The Fc receptor-mediated effector cell functions, such as for example ADCC, were believed to be an essential therapeutic mechanism of Rituxan33,34. antibodies (mAb) to treat non-Hodgkin lymphoma1. Rituxan is usually a chimeric immunoglobulin G1 (IgG1) mAb against the human CD20 antigen, which Resminostat hydrochloride is usually uniquely expressed on B cell surface. Previous studies found that Rituxan acts through several mechanisms contributing to tumor clearance. This includes complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and the direct induction of programmed cell death, which together can effectively reduce the circulating B-cell counts in patients with lymphoma2,3. The clinical success of Rituxan has significantly increased the market demand and generated huge profits, making Rituxan one of the top 10 10 best-selling antibody drugs with over $7.5 billion annual sale worldwide in 2014. Rituxan therapies require generally high doses, leading to high demand in Rituxan manufacturing capacity4. Rituxan is currently produced via mammalian cell culture, which is usually high cost and thus limits its mass production capacity5. On the other hand, patient population that need anti-CD20 Resminostat hydrochloride mAb treatment remains large. For example, in China the incidence of non-Hodgkin lymphoma has increased sharply in recent years. However, the penetration rate of Rituxan is usually less than 10% in China owning to its high cost. Therefore, the development of new methods for enhanced mAb production capacity is highly demanded. Over the past decade, recombinant anti-CD20 Cdh15 mAbs have been successfully produced in silkworms and herb seeds, but neither of these hosts has enabled large-scale production that meets the market demand68. Mammary gland bioreactors derived Resminostat hydrochloride from transgenic animals have been considered as efficient and attractive systems for producing recombinant pharmaceutical proteins, because their extremely high production capacities with low manufacturing costs9. Eukaryotic proteins, particularly those from mammalians, undergo complex post-translational modifications, such as glycosylation, which can widely vary among cell types and play a major role in protein functions10,11. Noteworthy, a recombinant human anti-thrombin III antibody (ATryn) that is produced in milk of transgenic goats and a C1 esterase inhibitor (Ruconest) that is produced in milk of transgenic rabbits have been approved for commercial use by the European Medicines Agency and the US FDA. These approved products demonstrate the commercial ability and broad market potential of mAbs mass-produced by transgenic animals1214. Here, we reported a new approach to cost-effectively produce recombinant anti-CD20 mAb in large scale using the mammary gland bioreactors of transgenic cattle. The mAb can be easily recovered from milk to a high purity. The recombinant mAb shared similar structure, antigen-binding capacity and CDC effect with Rituxan, but different in glycosylation pattern. Importantly, this different glycosylation may contribute to the observed higher ADCC effects andin vivotherapeutic efficacy. == Results == == Generation of transgenic cattle expressing an anti-CD20 mAb == Two cassettes, each made up of the IgG heavy chain and light chain genes under the control of the goat -casein promoter were constructed to generate transgenic cattle that expressed the anti-CD20 mAb in milk (Fig.1a). Two positive cell lines (FOV-3 and BFF-6) were used to produce cloned embryos for somatic cell nuclear transfer (SCNT). Ten calves were given birth to while 5 calves, referred as #0802, #1231, #1232, #0216, and #0220, remained healthy during the study. Among them, one (#0802) developed with the FOV cell type, whereas the remaining four developed with the BFF cell type (Supplementary Fig.1a). PCR and Southern blotting detected both the HC and LC transgenes in the genome of all five calves (Fig.1b,c). Next-generation sequencing showed a unique transgene-integration site in the intergenic region of chromosome 3 with the copy numbers of 20 for both the HC and LC Resminostat hydrochloride transgenes (Supplementary Fig.1b). A fluorescencein situhybridization (FISH) analysis detected a single integration site of the transgenes, confirming the sequencing results (Supplementary Fig.1c,d). == Physique 1. == Generation of transgenic cattle expressing a recombinant anti-CD20 mAb in their milk. (a) Schematic illustration of the two transgene constructs encoding heavy chain (17.2 kb) and.