Monoclonal antibodies as therapeutics in oncology

The specificity of antibodies has been harnessed to target cancer cells and the first therapeutic antibodies for use in oncology are now finding application in the clinic. Studies are currently under way to develop new and improved antibodies. Recent developments have been made in the identification of novel targets, including the use of genomic and proteomic technologies. Several methods are also being developed to enhance antibody efficacy.     

Monoclonal antibodies to rabbit liver cathepsin B

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein includihg the cysteine proteinases, catbepsins H and L.Abbreviations ELISA Enzyme-linked immunoadsorbent assay - EIP Enzyme immunoprecipitation - PAGE polyacrylamide gel electrophoresis - Ep-475 L-trans-epoxysuccinyl-leucylamido (-methyl) butane - Z benzyloxycarbonyl - NMec N-methylcoumarin - PEB phosphate-EDTA-Brij 35 - IAA iodoacetic acid - PBS phosphate-buffered saline - DMEM Dulbecco's Minimal Essential Medium - FITC fluorescein isothiocyanate     

Monoclonal antibody production in dialyzed continuous suspension culture

Hybridoma cell growth and monoclonal antibody production in dialyzed continuous suspension culture were investigated using a 1.5-L Celligen bioreactor. Medium supplemented with 1.5% fetal bovine serum was fed directly into the reactor at a dilution rate of 0.45 d(-1). Dailysis tubing with a molecular weight cut-off (MWCO) of 1000 was coiled inside the bioreactor. Fresh medium containing no serum or serum substitues passed through the dialysis tubing at flow rates of 2 to 5 L/d. The objective was to remove low molecular weight inhibitors, such as lactic acid and ammonia, by diffusion through the tubing, while continuoulsy replenishing essential nutrients by the same mechanism. Due to the low MWCO of the dialysis tubing high molecular weight components such as growth factors and antibody were not removed by the dialyzing stream. In the batch start-up phase, the monoclonal antibody (MAb) titer was almost 3 times that achieved in typical batch cultures (i.e., 170 to 180 mg/L). During dialyzed continuous operation, a substantial increase (up to 40%) in cell density, monoclonal antibody (MAb) titer, and reactor MAb productivity was observed, as compared with a conventional continuous suspension culture. The cell viability and the specific MAb productivity remained practically constant at different dialysis rates. This finding suggests that the steady state growth and death rate in continuous suspension hybridoma cultures are not direct functions of the nutrient or inhibitor concentrations.     

噬菌体抗体库技术在肿瘤治疗中的研究进展及应用北大核心CSCD

肿瘤是严重威胁人类健康的疾病。目前肿瘤治疗策略以手术切除、放化疗、靶向治疗和免疫治疗为主。单克隆抗体药物因具备高效性和低毒性等特点,逐渐成为肿瘤临床治疗中不可或缺的药物类型。噬菌体抗体库技术(phage antibody library technology,PALT)是一种新型的单克隆抗体制备技术,其将免疫球蛋白可变区VH(variable region of heavy chain)/VL(variable region of light chain)基因重组后整合在噬菌体载体上,并以融合蛋白的形式将抗体表达到噬菌体表面,从而获得多样性抗体库。抗体库经过“吸附-洗脱-扩增”过程即可筛选获得到特异结合抗原的抗体分子及其基因序列。PALT具有抗体生产周期短、抗体结构可塑性强、抗体产量大、多样性高和可直接生产人源化抗体等优点,已应用于乳腺癌、胃癌、肺癌和肝癌等肿瘤标志物的筛选和抗体药物的制备等领域。文中综述了PALT在肿瘤治疗中的研究进展及应用。     

Selective gene therapy for prostate cancer cells using liposomes conjugated with IgM type monoclonal antibody against prostate-specific membrane antigen

Prostate cancer cells express prostate-specific membrane antigen (PSMA). We developed an IgM type monoclonal antibody against PSMA. The antibody was coupled to poly-L-lysine and thereafter this conjugate was mixed with cationic liposomes containing plasmid DNA. The antibody-liposome complex was tested whether it could deliver the gene of interest selectively to the PSMA positive cells. As assessed by beta-galactosidase reporter gene, the transfection efficiency was 13.2% with anti-PSMA-liposome complex as compared to 4% with control IgM liposome complex. In contrast, no such differences were observed in PSMA negative PC-3, DU145 and T24 cells. Furthermore, in the suicide gene therapy in vitro with thymidine kinase gene plus ganciclovir system, anti-PSMA liposome complex demonstrated a selective growth inhibitory effect on PSMA positive LNCaP cells but not on PSMA negative cell lines.     

双特异性抗体在肿瘤治疗中的研究

双特异性抗体(bispecific antibody,BsAb)有两个抗原结合位点,其中一个位点可与靶细胞表面抗原结合,另一个位点则可与载荷物(如效应细胞,分子等)结合。将BsAb应用于肿瘤治疗,发挥抗肿瘤效应的思想已有二十多年历史,随着对效应细胞生物学了解的加深和抗体工程的飞速发展,各种形式的BsAb相继出现,多种BsAb药物已进入临床初期试验或治疗使用阶段。本文就BsAb的各种新形式及其在肿瘤治疗中的应用新进展作简要概述。     

Prophylactic and therapeutic testing of Nicotiana-derived RSV-neutralizing human monoclonal antibodies in the cotton rat model

Severe lower respiratory tract infection in infants and small children is commonly caused by respiratory syncytial virus (RSV). Palivizumab (Synagis^®), a humanized IgG₁ monoclonal antibody (mAb) approved for RSV immunoprophylaxis in at-risk neonates, is highly effective, but pharmacoeconomic analyses suggest its use may not be cost-effective. Previously described potent RSV neutralizers (human Fab R19 and F2–5; human IgG RF-1 and RF-2) were produced in IgG format in a rapid and inexpensive Nicotiana-based manufacturing system for comparison with palivizumab. Both plant-derived (palivizumab-N) and commercial palivizumab, which is produced in a mouse myeloma cell line, showed protection in prophylactic (p < 0.001 for both mAbs) and therapeutic protocols (p < 0.001 and p < 0.05 respectively). The additional plant-derived human mAbs directed against alternative epitopes displayed neutralizing activity, but conferred less protection in vivo than palivizumab-N or palivizumab. Palivizumab remains one of the most efficacious RSV mAbs described to date. Production in plants may reduce manufacturing costs and improve the pharmacoeconomics of RSV immunoprophylaxis and therapy.     

Monoclonal antibodies against naturally occurring bioactive compounds

The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigen-BSA conjugate with HAT-sensitive mouse myeloma cells. The cross-reaction of anti-forskolin antibodies with 7-deacetyl forskolin was 5.6%. A very small cross-reaction appeared with other derivatives. The full measuring range of the assay extends from 5 ng to 5 μg/ml of forskolin. Immunoaffinity column chromatography using anti-forskolin MAbs appears to be far superior to previously published separation methods. The capacity of the immunoaffinity column as determined by ELISA is 9 μg/ml. Forskolin has been isolated directly from the crude extracts of tuberous roots and the callus culture of Coleus forskohlii. A MAb against tetrahydrocannabinolic acid (THCA) was produced. The cross-reaction of anti-THCA antibody against other cannabinoids was very wide. Many cannabinoids and a spiro-compound were reactive, but did not react with other phenolics. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. Anti-ginsenoside Rb1 MAbs were produced. New western blotting method of determination for ginsenosides was established. Ginsenosides separated by silica gel TLC were transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was treated with NaIO₄ solution followed by BSA, resulting in a ginsenoside-BSA conjugate. Immunostaining of ginsenosides was more sensitive compared to other staining. Immunostaining of ginsenosides in the fresh ginseng root was succeeded using anti-ginsenoside Rb1 (GRb1) MAb after blotting to PVDF membrane. This revised version was published online in August 2006 with corrections to the Cover Date.     

Monoclonal anti-androgen receptor antibodies: Production,characterization, and potential diagnostic applications

Several monoclonal antibodies (mAbs) and novel mAb-based assays for the androgen receptors (AR) have been developed. Large amounts of the recombinant human AR protein produced by a baculovirus expression system were used as an antigen to produce mAbs. Twenty-nine AR-specific mAbs were first confirmed by Western blot analysis and were then characterized for their immunoglobulin isotypes, epitopes, and epitope localization in AR. Novel assays using flow cytometry and sandwich enzyme-linked immunosorbent assays (ELISA) were established to detect AR-expressing cells and to quantify soluble AR protein, respectively. Using immunostaining, we identified several anti-AR mAbs exclusively recognizing AR within the nuclei of the prostate cancer cell line LNCaP and of prostate tissues in both frozen and paraffin-embedded sections, whereas other mAbs could detect AR in both nuclear and cytoplasmic compartments. Interestingly, certain mAbs, such as G122-25 and G122-77, could distinguish the androgen-bound AR from the unoccupied AR. In sum, many purified AR protein and anti-AR mAbs, together with the assays developed, could be powerful tools for the study of functional AR and for the diagnosis of prostatic cancers.     

Monoclonal antibody differentiates chicken A system alloantigens

A monoclonal antibody (ISU-cA) was produced that recognized certain alloantigens of the chicken A blood group locus. Antigens produced by alleles A3, A4 and A8 were positive, and those produced by A2 and A5 were negative, by haemagglutination. The specificity of ISU-cA for chicken A blood group antigens was demonstrated by serologic analyses, genetic crosses and competitive inhibition of binding by anti-A alloantisera. To our knowledge, this is the first reported monoclonal antibody against a chicken blood group alloantigen system other than the B complex.     

Improvement in the antibody productivity of human-human hybridomas by transfection with Tac gene

The presence of a highly purified recombinant interleukin-2 (rIL-2) increased the production of immunoglobulin (IgM or IgG) by human-human hybridomas to 1.5–2.0 times the production by untreated cells. However, these cells did not react with anti-Tac (IL-2 receptor ) antibody. To enhance the response of the hybridoma cells to rIL-2, Tac gene was introduced by co-transfection with Tac gene expression plasmid pTB459 and G418 resistant gene expression plasmid pRSVneo. Tac cDNA transfected hybridoma (HBW-4.16.459-6-126) was induced to produce 6 times as much IgG by rIL-2 as was the control. This antibody production promoting phenomenon mediated by rIL-2 was depressed by anti-Tac antibody.     

Structure,heterogeneity and developability assessment of therapeutic antibodies

Increasing attention has been paid to developability assessment with the understanding that thorough evaluation of monoclonal antibody lead candidates at an early stage can avoid delays during late-stage development. The concept of developability is based on the knowledge gained from the successful development of approximately 80 marketed antibody and Fc-fusion protein drug products and from the lessons learned from many failed development programs over the last three decades. Here, we reviewed antibody quality attributes that are critical to development and traditional and state-of-the-art analytical methods to monitor those attributes. Based on our collective experiences, a practical workflow is proposed as a best practice for developability assessment including in silico evaluation, extended characterization and forced degradation using appropriate analytical methods that allow characterization with limited material consumption and fast turnaround time.     

Genetically engineered intracellular single-chain antibodies in gene therapy

The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders. To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation. Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins.     

Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.     

Construction of recombinant monoclonal antibodies from a chicken hybridoma line secreting specific antibody

The chicken is a useful animal for the development of the specificantibodies against the mammalian conserved proteins. We generated twotypes of recombinant chicken monoclonal antibodies (mAbs), using a phagedisplay technique from a chicken hybridoma HUC2-13 which secreted themAb to the N-terminal of the mammalian prion protein (PrP). Althoughthe mAb HUC2-13 is a useful antibody for the prion research, thehybridoma produces a low level of antibody production. In order to producea large amount of the mAb, we have constructed a single chain fragmentvariable region (scF_V) mAb by using the variable heavy(V_H) and light (V_L)genes which were amplified by using the two primer pairs and theflexible linker. The two phage display mAbs (HUC2p3 and HUC2p5)expressed on a M13 filamentous phage and their soluble type mAbs(HUC2s3 and HUC2s5) were reacted with the PrP peptide antigen in theELISA. In the Western blot analysis, the mAbs HUC2p3 and HUC2s3 wereas reactive to PrP^c from mouse brains as the mAb HUC2-13 was. The nucleotide sequences of V_H and V_L genes from HUC2-13 and the two cloneswere identical except for only one residue. These results indicate that themethods presented here provide an effective tool for the improvement ofthe low levels of antibody production in the chicken hybridoma system.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

治疗性全抗体的表达及其研究进展

治疗性全抗体是一类前景良好的治疗药物,具有潜在的巨大市场。截至2003年6月,美国FDA批准的治疗性全抗体有20种。全抗体表达是抗体工程的研究重点之一,本对全抗体的表达及其研究进展进行了阐述。     

Attack on neoplastic cell membranes by therapeutic antibody

Mouse monoclonal antibody is not well fitted to destroying tumour cell targets. Complement and cellular effectors are inefficiently recruited, the cells can undergo antigenic modulation, antigen-negative mutants can arise, and the tumour-bearing subject can amount an immune response against the therapeutic antibody.This paper describes the preparation of two chimeric antibody derivatives designed to cirvumvent some of these problems. The first derivative is FabFc, prepared by linking Fab' from monoclonal antibody to Fc from human IgG. The bismaleimide linking agent forms a thioether bond with an SH group released by reduction of SS bonds in the hinge of each constituent. The second derivative is bisFabFc, formed by a bismaleimide in this case joining two FabFc molecules via a free SH in the Fc hinge of each. As regards antibody activity against target cells bisFabFc can be univalent (one active, one inactive Fab arm), bivalent, or bispecific (with each Fab arm directed against a different cell surface antigen). Its juxtaposed dual Fc regions are designed to promote cooperative binding of effectors.Some preliminary characterization in vitro has employed antibodies of anti-idiotypic specificity directed against guinea-pig L₂C leukaemic B lymphocytes. The parent mouse IgG1 antibody failed to invoke complement cytotoxicity or antibody-dependent cellular cytotoxicity, while the chimeric derivatives yielded good killing in both systems. In complement lysis bivalent bicFabFc outperformed univalent, which in turn outperformed the FabFc monomer.     

Monoclonal antibodies: technology around the clock for new therapeutic hopes

Engineering monoclonal antibodies, now widely used in the clinic, has made it possible to develop a new generation of antibodies with optimized functional properties. These antibodies should allow a significant improvement of the treatment of diseases where only few drugs are available, if any. However, the cost of treatments with monoclonal antibodies requires further improvements in production and purification technologies, and raises the question of generic antibodies. The present review summarizes some of the technological past and present challenges in the field.