Control IgG was a Nicotiana-derived human IgG against toxin A

Control IgG was a Nicotiana-derived human IgG against toxin A. respectively). The additional plant-derived human mAbs directed against option 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. in which plant-specific N-glycans (with core 1,3 fucose and 1,2 xylose) are reduced by RNAi inhibition of plant-specific glycosyl-transferases,25 were used as the host plant. Plants (~1 kg) were infiltrated with the mAb vectors and after 7C8 d, the plants were homogenized with a commercial juicer. Extract was clarified and IgG purified by protein A chromatography (Table 1). Purified mAbs were greater than 95% real as assessed by SDS-PAGE and HPLC-SEC (Fig. S1), and all mAbs except for F2C5 had low levels of aggregate ( 3%). An additional purification step was performed on F2C5 resulting in elimination of detectable aggregates (Table CX-4945 sodium salt 1). Palivizumab (MedImmune) was purchased from a commercial supplier. Table?1. Purification yield and % aggregate of Nicotiana-derived mAbs line was used that yields mAbs with mammalian N-glycans predominantly of CX-4945 sodium salt the GnGn glycoform.25 Although this glycoform can enhance antibody-dependent cell-mediated cytotoxicity via higher affinity binding to the FcRIII receptor,32 it does not influence other effector functions or the long serum half-life of IgG conferred by binding to the FcRn receptor. This pattern was confirmed by the pharmacokinetics seen in the cotton rat (Fig.?2); rodent FcRn SLIT1 is known to be promiscuous in its binding of IgG from other species.33 Although there are an estimated 175,000 adults hospitalized annually with RSV infection in the US alone,5 data around the therapeutic usefulness of palivizumab in this setting are very limited.34 Studies in hematopoietic stem cell transplant patients (where mortality rates from RSV contamination can be extremely high) are inconclusive and likely have been limited by dosing costs.34 With current pricing, a 15 mg/kg dose of palivizumab for a 50 kg adult costs ~$125,000. We are unaware of any reports of therapeutic testing of palivizumab in CX-4945 sodium salt the cotton rat model. In this study, we found palivizumab and palivizumab-N both provided significant reductions in lung viral titer; however, doses of 6x and 20x the prophylactic dose (5 mg/kg) were required to provide comparable reductions of viral lung titer (100-fold) when delivered 1 and 2 d, respectively, after viral challenge. This is not surprising because the therapeutically applied mAb was targeting an established viral contamination. A large proportion of the worlds populace does not have access to palivizumab, and for those populations that do, the pharmacoeconomic benefit CX-4945 sodium salt is questionable. At commercial scale, production of mAbs in the Nicotiana transient expression system is expected to reduce costs 2C5-fold compared with production in mammalian cell culture.24,35 We have found contract manufacturing of plant mAb for preclinical and Phase 1 testing to be 3C5 times less expensive than contract manufacturing in cell culture. The reduced costs are primarily the result of the comparatively inexpensive infrastructure associated with producing the herb natural material. Within the last several years, Nicotiana-derived mAbs have been made under Good Manufacturing Practices (GMPs) and tested clinically.26,36 This manufacturing platform may serve as an alternative production system that could reduce costs and thus expand the patient populations that can benefit from this potent mAb product. Methods expression vectors RF-1 and RF-2 variable region amino acid sequences (VR) were used as reported and codon-optimized for and synthesized (GeneArt, AG; RF-1 VL, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283069″,”term_id”:”452883305″,”term_text”:”KC283069″KC283069; RF-1 VH, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283070″,”term_id”:”452883307″,”term_text”:”KC283070″KC283070; RF-2 VL, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283071″,”term_id”:”452883309″,”term_text”:”KC283071″KC283071; RF-2 VH, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283072″,”term_id”:”452883311″,”term_text”:”KC283072″KC283072).37 The F2C5 variable regions were originally reported by Crowe et al.19 and deposited in GenBank (accession nos. “type”:”entrez-nucleotide”,”attrs”:”text”:”L41061″,”term_id”:”9864840″,”term_text”:”L41061″L41061 and “type”:”entrez-nucleotide”,”attrs”:”text”:”L41062″,”term_id”:”2231130″,”term_text”:”L41062″L41062). The VH gene sequence was used as reported. The VL (lambda) FR1 residues 1C3 and 8 were altered to be consistent with genomic sequences according to Kabat database. Both genes were codon-optimized for (VL, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283073″,”term_id”:”452883313″,”term_text”:”KC283073″KC283073; VH, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283074″,”term_id”:”452883315″,”term_text”:”KC283074″KC283074). The RSV19 VRs were derived from RSV Fab 19.18 The VH sequence was used as reported. The VL CX-4945 sodium salt (kappa) FR1 residues 1C4 and FR4 residues 103C107 were altered to be consistent with Kabat, and both genes were codon-optimized for (VL, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283075″,”term_id”:”452883317″,”term_text”:”KC283075″KC283075; VH, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283076″,”term_id”:”452883319″,”term_text”:”KC283076″KC283076). Palivizumab VR sequences were used as reported and codon-optimized as above (VL, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283077″,”term_id”:”452883321″,”term_text”:”KC283077″KC283077; VH, “type”:”entrez-nucleotide”,”attrs”:”text”:”KC283078″,”term_id”:”452883323″,”term_text”:”KC283078″KC283078).21 Genes were codon optimized and synthesized and subsequently cloned into herb (TMV and PVX).