Kappa Constant Region Research Articles

Affinity purification of mAb from serum-containing hybridoma culture supernatant through a novel nanobody that discriminates mouse IgG from bovine IgG by recognizing the mouse kappa constant region (mCK)

When purifying mAb from serum-containing hybridoma culture supernatant, it is essential that mouse IgG remains free from contaminations of bovine IgG. However, the broadly used Protein A resin cannot achieve this goal due to binding between both mouse and bovine IgG. Here, a novel nanobody-based affinity purification magnetic beads that discriminates mouse IgG from bovine IgG was developed. To bind all subtypes of mouse IgG (IgG1, IgG2a, IgG2b and IgG3) that contain the kappa light chain, mCK (mouse kappa constant region)-specific nanobody binders were selected from an immune phage display VHH library; this library was constructed with peripheral blood mononuclear cells (PBMCs), which were collected from Bactrian camels immunized with a mix of intact mouse IgGs (IgG1, IgG2a, IgG2b and IgG3). A novel clone that exhibited a higher expression level and a higher binding affinity was selected (4E6). Then, the 4E6 nanobody in the format of VHH-hFC (human Fc) was conjugated on magnetic beads with a maximal binding capacity of 15.41±0.69 mg mouse IgG/mL beads. Furthermore, no bovine IgG could be copurified from hybridoma culture supernatant with immunomagnetic beads. This approach is valuable for the large-scale in vitro production of highly pure antibodies by hybridoma cells.

Humanization and expression of IgG and IgM antibodies in plants as potential diagnostic reagents for Valley Fever.

Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in glycoengineered Nicotiana benthamiana plants via transient expression as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.

Abstract 2779: NGS characterization of multiple immune receptors from a single multiplex PCR reaction

Abstract Background: Immune cell analysis by next-generation sequencing (NGS) has shown utility in the fields of hematology-oncology and immuno-oncology research. In hemato-oncology research, advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, as well as eliminate sample-specific primer design often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR immune cell assays require separate reactions to survey each receptor (TCRB, TCRG, IGH, IGK, IGL,⋯), depleting samples and leading to a longer time-to-answer. Two assays for immune receptor analysis based on Ion AmpliSeq technology have been developed to circumvent these issues, allowing for the effective use of up to thousands of primers in a single reaction. These two highly multiplexed NGS assays provide for efficient single-reaction library preparation for detection of IGH, IGK, and IGL chains and the TCRB and TCRG chains, respectively. Methods: Primer panels target the variable gene and joining gene regions of the individual loci (IGH, IGK, IGL and TCRB/TCRG) for all alleles found within the IMGT database, which allows readout of the complementary-determining region 3 (CDR3) sequence of each receptor. The B cell assay includes primers to amplify IGK loci rearrangements involving the kappa deletion and constant region intronic elements. To evaluate the performance of each assay, clonality detection assessment and limit-of-detection testing used gDNA from a total of 65 research samples representing common B and T cell malignancies. Sequencing was performed using the Ion GeneStudio S5 System and analysis was completed using Ion Reporter 5.16. Results: Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, DLBCL, and T-ALL) show a >90% overall positive detection rate. Measurements of linearity-of-response and limit-of-detection were carried out using T and B cell lines and clinical research samples diluted in PBL gDNA to between 10-2 and 10-6 by mass. Both the TCR and BCR multi-receptor assays perform as expected, with linear response of detection to the sample dilution, including the ability to detect clones of interest at 10-6. Conclusions: These results demonstrate the robustness of newly developed Ion AmpliSeq assays for multi-receptor B and T cell analysis. These assays will cut the time-to-answer for translational research studies as well as simplify clonality assessment and rare clone detection in hematology research. For research use only. Citation Format: Geoffrey Marc Lowman, Michelle Toro, Loni Pickle, Stephanie Ostresh, Shrutii Sarda, Chenchen Yang. NGS characterization of multiple immune receptors from a single multiplex PCR reaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2779.

Bispecific anti-mPDGFRβ x cotinine scFv-Cκ-scFv fusion protein and cotinine-duocarmycin can form antibody-drug conjugate-like complexes that exert cytotoxicity against mPDGFRβ expressing cells

Antibody selection for antibody-drug conjugates (ADCs) has traditionally depended on its internalization into the target cell, although ADC efficacy also relies on recycling of the receptor-ADC complex, endo-lysosomal trafficking, and subsequent linker/antibody proteolysis. In this study, we observed that a bispecific anti-murine platelet-derived growth factor receptor beta (mPDGFRβ) x cotinine single-chain variable fragment (scFv)-kappa constant region (Cκ)-scFv fusion protein and cotinine-duocarmycin can form an ADC-like complex to induce cytotoxicity against mPDGFRβ expressing cells. Multiple anti-mPDGFRβ antibody candidates can be produced in this bispecific scFv-Cκ-scFv fusion protein format and tested for their ability to deliver cotinine-conjugated cytotoxic drugs, thus providing an improved approach for antibody selection in ADC development.

Novel ROR1 Antibody Is Able to Trigger Specific and Superior Complement-Dependent Cytotoxicity (CDC) on Primary CLL Cells

Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia of the elderly, remains largely incurable despite chemotherapy and small molecule inhibitors. Treatment targeting CD19 and CD20 has achieved important clinical benefits; however, these antigens are also present on normal B-cells rendering the patients immunodeficient and at a higher risk of infections. In view of this, our immunotherapy strategy is to produce therapeutic antibodies targeting the receptor-tyrosine-kinase-like orphan receptor 1 (ROR1), a protein present on the surface of malignant B-cells and cancer stem cell-like cells (CSC), but absent on most normal tissue.In order to produce new ROR1 monoclonal antibodies (MAbs), we generated a rat hybridoma library and screened >150 anti-ROR1+clones. We then cloned 16 of our own novel antibodies as well as previously published clones: 4a5 and R12 to human IgG1, kappa constant regions. A total of 13 chimeric antibodies recognised human ROR1 on different cell types, out of which, clone SA1 showed superior complement-dependent cytotoxicity (CDC) than other ROR1 MAbs, including clones 4a5 and R12.To determine the binding domain of our ROR1 MAbs, we generated stable cell lines expressing either full length-ROR1 or various forms of truncated ROR1 extracellular domains. Flow cytometry evaluation using these cells, and Epitope Mapping ELISA using a ROR1 peptide library, showed that our antibodies preferentially bind the Ig-like and Frizzled domains. Additionally, we fused clone SA1 to a mouse IgG2a, kappa constant regions in order to perform competition assays amongst our ROR1 MAbs and previously reported clones. We found that there was no epitope overlap amongst any of the tested clones. Moreover, by substituting single amino acids in a relevant region within the Ig-like domain, which we previously determined to be critical for SA1 binding, we found that this epitope is unique to SA1 and, notably is not shared by other clones previously reported in the literature; including clone D10, the prototype of Cirmtuzumab. Importantly, further characterisation of clone SA1 revealed that it can get internalised by both SKW 6.4 cells and CLL cells.As a whole, we have generated and identified a novel anti-human ROR1 monoclonal antibody able to trigger specific CDC of ROR1+ cells. More than that, clone SA1 has a unique epitope and it is able to get internalised by both cell lines and CLL cells that endogenously express ROR1. Additional characterisation and functional analyses are ongoing in order to also develop this antibody as a drug-antibody conjugate (ADC). Importantly, effective targeting of ROR1 expressed on malignant cells and CSC cells but not healthy tissue would provide a safer and more effective treatment of CLL and, in turn, other ROR1+ malignancies. DisclosuresParedes-Moscosso:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Gohil:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies.

Construction and characterization of a new chimeric antibody against HER2

Event Abstract Back to Event Construction and characterization of a new chimeric antibody against HER2 Mohammad M. Amiri1, Fazel Shokri1, 2*, Mahmood Jeddi-Tehrani2, Tohid Kazemi3, Motahhare Bahadori2, Mahshid Maddah2, Mohammad Hojjat-Farsanghi4, Jalal Khoshnoodi1 and Hodjatallah Rabbani2 1 Tehran University of Medical Sciences, Department of Immunology, School of Public Health, Iran 2 Avicenna Research Institute, ACECR, Monoclonal Antibody Research Center, Iran 3 Tabriz University of Medical Sciences, Department of Immunology, Faculty of medicine, Iran 4 Cancer Center Karolinska, Karolinska Hospital, Immune and Gene Therapy Lab, Sweden Aims HER2 proto-oncogene encodes a receptor tyrosine kinase overexpressed in a variety of solid tumors. Amplification of this gene has been shown to correlate with poor prognosis in breast cancer patients. Immunotherapy with anti-HER2 antibody has shown promising results in patients with HER2-positive breast cancer. We have recently reported characterization of a mouse monoclonal antibody (mAb) (1T0) directed against HER2, which binds to an epitope different from that recognized by Trastuzumab and specifically inhibits proliferation of tumor cells overexpressing HER2. In the present study, we report chimerization of this antibody. Materials and Methods The immunoglobulin variable region heavy (VH) and light (VL) chain genes of 1T0 hybridoma cells were amplified and inserted into a mammalian expression vector containing cDNA of human gamma-1 and kappa constant regions using Splice Overlap Extension (SOE) PCR. The construct was subsequently transfected in the mammalian CHO cell line and the chimeric antibody was expressed, purified and characterized by ELISA, Western blot and flow cytometry. Results The purified chimeric antibody specifically binds to recombinant HER2 and HER2 positive tumor cells. Binding of this antibody to tumor cells inhibited proliferation of these cells. The binding affinity of the chimeric mAb was comparable to the parental mouse mAb. Conclusion This chimeric anti-HER2 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive malignancies. Keywords: Chimeric antibody, breast cancer, HER2, Monoclonal antibody, Immunotherapy Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Amiri MM, Shokri F, Jeddi-Tehrani M, Kazemi T, Bahadori M, Maddah M, Hojjat-Farsanghi M, Khoshnoodi J and Rabbani H (2013). Construction and characterization of a new chimeric antibody against HER2 . Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00069 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Fazel Shokri, Avicenna Research Institute, ACECR, Monoclonal Antibody Research Center, Tehran, Iran, fazshok@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mohammad M Amiri Fazel Shokri Mahmood Jeddi-Tehrani Tohid Kazemi Motahhare Bahadori Mahshid Maddah Mohammad Hojjat-Farsanghi Jalal Khoshnoodi Hodjatallah Rabbani Google Mohammad M Amiri Fazel Shokri Mahmood Jeddi-Tehrani Tohid Kazemi Motahhare Bahadori Mahshid Maddah Mohammad Hojjat-Farsanghi Jalal Khoshnoodi Hodjatallah Rabbani Google Scholar Mohammad M Amiri Fazel Shokri Mahmood Jeddi-Tehrani Tohid Kazemi Motahhare Bahadori Mahshid Maddah Mohammad Hojjat-Farsanghi Jalal Khoshnoodi Hodjatallah Rabbani PubMed Mohammad M Amiri Fazel Shokri Mahmood Jeddi-Tehrani Tohid Kazemi Motahhare Bahadori Mahshid Maddah Mohammad Hojjat-Farsanghi Jalal Khoshnoodi Hodjatallah Rabbani Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A TPO Receptor Agonist, ALXN4100TPO, Mitigates Radiation-Induced Lethality and Stimulates Hematopoiesis in CD2F1 Mice

Thrombopoietin (TPO) receptor agonists lacking sequence homology to TPO were designed by grafting a known peptide sequence into the hinge and/or kappa constant regions of a human anti-anthrax antibody. Some of these proteins were equipotent to TPO in stimulating cMpl-r activity in vitro and in increasing platelet levels in vivo. ALXN4100TPO (4100TPO), the best agonist in this series with a K(d) of 30 nM for cMpl-r, exhibited potent activity as a radiation countermeasure in CD2F1 mice exposed to lethal total-body radiation from a cobalt-60 γ-ray source. 4100TPO (2 mg/kg, s.c.) administered once either 24 h before or 6 h after TBI showed superior protection to five daily doses given before or after TBI. Prophylactic administration (69 to 94% survival) was superior to therapeutic schedules (60% survival). 4100TPO conferred a significant survival benefit (P < 0.01) when administered 4 days before or even 12 h after exposure and across a dose range of 0.1 to 8 mg/kg. The dose reduction factors (DRFs) with a single dose of 1 mg/kg 4100TPO 24 h before or 12 h after TBI were 1.32 and 1.11, respectively (P < 0.0001). Furthermore, 4100TPO increased bone marrow cellularity and megakaryocytic development and accelerated multi-lineage hematopoietic recovery in irradiated mice, demonstrating the potential of 4100TPO as both a protector and a mitigator in the event of a radiological incident.

Interleukin‐1β modulation using a genetically engineered antibody prevents adverse cardiac remodelling following acute myocardial infarction in the mouse

Division of Cardiology/VCU Pauley Heart Center, Virginia Commonwealth University, 1200 East Broad Street West Hospital, 10th Floor, East Wing, Room 1041, PO Box 980281, Richmond, VA 23298-0281, USA; Victoria Johnson Center, Virginia Commonwealth University, Richmond, VA, USA; School of Pharmacy, Virginia Commonwealth University, Richmond, VA, USA; and School of Medicine, University of Colorado, Aurora, CO, USA

Characterizing an engineered chimeric anti‐ricin A chain antibody

Ricin toxin, derived from the castor bean, is a potent toxin that inhibits protein synthesis. It has been considered a possible agent of bioterrorism for many years. In experimental studies antibodies have been shown to be an effective treatment against ricin poisoning. Maddaloni et al, described a murine monoclonal anti‐ricin A chain antibody, mRAC 18, that was shown to neutralize in vitro and protect in vivo. This antibody has been found effective, even when administered 12 hours post exposure. The use of monoclonal antibodies in therapy has steadily increased since their development. A limitation to the utility of mAb therapy is their immunogenicity. In an effort to create an antibody for use in post‐exposure human therapy of ricin intoxication, we have created a chimeric ricin A chain antibody. We combined the antigen‐specific variable region genes of the mouse antibody RAC 18 to human gamma‐1 and kappa constant region genes. The resultant chimeric antibody retained the same specificity for ricin as the parental antibody, offered passive protection in vivo, and had enhanced in vitro neutralizing ability. A lectin binding assay showed the chimeric antibody had significantly less sialic acid than the parental antibody. We conclude that differences in sialic acid content may be responsible for the increased in vitro neutralizing ability, and that this effect is independent of Fc receptor binding.

Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus

Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.

Chimeric monoclonal antibodies to hypervariable region 1 of hepatitis C virus

Two chimeric monoclonal antibodies (cAbs), 2P24 and 15H4, to hypervariable region 1 (HVR1) of hepatitis C virus (HCV) were constructed by grafting the variable regions of murine monoclonal antibodies (mAbs) 2P24 and 15H4 to a human IgG1 kappa constant region. Two cAb-producing cell lines were adapted to serum-free media. Both cAb 2P24 and cAb 15H4 cell lines produced 3-5 microg antibodies ml(-1) after 3-5 days culture. cAbs retained binding characteristics similar to those observed in the original mAbs. There was no clear difference in affinity between binding of cAbs and mAbs to seven HVR1 peptides. Mixtures of biotinylated cAbs or mAbs reacted with 32 (86 %) and 31 (84 %) of 37 HVR1 peptides, respectively, but not with non-HVR1 control peptides. HCV from 16 out of 18 (89 %) random HCV-containing plasmas was captured by the mixture of biotinylated cAbs. The capture from IgG-depleted plasmas suggested that cAbs captured mainly free rather than complexed HCV, irrespective of genotype. A mixture of the two cAbs inhibited HCV binding to Molt-4 cells in a dose-dependent manner. These cAbs may be useful for prevention of nosocomial HCV infection and passive immunization to prevent HCV reinfection after liver transplantation.

A Chimeric Antibody to Varicella-Zoster Virus Glycoprotein E

Varicella-Zoster virus (VZV) immune globulin (VZIG) derived from pooled human serum is currently used in immunotherapy of VZV-associated complications of chickenpox and shingles. We developed a mouse-human chimeric antibody against a VZV glycoprotein E (gE) epitope as a safer replacement for VZIG. Variable (V) heavy- and V kappa light-chain exons, derived from an anti-VZV gE antibody secreting mouse hybridoma cell line, were cloned into expression vectors containing an immunoglobulin promoter and enhancer, and human IgG1 or kappa constant (C) region genes. The expression vectors were cotransfected into mouse myeloma cell line (NSO), generating transformants that secreted chimeric human-mouse IgGs. The chimeric and the parent mouse antibody were indistinguishable in their antigen binding specificity. VZV gE chimeric antibody may prove to be a prophylactic antibody that could provide significant advantages over VZIG in having defined specificity, lessened possibility of contamination with viral pathogens, and consistent availability.

Expression of immunoglobulin kappa light chain constant region in abnormal human cervical epithelial cells

Although it is generally believed that, under normal conditions, the only source of immunoglobulin is mature B lymphocytes, we recently found several epithelium-derived carcinoma cell lines also express immunolglobulin-like protein. We extended our study to biopsy samples of human cervical tissues with various epithelial lesions. By in situ hybridization, we only detected a low level of mRNA for the immunoglobulin kappa light chain constant region in epithelia with cervicitis. However, in epithelia with dysplasia and carcinoma, the expression of mRNA for the kappa constant region was markedly increased. There was no significant difference in the level of mRNA for the kappa constant region between epithelial dysplasia and carcinoma. The aberrant expression of immunoglobulin kappa light chain constant region in dysplastic and cancerous cervical epithelial cells may serve as a marker for malignant cell transformation.

Characterization of a phage display-derived human monoclonal antibody (NHS76) counterpart to chimeric TNT-1 directed against necrotic regions of solid tumors.

To eliminate the human anti-mouse antibody (HAMA) response seen in patients treated with murine and chimeric antibodies, fully human monoclonal antibodies (MAbs) are now being developed. Tumor Necrosis Therapy (TNT) is an approach to tumor targeting that utilizes MAbs directed against common intracellular antigens such as nucleic acids, accessible only in necrotic areas of solid tumors. By binding to the necrotic core of tumors, these TNT MAbs can circumvent many of the limitations of MAbs directed against tumor cell surface antigens. Chimeric TNT-1 (chTNT-1) was first developed from the parent murine antibody by genetically engineering the murine variable regions to the human IgG(1) and kappa constant regions. Although the chimeric antibody's behavior was similar to that of the murine version, the 35% murine homology it shares allows for the potential of a HAMA response. A human antibody derived from a phage display library, designated NHS76, has been developed with similar binding characteristics to the TNT-1. To demonstrate that this genetically engineered human counterpart to chTNT-1 has similar pharmacokinetic characteristics, in vivo behavior, and targeting abilities, both antibodies were rigorously tested in parallel. For these studies, biodistribution analysis in LS174T human colon tumor-bearing nude mice was performed to compare the uptake levels in tumor and normal organs. In addition, mouse imaging and autoradiographic studies were conducted to demonstrate positive uptake in necrotic regions of tumor and negative uptake in viable tissues and organs. The results of these studies confirm the comparable nature of both antibodies and provide the necessary preclinical data to show the suitability of NHS76 as an improved product for the therapy of solid tumors in man.

Chimaeric Lym-1 monoclonal antibody-mediated cytolysis by neutrophils from G-CSF-treated patients: stimulation by GM-CSF and role of Fc gamma -receptors.

Chimaeric Lym-1 (chLym-1) is a monoclonal antibody generated by fusing the variable region genes of murine Lym-1 to human gamma1 and kappa constant regions. Owing to its selectivity and avidity for human malignant B cells, it is an attractive candidate for developing immune-interventions in B-lymphomas. In the attempt to identify rational bases for optimizing potential chLym-1 related therapeutic approaches, we studied the ability of this ch-mAb to trigger neutrophil-mediated Raji cell cytolysis in cooperation with two neutrophil-related cytokines, G-CSF and GM-CSF. ChLym-1 triggered low levels of cytolysis by normal neutrophils but induced consistent cytolysis in neutrophils from individuals treated with G-CSF. When exposed to GM-CSF, neutrophils from subjects treated with G-CSF became potent effectors, also leading to 75% lysis. By using mAbs specific for distinct FcgammaRs, normal neutrophils were inhibited by mAb IV.3, suggesting the intervention of FcgammaRII, constitutively expressed on the cells. On the other hand, neutrophils from patients treated with G-CSF were inhibited by mAb IV.3 plus mAb 197, a finding consistent with a cooperative intervention of FCgammaRII and G-CSF-induced FcgammaRI. The anti-FcgammaRIII mAb 3G8 promoted significant enhancement of the neutrophil cytolytic efficiency. Therefore, neutrophil FcgammaRIII behaves as a down-regulator of the cytolytic potential. The present findings suggest new attempts to develop mAb-based and G-CSF/GM-CSF combined immune-interventions in B lymphomas.

Treatment of B-cell non-Hodgkin's lymphoma with anti CD 20 monoclonal antibody Rituximab

Rituximab is a chimeric anti CD-20 monoclonal antibody containing human IgG1 kappa constant regions, with murine variable regions. The anti-lymphoma effects of Rituximab are probably due to complement and antibody-dependent cell-mediated cytotoxicity, and induction of apoptosis. Phase II trials have demonstrated a strong activity of rituximab alone in indolent B non-Hodgkin lymphoma, especially in patients with follicular lymphoma. The most utilized dose-schedule is 375 mg/m 2 weekly×4. The association with chemotherapy or with interferon-alpha increases Rituximab efficacy. More recently, Rituximab have showed activity also in diffuse large cell lymphoma, mantle cell lymphoma and in other B-malignancies. Good results have also been obtained utilizing Rituximab for in vivo purging. However, we are still far from having found a definite position for Rituximab in the treatment of lymphoproliferative disorders. The aim of future studies should be to develop new strategies that will hopefully produce the most effective Rituximab-based regimens in order to find the Rituximab key position in the treatment of B-malignancies

Molecular cloning and chimerisation of CDI 315B monoclonal antibody

A chimeric mouse-human antibody has been created that recognizes an antigen found on breast cancer cells and melanoma cells. Immunoglobulin constant domains of mouse monoclonal antibody CDI 315B Cgamma1 and CK, were substituted by the human Cgamma1 and Ckappa. The CDI 315B variable heavy and light chain regions were PCR amplified from hybridoma RNA and sequenced. Mouse variable VH and VL regions were joint to human IgG1 and kappa constant regions and subcloned into pcDNA3 expression vectors. The Sp2/0 murine myeloma cells were transfected with expression vectors pcDNA3L and pcDNA3H and the reactivity of chimeric antibodies was tested by indirect ELISA using B16F1 murine melanoma cells as well as MCF7 human breast cancer cells, as antigen.

Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries

Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c- myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC L for expression as fusion proteins with human kappa constant region (scFv-C L). Expression of these constructs in Escherichia coli yielded 0.2–1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c- myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.

Construction and Characterization of a Chimeric Fusion Protein Consisting of an Anti-idiotype Antibody Mimicking a Breast Cancer-Associated Antigen and the Cytokine GM-CSF

Anti-idiotype antibody, 11D10 mimics biologically and antigenically a distinct and specific epitope of the high molecular weight human milk fat globule (HMFG), a cancer-associated antigen present in over 90% of breast tumor samples. To augment the immunogenicity of 11D10 without the aid of a carrier protein or adjuvant, we made a chimeric 11D10-GM-CSF fusion protein for use as a vaccine. An expression plasmid for 11D10 was made by ligation of the DNA sequences of the 11D10 light-chain variable region upstream of the human kappa constant region. The heavy-chain plasmid carrying GM-CSF was made by ligation of the heavy-chain variable region sequences upstream of the human gamma1 constant region CH1 fused to the DNA fragment encoding the mature GM-CSF peptide 3' to the CH3 exon. NS1 plasmacytoma cells were transfected with the light and heavy-chain vectors by electroporation. Fusion protein secreted in the culture medium was purified and was characterized by gel electrophoresis as well as by determination of the biological activity of the fused GM-CSF. In nonreducing SDS-polyacrylamide gels, a single band approximately 200 Kd reacted with anti-human kappa, anti-human lambda1 and anti-GM-CSF antibodies. In reducing polyacrylamide gels, a approximately 74 kd protein reacted with anti-human lambda1 and anti-GM-CSF antibodies. The fusion protein induced proliferation of GM-CSF dependent NFS-60 cells. These results suggest that the protein is a chimeric anti-idiotype antibody consisting of 11D10 variable domains, human kappa and lambda1 constant domains and that the GM-CSF moiety fused to the constant region lambda1 is biologically active.

Human IgA Monoclonal Antibodies Specific for a Major Ragweed Pollen Antigen

Human hybridoma cell lines secreting IgG specific for the major allergen in the pollen of short ragweed, Amb a I, were established from patients who had been receiving antigen injections for immunotherapy. Recombinant Ig genes were then constructed by cloning the heavy and light chain variable region genes of the human hybridoma cell line and joining them to the human alpha or kappa constant region genes in mammalian expression vectors. Amb a I-specific IgA was expressed in two mouse myeloma cell lines, NS0 and Sp2/0. In both systems, transfected alpha and kappa chains were assembled into IgA monomers or into dimers covalently linked by the endogenous murine J chains. We propose that recombinant IgA monoclonal antibodies specific for airborne allergens may be applied to the mucosal surface of the nasal linings or of the lower airway of sensitized individuals to inhibit the entry of allergenic molecules across the mucosal epithelium and, therefore, to prevent the development of allergic responses.