/typhlitis. Two patients (12.5%) had disease progression and dysplastic changes were found in one. One patient died 3 years after treatment for an unrelated medical reason. Persistent cytopenias were reported in 4 patients (25%) during the follow-up period. ConclusionOverall, consolidation RIT with 131I-tositumomab after first remission appears to be a feasible approach and may provide the potential benefit of converting PR to CR or eliminating MRD in CLL/SLL patients. Further long–term follow-up to assess possible prolonged side effects and clinical effectiveness of this approach remain on-going. [Display omitted] Disclosures:Off Label Use: Bexxar is not FDA approved for consolidation of CLL. Gopal:GSK : Research Funding. Becker:Pfizer: Consultancy. Maloney:GSK: Consultancy. Press:Roche/Genentech: Consultancy.