AbstractIncreased use of therapeutic monoclonal antibodies and the relatively high manufacturing costs fuel the need for more efficient production methods. Here we introduce a novel, fast, robust, and safe isolation platform for screening and isolating antibody-producing cell lines using a nanowell chip and an innovative single-cell isolation method. An anti-Her2 antibody producing CHO cell pool was used as a model. The platform; (1) Assures the single-cell origin of the production clone, (2) Detects the antibody production of individual cells and (3) Isolates and expands the individual cells based on their antibody production. Using the nanowell platform we demonstrated an 1.8–4.5 increase in anti-Her2 production by CHO cells that were screened and isolated with the nanowell platform compared to CHO cells that were not screened. This increase was also shown in Fed-Batch cultures where selected high production clones showed titers of 19–100 mg/L on harvest day, while the low producer cells did not show any detectable anti-Her2 IgG production. The screening of thousands of single cells is performed under sterile conditions and the individual cells were cultured in buffers and reagents without animal components. The time required from seeding a single cell and measuring the antibody production to fully expanded clones with increased Her-2 production was 4–6 weeks.

Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments.

Purpose Modulating sialylation of therapeutic glycoproteins may be used to influence their clearance and systemic exposure. We studied the effect of low and high sialylated IL4–10 fusion protein (IL4–10 FP) on in vitro and in vivo bioactivity and evaluated the effect of differential sialylation on pharmacokinetic parameters. Methods CHO cell lines producing low (IL4–10 FP lowSA) and high sialylated (IL4–10 FP highSA) fusion protein were generated. Bioactivity of the proteins was evaluated in an LPS-stimulated whole blood assay. Pharmacokinetics were studied in rats, analyzing plasma levels of IL4–10 FP upon intravenous injection. In vivo activity was assessed in an inflammatory pain mice model upon intrathecal injection. Results IL4–10 FP lowSA and IL4–10 FP highSA had similar potency in vitro . The pharmacokinetics study showed a 4-fold higher initial systemic clearance of IL4–10 FP lowSA, whereas the calculated half-life of both IL4–10 FP lowSA and IL4–10 FP highSA was 20.7 min. Finally, both IL4–10 FP glycoforms inhibited persistent inflammatory pain in mice to the same extent. Conclusions Differential sialylation of IL4–10 fusion protein does not affect the in vitro and in vivo activity, but clearly results in a difference in systemic exposure. The rapid systemic clearance of low sialylated IL4–10 FP could be a favorable characteristic to minimize systemic exposure after administration in a local compartment.

Based on their mechanisms-of-action, CD20 monoclonal antibodies (mAbs) are grouped into Type I [complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)] and Type II [programmed cell death (PCD) and ADCC] mAbs. We generated 17 new hybridomas producing CD20 mAbs of different isotypes and determined unique heavy and light chain sequence pairs for 13 of them. We studied their epitope binding, binding kinetics and structural properties and investigated their predictive value for effector functions, i.e. PCD, CDC and ADCC. Peptide mapping and CD20 mutant screens revealed that 10 out of these 11 new mAbs have an overlapping epitope with the prototypic Type I mAb rituximab, albeit that distinct amino acids of the CD20 molecule contributed differently. Binding kinetics did not correlate with the striking differences in CDC activity among the mIgG2c mAbs. Interestingly, chimerization of mAb m1 resulted in a mAb displaying both Type I and II characteristics. PCD induction was lost upon introduction of a mutation in the framework of the heavy chain affecting the elbow angle, supporting that structural changes within this region can affect functional activities of CD20 mAbs. Together, these new CD20 mAbs provide further insights in the properties dictating the functional efficacy of CD20 mAbs.

Background Cow's milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow's milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays. Objective An oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays. Methods Mice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains. Results After sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5–10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow's milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow's milk allergic children. Conclusion Usage of our 'unlimited' source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow's milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.

Therapeutic monoclonal antibodies, a highly successful class of biological drugs, are conventionally manufactured in mammalian cell lines. A recent approach to increase the therapeutic effectiveness of monoclonal antibodies has been to combine two or more of them; however this increases the complexity of development and manufacture. To address this issue a method to efficiently express multiple monoclonal antibodies from a single cell has been developed and we describe here the generation of stable cell clones that express high levels of a human monoclonal antibody mixture. PER.C6® cells were transfected with a combination of plasmids containing genes encoding three different antibodies. Clones that express the three corresponding antibody specificities were identified, subcloned, and passaged in the absence of antibiotic selection pressure. At several time points, batch production runs were analyzed for stable growth and IgG production characteristics. The majority (11/12) of subclones analyzed expressed all three antibody specificities in constant ratios with total IgG productivity ranging between 15 and 20 pg/cell/day under suboptimal culture conditions after up to 67 population doublings. The growth and IgG production characteristics of the stable clones reported here resemble those of single monoclonal antibody cell lines from conventional clone generation programs. We conclude that the methodology described here is applicable to the generation of stable PER.C6® clones for industrial scale production of mixtures of antibodies. Biotechnol. Bioeng. 2010;106: 741–750. © 2010 Wiley Periodicals, Inc.

To the Editor: Streptococcus pneumoniae is a major cause of pneumonia, septicemia, and meningitis. It causes great morbidity and mortality throughout the world. In particular, young children, the elderly, and immunocompromised individuals are susceptible to pneumococcal infections. Host protection against pneumococcal infections is mediated by antibodies against pneumococcal surface proteins and capsular polysaccharides (caps-PSs).1Bruyn G.A. Zegers B.J. van Furth R. Mechanisms of host defense against infection with Streptococcus pneumoniae.Clin Infect Dis. 1992; 14: 251-262Crossref PubMed Scopus (166) Google Scholar Caps-PSs are a main determinant of virulence of S pneumoniae, and antibodies to caps-PSs are protective against infection with S pneumoniae. Some pneumococcal surface proteins, such as pneumococcal surface protein A (PspA), are also considered virulence factors. Whereas proteins are T lymphocyte–dependent antigens, caps-PSs are considered T lymphocyte–independent type 2 (TI-2) antigens. Although T lymphocytes are not needed for induction of the antibody response to TI-2 antigens, they are involved in the regulation of this antibody response.2Mond J.J. Lees A. Snapper C.M. T cell-independent antigens type 2.Annu Rev Immunol. 1995; 13: 655-692Crossref PubMed Scopus (697) Google Scholar The role of T lymphocytes in the regulation of the antibody production to TI-2 antigens is poorly understood. Little is known regarding the mechanisms by which intact S pneumoniae elicits protein- and caps-PS–specific antibodies in human subjects. In the present study we immunized humanized mice with severe combined immunodeficiency (SCID) with heat-inactivated S pneumoniae as a model to study the human antibody response to S pneumoniae. To evaluate the role of CD4+ T cells in the antibody response to pneumococcal caps-PSs and PspA, 6- to 8-week-old SCID mice were treated with TMβ1 (an anti-murine IL-2 receptor β-chain antibody used to deplete murine natural killer cells and to improve transplant survival3Tournoy K.G. Depraetere S. Meuleman P. Leroux-Roels G. Pauwels R.A. Murine IL-2 receptor beta chain blockade improves human leukocyte engraftment in SCID mice.Eur J Immunol. 1998; 28: 3221-3230Crossref PubMed Scopus (31) Google Scholar) and transplanted (in peritoneum) either with PBMCs (containing 4 × 106 B lymphocytes, n = 6) or with PBMCs depleted of CD4+ cells (by using microbeads; Miltenyi Biotec, Bergisch Gladbach, Germany; n = 6). After transplantation, the mice were immunized with heat-inactivated S pneumoniae serotype 3 (2 × 108 colony-forming units). Two weeks after immunization, antibodies were measured by means of ELISA (with preabsorption of the sera with caps-PS serotype 22F [5 mg/L] for anti–caps-PS).4Jeurissen A. Wuyts G. Kasran A. Ramdien-Murli S. Blanckaert N. Boon L. et al.The human antibody response to pneumococcal capsular polysaccharides is dependent on the CD40-CD40 ligand interaction.Eur J Immunol. 2004; 34: 850-858Crossref PubMed Scopus (23) Google Scholar, 5Khan A.Q. Lees A. Snapper C.M. Differential regulation of IgG anti-capsular polysaccharide and antiprotein responses to intact Streptococcus pneumoniae in the presence of cognate CD4 (+) T cell help.J Immunol. 2004; 172: 532-539PubMed Google Scholar SCID/SCID mice reconstituted with PBMCs, but not with PBMCs depleted of CD4+ cells, produced high levels of IgM and IgG antibodies against caps-PSs and PspA (data not shown; P < .03, Mann-Whitney U test). SCID/SCID mice were reconstituted with human PBMCs and immunized with S pneumoniae serotype 3 in the presence (n = 6) or absence of antagonistic anti-CD40 (ch5D12, 500 μg; PanGenetics, Utrecht, The Netherlands; n = 6) to investigate whether the CD40–CD40 ligand (CD40L) interaction is important for the human IgM and IgG anti–caps-PS antibody production in vivo. Anti-CD40 totally blocked the IgM and IgG anti-PspA antibody response, as well as the anti–caps-PS antibody response (P < .03). Human polyclonal control antibodies did not affect the antibody response (data not shown). Similarly, anti-CD40 blocked the antibody response to S pneumoniae serotype 1 and serotype 14 and the antibody response (IgM and IgG) to dextran, another TI-2 antigen (data not shown; P < .03, Mann-Whitney U test). Next we examined whether the stimulatory effect of CD4+ T lymphocytes on the human polysaccharide- and protein-specific antibody response to S pneumoniae was the result of a direct effect of T lymphocytes on B lymphocytes or of an indirect effect through interaction of T lymphocytes with CD14+ mononuclear cells. SCID/SCID mice were reconstituted with (1) purified B lymphocytes, (2) purified B lymphocytes in combination with purified CD4+ T lymphocytes, or (3) purified B lymphocytes in combination with purified CD4+ T lymphocytes and purified CD14+ cells. The results are shown in Fig 1 and indicate that transfer of B lymphocytes, CD4+ T lymphocytes, and CD14+ antigen-presenting cells was needed for SCID/SCID mice to be able to produce polysaccharide- and protein-specific antibodies. Transfer of purified B and T lymphocytes was not sufficient to induce anti–caps-PS and anti-protein antibody production. The presence of CD14+ monocytes was needed. The anti–caps-PS and anti-PspA antibody production by B lymphocytes, T lymphocytes, and CD14+ mononuclear cells could be totally blocked with anti-CD40 mAbs (P < .0003). Taken together, these data suggest that CD40L expressing CD4+ T lymphocytes stimulate the anti–caps-PS and anti-protein antibody production through interaction with CD40-expressing non-B antigen-presenting cells. Patients with X-linked hyper-IgM (XHIM) syndrome have a mutation at the CD40L gene and therefore lack expression of functional CD40L. Patients with XHIM syndrome have mainly IgM-producing CD27+IgD+IgM+ B lymphocytes and lack IgG-producing CD27+IgD−IgM− B lymphocytes.6Agematsu K. Nagumo H. Shiozaki K. Hokibara S. Yasui K. Terada K. et al.Absence of IgD-CD27+ memory B cell population in X-linked hyper-IgM syndrome.J Clin Invest. 1998; 102: 853-860Crossref PubMed Scopus (166) Google Scholar SCID/SCID mice reconstituted with CD19+ B lymphocytes originating from a healthy blood donor and with PBMCs depleted of CD19+ B lymphocytes from a patient with XHIM syndrome completely failed to produce anti–caps-PS and anti-PspA antibodies after immunization with S pneumoniae. By contrast, SCID/SCID mice repopulated with CD19+ B lymphocytes from a patient with XHIM syndrome in combination with PBMCs depleted of CD19+ B lymphocytes from a healthy blood donor produced IgM anti–caps-PS and anti-PspA antibody levels comparable with those seen in the control condition after immunization with S pneumoniae (data not shown). Only very low levels of IgG anti–caps-PS and anti-PspA antibodies were formed (data not shown). In a previous study we demonstrated that the human antibody response to soluble caps-PS (pneumococcal vaccine) was dependent on the CD40-CD40L interaction.3Tournoy K.G. Depraetere S. Meuleman P. Leroux-Roels G. Pauwels R.A. Murine IL-2 receptor beta chain blockade improves human leukocyte engraftment in SCID mice.Eur J Immunol. 1998; 28: 3221-3230Crossref PubMed Scopus (31) Google Scholar In the present study we evaluated the regulation of the human antibody response to S pneumoniae. More specifically, we investigated the role of CD4+ T cells and the CD40-CD40L costimulatory interaction in the protein (PspA)– and caps-PS-specific antibody responses to S pneumoniae. Consistent with studies in mice,5Khan A.Q. Lees A. Snapper C.M. Differential regulation of IgG anti-capsular polysaccharide and antiprotein responses to intact Streptococcus pneumoniae in the presence of cognate CD4 (+) T cell help.J Immunol. 2004; 172: 532-539PubMed Google Scholar we found that the IgG polysaccharide-specific and protein-specific response to S pneumoniae was dependent on CD4+ T lymphocytes and the CD40-CD40L interaction. In contrast to a previous study in mice,5Khan A.Q. Lees A. Snapper C.M. Differential regulation of IgG anti-capsular polysaccharide and antiprotein responses to intact Streptococcus pneumoniae in the presence of cognate CD4 (+) T cell help.J Immunol. 2004; 172: 532-539PubMed Google Scholar the human IgM anti-polysaccharide response to S pneumoniae was dependent on CD4+ T cells, CD14+ mononuclear cells, and the CD40-CD40L interaction. The IgM protein– and caps-PS specific antibody response was blocked by anti-CD40, and adoptive transfer of CD4+ T cells obtained from a patient with XHIM syndrome failed to stimulate the IgM anti-protein and anti–caps-PS antibody response. Finally, we revealed that CD40L-expressing CD4+ T lymphocytes stimulate the human anti–caps-PS antibody response in an indirect way through interaction with CD40-expressing CD14+ monocytes. In murine studies on the antibody response to soluble caps-PS, a similar interaction between CD4+ T cells and antigen-presenting cells has been proposed.7Jeurissen A. Billiau A.D. Moens L. Shengqiao L. Landuyt W. Wuyts G. et al.CD4+ T lymphocytes expressing CD40 ligand help the IgM antibody response to soluble isolated pneumococcal polysaccharides via an intermediate cell type.J Immunol. 2006; 176: 529-536PubMed Google Scholar We hypothesize that the CD40-CD40L interaction between CD14+ mononuclear cells and CD4+ T cells could induce the release of soluble factors that can further stimulate B lymphocytes to produce IgM and IgG anti–caps-PS antibodies. A role for dendritic cells in the anti–caps-PS immune response has previously been suggested. Meltzer and Goldblatt8Melzer U. Goldblatt D. Pneumoccal polysaccharides interact with human dendritic cells.Infect Immun. 2006; 74: 1890-1895Crossref PubMed Scopus (30) Google Scholar showed that pneumococcal polysaccharides interact with human dendritic cells. Colino et al9Colino J. Shen Y. Snapper C.M. Dendritic cells pulsed with intact Streptococcus pneumoniae elicit both protein- and polysaccharide-specific immunoglobulin isotype responses in vivo through distinct mechanisms.J Exp Med. 2001; 195: 1-13Crossref Scopus (117) Google Scholar reported that bone marrow–derived dendritic cells pulsed with S pneumoniae could stimulate the anti–caps-PS antibody response when injected into naive mice and that endogenous T lymphocytes were critical for the development of IgG antibodies specific for both protein and polysaccharide antigens. In conclusion, the data described in this report implicate a role for endogenous CD40-CD40L interactions in the human antibody response to TI-2 antigens in general and to intact heat-inactivated S pneumoniae in particular. CD40L-expressing CD4+ T lymphocytes stimulate the antibody response to S pneumoniae through interaction with CD14+ mononuclear cells.

Clinical trials designed to achieve tolerance in humans by selectively antagonizing one of the T-cell costimulatory pathways, CD40-CD40L or CD80/CD86-CD28, are pending. However, simultaneous blockade of both pathways synergistically prevented graft rejection and successfully induced donor-specific tolerance in animal models. Synergism is also supported in human T-cells in vitro following anti-CD86 mAb and anti-CD40 mAb blockade. Therefore, in our view the most promising clinical strategy would be to antagonize both CD40 and CD86. Fast clinical entrance of this anti-CD86 and anti-CD40 bidirectional concept is highly facilitated by a single molecule approach. In the present study, a single bispecific fusion protein was constructed that specifically binds human CD40 and CD86 and which combines the antagonistic activities of both anti-CD40 and anti-CD86 humanized mAb. The anti-CD40/86 fusion protein showed tolerance inducing potential as it prevented both allogeneic T-cell expansion and generation of cytotoxic effector T cells and induced anergic antigen specific regulatory T cells. These data provide proof of concept in successfully combining the antagonistic activity of two humanized mAb with great clinical potential in transplantation and autoimmunity, in one single molecule.

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.

Monoclonal antibody (Mab) 5D12 is a potent antagonist of the CD40–CD40L pathway. This cellular interaction has been validated in a large number of experimental animal models where dys-regulation of the immune system plays a role. Chimeric 5D12 (ch5D12) was constructed to reduce the potential immunogenicity and enhance the in vivo half-life when used in humans. ch5D12 is a molecularly engineered human IgG4 antibody containing the variable domains of the heavy and light chains of the murine version of 5D12 (mu5D12). This new chimeric Mab was tested in a marmoset experimental autoimmune encephalomyelitis model and was shown to effectively prevent disease symptoms. The results of this in vivo evaluation supported clinical use of ch5D12 for immune targeted diseases. Therefore GMP material was prepared and a GLP-compliant tissue cross-reactivity study on human tissues (3 donors/37 tissues) and cynomolgus tissues (2 donors/37 tissues) was performed. ch5D12 stained on the surface of B cells and selected dendritic cells and no unexpected cross-reactivity was observed. The identical staining patterns in human and cynomolgus tissues justified the use of cynomolgus monkeys as a relevant model for humans. A GLP-compliant safety and tolerability evaluation for ch5D12 in cynomolgus monkeys was performed using the GMP produced material. Weekly administration of ch5D12 at two dose levels for 4 weeks was shown to be safe and without any side effects in all monkeys.