Expression Of Chimeric Antibodies Research Articles

2A Peptide-based, Lentivirus-mediated Anti-death Receptor 5 Chimeric Antibody Expression Prevents Tumor Growth in Nude Mice

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces tumor cell death via death receptors on target cells, without adverse effects on most normal cells. Its receptors are therefore an attractive target for antibody-mediated tumor therapy. Here, we report the creation of a lentivirus vector constructed by linking the heavy chain and the light chain of the antibody with a 2A/furin self-processing peptide in a single open reading frame that expresses a novel chimeric antibody (named as zaptuximab) with tumoricidal activity, which is consisted of the variable region of a mouse anti-human DR5 monoclonal antibody, AD5-10, and the constant region of human immunoglobulin G1. Lentivirus-expressed zaptuximab bound specifically to its antigen, DR5, and exhibited significant apoptosis-inducing activity in various tumor cell lines. The packaged recombinant virus lenti-HF2AL showed strong apoptosis-inducing activity in vitro. Meanwhile, inoculated subcutaneous human colon HCT116 tumor formation in nude mice were inhibited significantly. Moreover, there was a synergistic effect of mitomycin C (MMC) on the observed tumoricidal efficacy, prolonging the life span of nude mice with orthotopic human lung tumor cancers. These data suggest that lentivirus-mediated, 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility as an anticancer treatment and may represent a rational adjuvant therapy in combination with chemotherapy.

537. Lentivirus-Mediated DR5 Agonistic Chimeric Antibody Expression for Cancer Gene Therapy

537. Lentivirus-Mediated DR5 Agonistic Chimeric Antibody Expression for Cancer Gene Therapy

Expression and tumoricidal activity of a human-mouse chimeric anti-DR5 antibody through a Furin/2A peptide

To express a full-length human-mouse chimeric anti-DR5 antibody from a single open reading frame with tumoricidal activity to various cancer cells. The heavy and light chains of chimeric antibody were joined by the Furin and 2A (F/2A) self-cleavage peptide and cloned into a lentiviral vector of pWPXL. Then the HEK293 cells were infected with the constructed expression vector pWPXL-HF2AL. Western blot, enzyme-linked immunosorbent assay (ELISA) and MTS assay were used to detect the chimeric antibody expression, cleavage, binding affinity to the antigen and tumoricidal activity to various tumor cells. The recombinant chimeric antibody was successfully expressed from a single open reading frame in pWPXL-HF2AL construct. And it possessed a similar binding affinity to the parental murine counterpoint and strong tumoricidal activity to various cancer cells. For example, on the concentration of 3 µg/ml, it made the relative cells viability of HCT116, SMMC7721, A549 and U251 down to 20.6% ± 2.6%, 35.1% ± 2.7%, 76.1% ± 6.1% and 15.6% ± 2.0% respectively. The human-mouse chimeric anti-DR5 antibody of F/2A peptide is successfully expressed. Possessing a strong tumoricidal activity in various cancer cells, it may provide a novel strategy for cancer biotherapy.

Adeno-associated virus-mediated anti-DR5 chimeric antibody expression suppresses human tumor growth in nude mice

In the present study we demonstrate that adeno-associated virus (AAV)-mediated anti-DR5 (death receptor 5) mouse–human chimeric antibody (shorten as Adximab) expression significantly suppressed tumor cell growth by inducing apoptosis both in vitro and in vivo. The viral-expressed and cell-secreted Adximab efficiently bound DR5 with an affinity of 0.7 nM and induced apoptosis of various tumor cells, but not normal cells. A single intramuscular injection of recombinant AAV particles resulted in a stable expression of Adximab in mouse serum for at least 70 days. AAV-mediated Adximab expression led to a significant suppression of tumor growth in nude mice receiving xenografts of human liver and colon cancer. These data suggest that chimeric antibody gene transfer may provide an alternative strategy for the therapy of varieties of cancers.

The Construction and Expression of a Novel Chimeric Anti-DR5 Antibody

Human tumor necrosis factor related apoptosis inducing ligand (TRAIL) could selectively induce apoptosis in a variety of transformed cells and is currently being developed as a cancer therapeutic drug. Unfortunately it has been reported that some forms of TRAIL could cause damage to normal liver cells. In order to avoid this side effect, many groups are now developing agonistic monoclonal antibody (MAb) against DR5. Previously, we developed a MAb (mDRA6) against DR5 with apoptosis inducing ability. The application of mouse origin antibody is limited in clinical use because it can induce human anti-mouse antibody (HAMA) responses that can cause allergic reaction and damage in the human body. Therefore, the variable region genes of this MAb were cloned and ligated into chimeric antibody expression vector (pCMV-VH and pCMV-VL), generating chimeric anti-human DR5 MAb (cmDRA6) expression vectors. The two plasmids were introduced into 293T cells with Lipofectamine 2000. The cell culture supernatant was collected and the expression level of cmDRA6 was detected with standard ELISA method. The chimeric mDRA6 could bind to its target antigen as demonstrated by the results of both ELISA and Western blot. This study of mDRA6 has laid the solid foundation for further application research in the future.

Expression of Chimeric Antibody in Mammalian Cells using Dicistronic Expression Vector

A dicistronic expression vector was constructed for Chinese hamster ovary (CHO) cells that produce both selectable marker-DHFR (dihydrofolate reductase) gene and recombinant antibody cDNA from a single primary transcript via differential splicing. The vector was derived from a pDHL vector and contained the human constant region cDNA so that any human-mouse chimeric antibodies could be expressed. The expression vector produced stable CHO cell clones that secreted nearly double the amount of chimeric antibodies than produced by conventional expression approaches, where the DHFR gene and relevant cDNA are controlled by separate transcription cassettes. Clones with increased expression of interested genes can be efficiently generated by selection in medium containing a gradually increasing amount of methotrexate. The dicistronic expression system using incomplete splicing DHFR gene strategy thus provides a convenient, high-level, and rapid expression of chimeric antibodies.

Construction and expression of a human-mouse chimeric antibody against human bladder cancer

Objective: To construct and express a human-mouse chimeric antibody against human bladder cancer. Method: The variable region genes of anti-human bladder cancer monoclonal antibody BDI-1 were cloned by RT-PCR. A human-mouse chimeric antibody expression vector was constructed and transfected into CHO cells. The chimeric antibody against bladder cancer was expressed and characterized. Result: Eukaryotic expression vector of the chimeric antibody against human bladder carcinoma was successfully constructed, and was expressed in eukaryotic cells; the expressed chimeric antibody ch-BDI showed same specificity as its parent McAb against human bladder cancer cells. Conclusion: The constructed chimeric antibody was expressed successfully in eukaryotic cells, and the chimeric antibody had desired affinity against human bladder cancer cells.

Improving the expression of chimeric antibodies following homologous recombination in hybridoma cells

Mouse/human chimeric antibodies can easily be generated by exchanging the immunoglobulin constant gene segments with human sequences in mouse hybridoma cells by gene targeting. This obviates the need to isolate the variable gene regions from the hybridoma and permits high-level expression of chimeric antibodies, because the production rate of the hybridoma is often maintained. Here we show that the efficiency of this strategy can be further improved by increasing the number of high-producer clones arising from a recombination experiment. In principle, antibody production can be enhanced by removing the selection marker genes from the targeted cells.

Construction and expression of chimeric antibodies by a simple replacement of heavy and light chain V genes into a single cassette vector.

A novel vector pMH-gpt, which is proved useful for cloning mouse immunoglobulin heavy and light chain V genes and for expressing mouse-human chimeric antibody, was constructed. The vector contains human genomic C gamma 1 and C kappa genes, cloning sites for immunoglobulin V region genes, murine Ig promoters, a human Ig heavy chain enhancer, and the selection marker gene Eco-gpt. Because VH and V kappa genes can be cloned into a single vector, a chimeric antibody gene is easily constructed by this simple insertion procedure. The usefulness of the vector was confirmed by construction of two mouse-human chimeric antibodies. Mouse monoclonal antibody (MAb) 196-14 recognizes the ovarian cancer-associated antigen (CA125), and MoAb 2-18 reacts with carcinoembryonic antigen (CEA). Mouse-human chimeric 196-14 and 2-18 antibodies were readily constructed and efficiently produced in a mouse myeloma cell line by utilizing the vector. Both chimeric antibodies retained binding activity to their respective antigens. In biodistribution and immunoscintigraphy studies, specificity of radiolabeled chimeric 196-14 antibody was identical to that of its murine counterpart and significant accumulation at the tumor site was observed. The pMH-gpt vector is useful for constructing and producing mouse-human chimeric antibodies.

A new vector for the high level expression of chimeric antibodies in myeloma cells

We previously reported the expression of a mouse/human chimeric anti-ganglioside G D3 antibody, KM871 (IgG1,κ) in mouse myeloma SP2/0 cells under the control of the ecotropic Moloney virus long terminal repeat by the co-transfection of chimeric heavy (H) and light (L) chain vectors (Shitara et al. (1993) Cancer Immunol. Immunother.). To establish an efficient and high level expression system for the chimeric antibody, we did comparative study on vector systems and host cells. An improved expression vector, named ‘a tandem vector, pChi641HLGM4’ was constructed, in which both of the chimeric H and L chain gene transcription units and a dihydrofolate reductase (dhfr) gene transcription unit were inserted. When two kinds of mouse myeloma cell lines, SP2/0 and P3U1, were used as host cells, frequency of the incidence of antibody-producing transfectants was markedly increased by the use of the tandem vector compared with the use of the mixture of each chimeric H vector and L chain vector. To select out appropriate host cells, transfection frequency and antibody production level were compared among SP2/0, P3U1 and rat myeloma YB2/0 cells by transfection of the tandem vector. YB2/0 cell was shown to have the highest potential in both the transfection frequency and the antibody production. Introduction of the tandem vector into YB2/0 cells and the subsequent amplification with 50–200 nM methotrexate gave rise to several clones that stably secreted 70–100 μg/10 6 cells per 24 h of the chimeric antibody. This productivity of the antibody is one of the highest levels which have been achieved by other investigators using transfected myeloma cells. Using this system it took only 2–3 months to establish the transfectant clones which stably produced the chimeric antibody.

High-level expression of chimeric antibodies using adapted cDNA variable region cassettes

A rapid and generally applicable method for the modification of immunoglobulin cDNAs was developed so that the variable (V) regions could be expressed as cassettes, together with a variety of constant regions. Murine cDNAs were isolated, seuqenced and the V regions joined to short ogligonucleotides providing both splice donor sites and unique restriction sites for insertion into an expression vector. Using this strategy we have expressed the V regions of several murine antibodies, together with the human gamma 1 constant region. Although most of these chimeric antibodies were readily expressed, one murine light-chain cDNA sequence could not be expressed in transfected hybridoma cells. Reconstruction experiments indicate that the sequence created by the fusion of the murine leader and variable region blocked expression at the level of RNA accumulation. The methods described, as well as the potential problems of expression, are applicable to both traditional cDNA fragments and those obtained by in vitro amplification techniques.