The recombinant chimeric antibody chHAb18 against hepatocellular carcinoma can be produced in milk of transgenic mice

Biologically active recombinant monoclonal antibodies (mAbs) and their derivatives are in demand as therapeutic agents against a variety of cancers. The antibodies are generally produced by mammalian cell culture, but their production in the milk of transgenic animals would help meet the increasing demand. The mouse-human chimeric antibody chHAb18 has been proven to inhibit the invasion and metastasis of human hepatocellular carcinoma (HCC) cells by recognizing the HAb18G/CD147 molecule that is highly expressed on the surface of HCC tissue. Here, we report that transgenic mice generated by co-microinjection of two cassettes encoding the heavy and light chain genes of chHAb18 could highly express functional chHAb18 in their mammary glands. The expression level range of 1.1–7.4 mg ml⁻¹ was independent of transgenic copy number. Immunoassays demonstrated the ability and specificity of chHAb18 to bind purified antigen (i.e., HAb18G) or HCC cells. Recombinant chHAb18 from transgenic milk exhibited affinity almost equal to chHAb18 derived from CHO cells, and was 68% of that of the parental murine antibody, HAb18. In light of successful clinical application of HAb18, the chHAb18 expressed in mammary glands of transgenic mice constitutes an important step towards high-yield and scaled-up production of this antibody.     

Expression of a functional mouse-human chimeric anti-CD19 antibody in the milk of transgenic mice

Human B cell lymphomas are suitable targets for immunotherapy. Clinical trials with mouse-human chimeric B cell-specific monoclonal antibodies (mAbs) have already shown promising results. However, limitations for their use in clinical trials can be the lack of sufficient amounts and high production costs. Expression of mAbs in the mammary gland of transgenic animals provides an economically advantageous possibility for production of sufficient quantities of a promising antibody for clinical trials and beyond. In this paper, we show the feasibility of this approach, by generating transgenic mice expressing mouse-human chimeric anti-CD19 mAbs in their milk. Mouse anti-CD19 variable (V) region genes were combined with human IgG1 heavy (H) and kappa light (L) chain constant (C) region genes and fused to the bovine -lactoglobulin (BLG) promoter in two separate expression cassettes. Co-injection resulted in five transgenic lines. In one of these lines completely assembled chimeric mAbs were secreted into the milk, at an approximate level of 0.5mg/ml. These mAbs were able to bind specifically to the CD19 surface antigen on human B cells.     

人促血小板生成素在转基因小鼠乳腺中定位表达的研究(英文)

通过转基因动物乳腺生物反应器大规模生产药用蛋白质已成为现代生物技术新的生长点之一。为研制表达人促血小板生成素的哺乳动物生物反应器的转基因小鼠模型,本论文以小鼠乳清酸蛋白 (mWAP) 基因5挾说骺厍团-s1-酪蛋白基因3挾说骺厍魑鹘谠菇擞糜诒泶锶舜傺“迳伤氐娜橄僮橹匾煨员泶镌靥錺WAPTPO(Fig.1)。通过常规显微注射的方法把mWAP启动子指导的hTPO表达载体导入小鼠受精卵,获得出生小鼠16只。经PCR检测,有6只为转基因阳性(Fig.2)。G0代小鼠中转基因整合率为37.5% (6/16),用ELISA方法在G0代转基因雌鼠的乳汁中检测了促血小板生成素的表达,表达量在0.8 mg/mL以上(Table 1)。这些结果表明我们已建立了乳腺表达hTPO 的转基因小鼠模型,为以后大型家畜乳腺生物反应器的研制提供了科学依据。     

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO− and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.     

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO? and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.     

Lactation performance of transgenic goats expressing recombinant human butyryl-cholinesterase in the milk

The production of recombinant proteins in the milk of transgenic animals has attracted significant interest in the last decade, as a valuable alternative for the production of recombinant proteins that cannot be or are inefficiently produced using conventional systems based on microorganisms or animal cells. Several recombinant proteins of pharmaceutical and biomedical interest have been successfully expressed in high quantities (g/l) in the milk of transgenic animals. However, this productivity may be associated with a compromised mammary physiology resulting, among other things, from the extraordinary demand placed on the mammary secretory cells. In this study we evaluated the lactation performance of a herd of 50 transgenic goats expressing recombinant human butyryl-cholinesterase (rBChE) in the milk. Our findings indicate that high expression levels of rBChE (range 1–5 g/l) are produced in these animals at the expense of an impaired lactation performance. The key features characterizing these transgenic performances were the decreased milk production, the reduced milk fat content which was associated with an apparent disruption in the lipid secretory mechanism at the mammary epithelium level, and a highly increased presence of leukocytes in milk which is not associated with mammary infection. Despite of having a compromised lactation performance, the amount of rBChE produced per transgenic goat represents several orders of magnitude more than the amount of rBChE present in the blood of hundreds of human donors, the only other available source of rBChE for pharmaceutical and biodefense applications. As a result, this development constitutes another successful example in the application of transgenic animal technology.     

Transgenic animal bioreactors

The production of recombinant proteins is one of the major successes of biotechnology. Animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animals are being used for this purpose. Milk, egg white, blood, urine, seminal plasma and silk worm cocoon from transgenic animals are candidates to be the source of recombinant proteins at an industrial scale. Although the first recombinant protein produced by transgenic animals is expected to be in the market in 2000, a certain number of technical problems remain to be solved before the various systems are optimized. Although the generation of transgenic farm animals has become recently easier mainly with the technique of animal cloning using transfected somatic cells as nuclear donor, this point remains a limitation as far as cost is concerned. Numerous experiments carried out for the last 15 years have shown that the expression of the transgene is predictable only to a limited extent. This is clearly due to the fact that the expression vectors are not constructed in an appropriate manner. This undoubtedly comes from the fact that all the signals contained in genes have not yet been identified. Gene constructions thus result sometime in poorly functional expression vectors. One possibility consists in using long genomic DNA fragments contained in YAC or BAC vectors. The other relies on the identification of the major important elements required to obtain a satisfactory transgene expression. These elements include essentially gene insulators, chromatin openers, matrix attached regions, enhancers and introns. A certain number of proteins having complex structures (formed by several subunits, being glycosylated, cleaved, carboxylated...) have been obtained at levels sufficient for an industrial exploitation. In other cases, the mammary cellular machinery seems insufficient to promote all the post-translational modifications. The addition of genes coding for enzymes involved in protein maturation has been envisaged and successfully performed in one case. Furin gene expressed specifically in the mammary gland proved to able to cleave native human protein C with good efficiency. In a certain number of cases, the recombinant proteins produced in milk have deleterious effects on the mammary gland function or in the animals themselves. This comes independently from ectopic expression of the transgenes and from the transfer of the recombinant proteins from milk to blood. One possibility to eliminate or reduce these side-effects may be to use systems inducible by an exogenous molecule such as tetracycline allowing the transgene to be expressed only during lactation and strictly in the mammary gland. The purification of recombinant proteins from milk is generally not particularly difficult. This may not be the case, however, when the endogenous proteins such as serum albumin or antibodies are abundantly present in milk. This problem may be still more crucial if proteins are produced in blood. Among the biological contaminants potentially present in the recombinant proteins prepared from transgenic animals, prions are certainly those raising the major concern. The selection of animals chosen to generate transgenics on one hand and the elimination of the potentially contaminated animals, thanks to recently defined quite sensitive tests may reduce the risk to an extremely low level. The available techniques to produce pharmaceutical proteins in milk can be used as well to optimize milk composition of farm animals, to add nutriceuticals in milk and potentially to reduce or even eliminate some mammary infectious diseases. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antibody manufacture in transgenic animals and comparisons with other systems

Various forms of recombinant monoclonal antibodies are being used increasingly, mainly for therapeutic purposes. The isolation and engineering of the corresponding genes is becoming less of a bottleneck in the process; however, the production of recombinant antibodies is itself a limiting factor and a shortage is expected in the coming years. Milk from transgenic animals appears to be one of the most attractive sources of recombinant antibodies. None of the production systems presently implemented (CHO cells, insect cells infected by baculovirus, or transgenic animals and plants) has yet been optimized. This review describes the advantages of using milk for antibody production in comparison with the other systems.     

Efficiency of human lactoferrin transgenic donor cell preparation for SCNT

The combination of somatic cell nuclear transfer (SCNT) and transgenic technology leads to the production of transgenic cloned animals, wherein the preparation of competent transgenic donor cells is the pivotal upstream step. The purpose of this study was to establish an efficient procedure to prepare human lactoferrin (hLTF) transgenic donor cells for SCNT. Thus, two cell culture systems were employed: caprine mammary epithelial cells (for evaluation of the hTLF transgenic expression in vitro), and fetal-derived fibroblast cells (for identification of competent transgenic donor cells). Induced by hormonal signals, recombinant hLTF was detected in the supernatant of transfected mammary epithelial cells by Western blot. Reliable hLTF transgenic fibroblast cell clones were identified by screening with multiple PCR amplification, EGFP fluorescence, and chromosomal counting (32.5+/-2.3%). This study may provide an effective upstream system to prepare SCNT donor cells for the production of human recombinant pharmaceuticals from the milk of transgenic animals.     

抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器的制备与验证

目的:构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体并制备和验证抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型。方法:利用PCR法扩增出抗人p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L,然后分别将嵌合抗体重链基因H和嵌合抗体轻链基因L连接到乳腺特异性表达质粒pBC1,从而构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L。分别将抗p185~(erbB2)人鼠嵌合抗体ChAb26乳腺特异表达载体pBC1-H和pBC1-L线性化,然后使用原核显微共注射法获得8只转基因FVB小鼠,通过鼠尾直接PCR鉴定其转基因阳性。通过RT-PCR、荧光定量PCR鉴定转基因小鼠乳腺组织中抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达。使用小鼠乳汁采集器收集其乳汁并通过Western blot和夹心ELISA等实验鉴定抗p185~(erbB2)人鼠嵌合抗体ChAb26是否获得表达。结果:经测序验证,抗p185~(erbB2)人鼠嵌合抗体ChAb26的嵌合重链基因H和嵌合轻链基因L分别与乳腺特异表达质粒pBC1正确正向连接。鼠尾直接PCR结果显示所获8只转基因FVB小鼠均为转基因双阳性小鼠,且抗p185~(erbB2)人鼠嵌合抗体ChAb26的重链基因H和轻链基因L在它们的后代中稳定遗传,它们的后代中转基因小鼠双阳性率约为30%; RT-PCR和荧光定量PCR的结果显示,转基因双阳性小鼠及其双阳性后代的乳腺组织中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的mRNA表达; Western blot和ELISA等实验结果显示,转基因双阳性小鼠乳汁中存在抗p185~(erbB2)人鼠嵌合抗体ChAb26的蛋白质表达,而且抗p185~(erbB2)人鼠嵌合抗体ChAb26与羊抗人κ链抗体和羊抗人Ig G Fc-HRP抗体均能特异性结合。结论:成功构建抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因动物乳腺特异性表达载体pBC1-H和pBC1-L和制备了抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因小鼠乳腺生物反应器模型,为今后抗p185~(erbB2)人鼠嵌合抗体ChAb26转基因牛乳腺生物反应器的研究奠定了理论和技术基础。     

Expression of human growth hormone in the milk of transgenic rabbits with transgene mapped to the telomere region of chromosome 7q

The advent of transgenic technology has provided methods for the production of pharmaceuticals by the isolation of these proteins from transgenic animals. The mammary gland has been focused on as a bioreactor, since milk is easily collected from lactating animals and protein production can be expressed at very high levels, including hormones and enzymes. We demonstrate here the expression pattern of recombinant human growth hormone (rhGH) in transgenic rabbits carrying hGH genomic sequences driven by the rat whey acidic protein (WAP) promoter. The transgene was mapped to the q26-27 telomere region of chromosome 7q by fluorescence in situ hybridization (FISH). Nearly 30 % of the F1 generation demonstrated the presence of transgene. The recombinant growth hormone was detected in the milk of the transgenic rabbit females, but not in serum, up to the level of 10???g/ml. Ectopic expression of the transgene in the brain, heart, kidney, liver, and salivary gland was not observed, indicating that a short sequence of rat WAP promoter (969 bp) contained essential sequences directing expression exclusively to the mammary gland. The biological activity of recombinant growth hormone was measured by immunoreactivity and the capability to stimulate growth of the hormone-dependent Nb211 cell line.     

Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits

The large-scale production of recombinant biopharmaceutical glycoproteins in the milk of transgenic animals is becoming more widespread. However, in comparison with bacterial, plant cell, or cell culture production systems, little is known about the glycosylation machinery of the mammary gland, and hence on the glycosylation of recombinant glycoproteins produced in transgenic animals. Here the influence is presented of several lactation parameters on the N-glycosylation of recombinant C1 inhibitor (rhC1INH), a human serum glycoprotein, expressed in the milk of transgenic rabbits. Enzymatically released N-glycans of series of rhC1INH samples were fluorescently labeled and fractionated by HPLC. The major N-glycan structures on rhC1INH of pooled rabbit milk were similar to those on native human C1 inhibitor and recombinant human C1 inhibitor produced in transgenic mouse milk, with only the degree of sialylation and core fucosylation being lower. Analyses of individual animals furthermore showed slight interindividual differences; a decrease in the extent of sialylation, core fucosylation, and oligomannose-type glycosylation with the progress of lactation; and a positive correlation between expression level and oligomannose-type N-glycan content. However, when large quantities of rhC1INH were isolated for preclinical and clinical studies, highly consistent N-linked glycan profiles and monosaccharide compositions were found.     

Functional recombinant human anti-HAV antibody expressed in milk of transgenic mice

Hepatitis A virus (HAV) is a wide spread pathogenic agent and is the common cause of acute Hepatitis A worldwide. Passive immunization of HAV plays an extremely important role in post-exposure prophylaxis with clinical applications often requiring large amounts of antibody. As an alternative to the in vitro production of recombinant proteins, expression of monoclonal antibodies (mAbs) in the milk of transgenic animals is currently used being associated with low production costs and high activity. In this paper, eight founder lines of transgenic mice were generated by co-microinjection of the two cassettes encoding the heavy- and light-chains of a neutralizing anti-HAV antibody, respectively. The expressed heavy- and light-chains of the mAb were correctly assembled and modified in the mammary gland as detected by western blotting. High expression levels of the antibody were achieved during the lactation period and found to be independent of the copy numbers of integrated transgenes. The highest level was up to 32.2 mg/ml. The binding specificity and neutralizing activity of the expressed mAb were assayed by ELISA and neutralizing test, showing that it is capable to neutralize the JN strain of Hepatitis A virus efficiently. Therefore, our results suggest that a large-scale and efficient production of the anti-HAV mAb in the milk of transgenic farm animals would be feasible in the future.     

Virus-neutralizing monoclonal antibody expressed in milk of transgenic mice provides full protection against virus-induced encephalitis

Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine beta-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis.     

Characterization of cell surface antigens of human mammary epithelial cells with monoclonal antibodies prepared against human milk fat globule

Hybridomas have been prepared that secrete monoclonal antibodies against three different surface antigens of normal human mammary epithelial cells by fusion of mouse myeloma cells with spleen cells from mice and rats immunized with delipidated human milk fat globules. Using a novel method for molecular weight determination, the three different monoclonal antibodies, BLMRL-HMFG-Mc3, BLMRL-HMFG-McR2, and BLMRL-HMFG-Mc5, were found to identify molecules with apparent molecular weights of 46,000, 70,000, and 400,000 daltons, respectively. The latter is a mucin-like glycoprotein with a high sugar content and has not previously been described as a component of the human milk fat globule or of human mammary epithelial cell membranes. Single-cell quantitation of binding of monoclonal BLMRL-HMFG-Mc5 to three breast tumor cell lines using a Microscope Spectrum Analyzer and indirect immunofluorescence revealed a heterogeneous expression. Further, using a competitive radioimmunoassay, it was found that breast tumor cell lines differed by at least 10-fold in the 400,000-molecular-weight antigen content. None of the three antigens are detectable on several nonbreast cell lines, including normal breast fibroblasts.     

The High-Level Expression of Human Tissue Plasminogen Activator in the Milk of Transgenic Mice with Hybrid Gene Locus Strategy

Transgene expression for the mammary gland bioreactor aimed at producing recombinant proteins requires optimized expression vector construction. Previously we presented a hybrid gene locus strategy, which was originally tested with human lactoferrin (hLF) as target transgene, and an extremely high-level expression of rhLF ever been achieved as to 29.8 g/l in mice milk. Here to demonstrate the broad application of this strategy, another 38.4 kb mWAP-htPA hybrid gene locus was constructed, in which the 3-kb genomic coding sequence in the 24-kb mouse whey acidic protein (mWAP) gene locus was substituted by the 17.4-kb genomic coding sequence of human tissue plasminogen activator (htPA), exactly from the start codon to the end codon. Corresponding five transgenic mice lines were generated and the highest expression level of rhtPA in the milk attained as to 3.3 g/l. Our strategy will provide a universal way for the large-scale production of pharmaceutical proteins in the mammary gland of transgenic animals.     

Quantitative collection of milk and active recombinant proteins from the mammary glands of transgenic mice

Summary

Transgenic mice expressing foreign genes specifically in their mammary glands have been obtained by several groups in the world. The mouse is generally considered as a good reference animal to evaluate the efficiency of gene constructs to be used in larger mammals for the preparation of the corresponding recombinant proteins at an industrial scale. The method described here shows that mammary glands from lactating mice separated from their pups for one day spontaneously released 1.5 ml milk when stored at O'C. The proteins of milk obtained by this method were essentially similar to those obtained after milking. Human growth hormone (hGH) gene under the control of the rabbit whey acidic (WAP) gene promoter was expressed at a high level in the milk of transgenic mice (4 mg/ml milk in the mice examined here). hGH was present in milk obtained after milking or after the incubation of the mammary glands at O'C. In both cases, the hormone was present in essentially similar concentration, undegraded and biologically active (as judged by its prolactin‐like activity). The method depicted here is very simple and can be applied easily to many mice. Its major limitation is that it implies the breeding and the sacrifice of a relatively large number of animals. One gram of crude recombinant protein can be virtually obtained in this way with about 200 lactating mice from their milk containing the proteins at the concentration of 3‐4 mg/ml. The milk of transgenic mice can therefore be considered as a practical source of recombinant proteins for biochemical and pharmaceutical studies.