A new approach to produce IgG4-like bispecific antibodies.

While achieving rapid developments in recent years, bispecific antibodies are still difficult to design and manufacture, due to mispair of both heavy and light chains. Here we report a novel technology to make bispecific molecules. The knob-into-hole method was used to pair two distinct heavy chains as a heterodimer. IgG4 S228P CH1-CL interface was then partially replaced by T-cell receptor α/ß constant domain to increase the efficiency of cognate heavy and light chain pairing. Following expression and purification, the bispecific antibody interface exchange was confirmed by Western blotting and LC-MS/MS. To ensure its validity, we combined a monovalent bispecific antibody against PD-1 (sequence from Pembrolizumab) and LAG3 (sequence from Relatlimab). The results showed that the molecule could be assembled correctly at a ratio of 95% in cells. In vitro functional assay demonstrated that the purified bispecific antibody exhibits an enhanced agonist activity compared to that of the parental antibodies. Low immunogenicity was predicted by an open-access software and ADA test.

Memory B Cells in Multiple Sclerosis: Emerging Players in Disease Pathogenesis.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Once thought to be primarily driven by T cells, B cells are emerging as central players in MS immunopathogenesis. Interest in multiple B cell phenotypes in MS expanded following the efficacy of B cell-depleting agents targeting CD20 in relapsing-remitting MS and inflammatory primary progressive MS patients. Interestingly, these therapies primarily target non-antibody secreting cells. Emerging studies seek to explore B cell functions beyond antibody-mediated roles, including cytokine production, antigen presentation, and ectopic follicle-like aggregate formation. Importantly, memory B cells (Bmem) are rising as a key B cell phenotype to investigate in MS due to their antigen-experience, increased lifespan, and rapid response to stimulation. Bmem display diverse effector functions including cytokine production, antigen presentation, and serving as antigen-experienced precursors to antibody-secreting cells. In this review, we explore the cellular and molecular processes involved in Bmem development, Bmem phenotypes, and effector functions. We then examine how these concepts may be applied to the potential role(s) of Bmem in MS pathogenesis. We investigate Bmem both within the periphery and inside the CNS compartment, focusing on Bmem phenotypes and proposed functions in MS and its animal models. Finally, we review how current immunomodulatory therapies, including B cell-directed therapies and other immunomodulatory therapies, modify Bmem and how this knowledge may be harnessed to direct therapeutic strategies in MS.

Targeting the membrane-proximal C2-set domain of CD33 for improved CD33-directed immunotherapy.

There is increasing interest in targeting CD33 in malignant and non-malignant disorders. In acute myeloid leukemia, longer survival with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this strategy. Still, GO benefits only some patients, prompting efforts to develop more potent CD33-directed therapeutics. As one limitation, CD33 antibodies typically recognize the membrane-distal V-set domain. Using various artificial CD33 proteins, in which this domain was differentially positioned within the extracellular portion of the molecule, we tested whether targeting membrane-proximal epitopes enhances the effector functions of CD33 antibody-based therapeutics. Consistent with this idea, a CD33V-set/CD3 bispecific antibody (BsAb) and CD33V-set-directed chimeric antigen receptor (CAR)-modified T cells elicited substantially greater cytotoxicity against cells expressing a CD33 variant lacking the entire C2-set domain than cells expressing full-length CD33, whereas cytotoxic effects induced by GO were independent of the position of the V-set domain. We therefore raised murine and human antibodies against the C2-set domain of human CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33PAN antibodies"). These antibodies internalized when bound to CD33 and, as CD33PAN/CD3 BsAb, had potent cytolytic effects against CD33+ cells. Together, our data provide the rationale for further development of CD33PAN antibody-based therapeutics.

Essentially Leading Antibody Production: An Investigation of Amino Acids, Myeloma, and Natural V-Region Signal Peptides in Producing Pertuzumab and Trastuzumab Variants.

Boosting the production of recombinant therapeutic antibodies is crucial in both academic and industry settings. In this work, we investigated the usage of varying signal peptides by antibody V-genes and their roles in recombinant transient production, systematically comparing myeloma and the native signal peptides of both heavy and light chains in 168 antibody permutation variants. We found that amino acids count and types (essential or non-essential) were important factors in a logistic regression equation model for predicting transient co-transfection protein production rates. Deeper analysis revealed that the culture media were often incomplete and that the supplementation of essential amino acids can improve the recombinant protein yield. While these findings are derived from transient HEK293 expression, they also provide insights to the usage of the large repertoire of antibody signal peptides, where by varying the number of specific amino acids in the signal peptides attached to the variable regions, bottlenecks in amino acid availability can be mitigated.

Discovery of IL-6 and Development of Anti-IL-6R Antibody.

A series of our studies on IL-6 have revealed that it has a pleiotropic activity in various tissues and cells and its deregulated expression is responsible for several chronic inflammations and hemopoietic malignancies.Humanized antibody against 80kd IL-6R (Tocilizumab) has shown significant therapeutic effect in RA, JIA, Castleman's diseases and several other autoimmune inflammatory diseases, such as, giant cell arteritis, reactive arthritis, polymyalgia rheumatica and adult still's disease. Cytokine storm induced by CAR-T cell therapy has been shown to be controlled by Tocilizumab.Therapeutic effect of Tocilizumab confirmed that over and constitutive-production of IL-6 is responsible for the pathogenesis of autoimmune diseases.Then, the question to be asked is how is IL-6 production regulated. We identified a novel molecule called Arid5a which binds with the 3'-UTR of IL-6 mRNA and protects its degradation by competing with Regnase-1. Interestingly, this molecule is present in nuclei and inflammatory stimulation induced translocation of Arid5a from nuclei into cytoplasm and it competes with Regnase-1 for the protection of mRNA of IL-6.Our study indicates that Arid5a is one of the key molecules for inflammation as well as the development of septic shock.The results also suggest the therapeutic potential of anti-agonistic agents for Arid5a in the prevention of various inflammatory diseases and septic shock.

Development of a Novel Humanized Monoclonal Antibody to Secreted Frizzled-Related Protein-2 That Inhibits Triple-Negative Breast Cancer and Angiosarcoma Growth In Vivo.

BACKGROUND: We previously reported that secreted frizzled-related protein-2 (SFRP2) is expressed in a variety of tumors, including sarcoma and breast carcinoma, and stimulates angiogenesis and inhibits tumor apoptosis. Therefore, we hypothesized that a humanized SFRP2 monoclonal antibody (hSFRP2 mAb) would inhibit tumor growth. METHODS: The lead hSFRP2 antibody was tested against a cohort of 22 healthy donors using a time course T-cell assay to determine the relative risk of immunogenicity. To determine hSFRP2 mAb efficacy, nude mice were subcutaneously injected with SVR angiosarcoma cells and treated with hSFRP2 mAb 4 mg/kg intravenously every 3 days for 3 weeks. We then injected Hs578T triple-negative breast cells into the mammary fat pad of nude mice and treated for 40 days. Control mice received an immunoglobulin (Ig) G1 control. The SVR and Hs578T tumors were then stained using a TUNEL assay to detect apoptosis. RESULTS: Immunogenicity testing of hSFRP2 mAb did not induce proliferative responses using a simulation index (SI) ≥ 2.0 (p < 0.05) threshold in any of the healthy donors. SVR angiosarcoma tumor growth was inhibited in vivo, evidenced by significant tumor volume reduction in the hSFRP2 mAb-treated group, compared with controls (n = 10, p < 0.001). Likewise, Hs578T triple-negative breast tumors were smaller in the hSFRP2 mAb-treated group compared with controls (n = 10, p < 0.001). The hSFRP2 mAb treatment correlated with an increase in tumor cell apoptosis (n = 11, p < 0.05). Importantly, hSFRP2 mAb treatment was not associated with any weight loss or lethargy. CONCLUSION: We present a novel hSFRP2 mAb with therapeutic potential in breast cancer and sarcoma that has no effect on immunogenicity.

Development and Characterization of a Potent Tumor Necrosis Factor-Alpha-Blocking Agent.

Tumor necrosis factor-α (TNFα), one of the major proinflammatory cytokines, plays a key role in an effective immune response. However, the chronic presence of TNFα can lead to several inflammatory disorders, such as rheumatoid arthritis, psoriasis, Crohn's disease, etc. Inhibition of TNFα by pharmacological inhibitors or antibodies has proven to be effective in palliative treatment to some extent. The aim of this study was to develop an anti-TNFα antibody, which may be used as a therapeutic option to inhibit TNFα-mediated cytotoxicity. We characterized several hybridoma clones secreting monoclonal antibodies (mAbs) to human-TNFα. Four mAbs rescued L929 fibroblast cells from TNFα-triggered cell death and one of these, namely C8, was found to have the highest affinity. To gain insights into the mechanism by which mAb C8 inhibits human TNFα-mediated toxicity, the epitope corresponding to the mAb was delineated. The antigenic determinant was found to comprise of the stretch of amino acids 99-120, of which, 102-104 (glutamine, arginine, glutamic acid) form the core epitope. The observation was supported by bioinformatics analyses of an antigen/antibody complex model. In addition, the binding affinity of mAb C8 to TNFα was found to be comparable with that of infliximab, which is a commercially available anti-TNFα mAb.

Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).

Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.

Quality and cost assessment of a recombinant antibody fragment produced from mammalian, yeast and prokaryotic host cells: A case study prior to pharmaceutical development.

Monoclonal antibody fragments (Fab) are a promising class of therapeutic agents. Fabs are aglycosylated proteins and so many expression platforms have been developed including prokaryotic, yeast and mammalian cells. However, these platforms are not equivalent in terms of cell line development and culture time, product quality and possibly cost of production that greatly influence the success of a drug candidate's pharmaceutical development. This study is an assessment of the humanized Fab fragment ACT017 produced from two microorganisms (Escherichia coli and Pichia pastoris) and one mammalian cell host (CHO). Following low scale production and Protein L-affinity purification under generic conditions, physico-chemical and functional quality assessments were carried out prior to economic analysis of industrial scale production using a specialized software (Biosolve, Biopharm Services, UK). Results show higher titer production when using E. coli but associated with high heterogeneity of the protein content recovered in the supernatant. We also observed glycoforms of the Fab produced from P. pastoris, while Fab secreted from CHO was the most homogeneous despite a much longer culture time and slightly higher estimated cost of goods. This study may help inform future pharmaceutical development of this class of therapeutic proteins.

Antibody engineering to improve manufacturability.

Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.

A BioDesign Approach to Obtain High Yields of Biosimilars by Anti-apoptotic Cell Engineering: a Case Study to Increase the Production Yield of Anti-TNF Alpha Producing Recombinant CHO Cells.

Recent developments in medical biotechnology have facilitated to enhance the production of monoclonal antibodies (mAbs) and recombinant proteins in mammalian cells. Human mAbs for clinical applications have focused on three areas, particularly cancer, immunological disorders, and infectious diseases. Tumor necrosis factor alpha (TNF-α), which has both proinflammatory and immunoregulatory functions, is an important target in biopharmaceutical industry. In this study, a humanized anti-TNF-α mAb producing stable CHO cell line which produces a biosimilar of Humira (adalimumab) was used. Adalimumab is a fully human anti-TNF mAb among the top-selling mAb products in recent years as a biosimilar. Products from mammalian cell bioprocesses are a derivative of cell viability and metabolism, which is mainly disrupted by cell death in bioreactors. Thus, different strategies are used to increase the product yield. Suppression of apoptosis, also called anti-apoptotic cell engineering, is the most remarkable strategy to enhance lifetime of cells for a longer production period. In fact, using anti-apoptotic cell engineering as a BioDesign approach was inspired by nature; nature gives prolonged life span to some cells like stem cells, tumor cells, and memory B and T cells, and researchers have been using this strategy for different purposes. In this study, as a biomimicry approach, anti-apoptotic cell engineering was used to increase the anti-TNF-α mAb production from the humanized anti-TNF-α mAb producing stable CHO cell line by Bcl-xL anti-apoptotic protein. It was shown that transient transfection of CHO cells by the Bcl-xL anti-apoptotic protein expressing plasmid prolonged the cell survival rate and protected cells from apoptosis. The transient expression of Bcl-xL using CHO cells enhanced the anti-TNF-α production. The production of anti-TNF-α in CHO cells was increased up to 215 mg/L with an increase of 160% after cells were transfected with Bcl-xL expressing plasmid with polyethylenimine (PEI) reagent at the ratio of 1:6 (DNA:PEI). In conclusion, the anti-apoptotic efficacy of the Bcl-xL expressing plasmid in humanized anti-TNF-α MAb producing stable CHO cells is compatible with curative effect for high efficiency recombinant protein production. Thus, this model can be used for large-scale production of biosimilars through transient Bcl-xL gene expression as a cost-effective method.

Chickens with humanized immunoglobulin genes generate antibodies with high affinity and broad epitope coverage to conserved targets.

Transgenic animal platforms for the discovery of human monoclonal antibodies have been developed in mice, rats, rabbits and cows. The immune response to human proteins is limited in these animals by their tolerance to mammalian-conserved epitopes. To expand the range of epitopes that are accessible, we have chosen an animal host that is less phylogenetically related to humans. Specifically, we generated transgenic chickens expressing antibodies from immunoglobulin heavy and light chain loci containing human variable regions and chicken constant regions. From these birds, paired human light and heavy chain variable regions are recovered and cloned as fully human recombinant antibodies. The human antibody-expressing chickens exhibit normal B cell development and raise immune responses to conserved human proteins that are not immunogenic in mice. Fully human monoclonal antibodies can be recovered with sub-nanomolar affinities. Binning data of antibodies to a human protein show epitope coverage similar to wild type chickens, which we previously showed is broader than that produced from rodent immunizations.

[Antibody Engineering: From the Idea to Its Implementation].

The late 1970s brought opportunities to create proteins with new properties and, in particular, various derivatives of mouse monoclonal antibodies (mAbs) owing to the discoveries in molecular and cell biology and the development of bioengineering. Studies of mouse/human "chimeric" antibodies, miniantibodies to be synthesized in bacterial cells, full-size single-chain antibodies, complexes of miniantibodies with intramolecular chaperones, and other approaches made it possible to create a multitude of multifunctional biopreparations with predefined properties. The review describes, with the example of one research team, how studies in the field began and what the basis for their progress was.

[Humanization of Murine Monoclonal anti-hTNF Antibody: The F10 Story].

Tumor necrosis factor (TNF) is a proinflammatory cytokine implicated in pathogenesis of multiple autoimmune and inflammatory diseases. Anti-TNF therapy has revolutionized the therapeutic paradigms of autoimmune diseases and became one of the most successful examples of the clinical use of monoclonal antibodies. Currently, anti-TNF therapy is used by millions of patients worldwide. At the moment, fully human anti-TNF antibody Adalimumab is the best-selling anti-cytokine drug in the world. Here, we present a story about a highly potent anti-TNF monoclonal antibody initially characterized more than 20 years ago and further developed into chimeric and humanized versions. We present comparative analysis of this antibody with Infliximab and Adalimumab.

5B9, a monoclonal antiplatelet factor 4/heparin IgG with a human Fc fragment that mimics heparin-induced thrombocytopenia antibodies.

Essentials No humanized monoclonal antibody was available to study heparin-induced thrombocytopenia (HIT). We developed the first anti-platelet factor 4 (PF4)/heparin antibody with a human Fc fragment. This antibody (5B9) fully mimics the effects of human HIT antibodies. 5B9 binds two regions within PF4 that may be critical for the pathogenicity of HIT antibodies. SUMMARY: Background The diagnosis of heparin-induced thrombocytopenia (HIT) is based on clinical and biological criteria, but a standard is lacking for laboratory assays. Moreover, no humanized HIT antibody is available for pathophysiological studies. Objective To characterise 5B9, a chimeric monoclonal antibody, which fully mimics the effects of human HIT antibodies. Methods/Results 5B9, a chimeric anti-platelet factor 4/heparin complexes IgG1 antibody, was obtained after immunizing specific transgenic mice. 5B9 induced heparin FcγRIIA-dependent platelet aggregation and tissue factor mRNA synthesis in monocytes. It also induced significant thrombocytopenia and thrombin generation in mice expressing human PF4 and FcγRIIA receptors. The binding of 5B9 to PF4/H complexes was inhibited by 15 of 25 HIT plasma samples and only three of 25 samples containing non-pathogenic anti-PF4/H antibodies. KKO, a murine IgG2b HIT antibody, also inhibited the binding of 5B9 to PF4/H, suggesting that epitopes recognized by both antibodies are close. A docking analysis based on VH and VL sequences of 5B9 showed that binding of 5B9 Fab to PF4 involved 12 and 12 residues in B and D monomers, respectively, including seven previously identified as critical to the formation of a PF4/KKO complex. Two regions (Asp-7 to Thr-15 and Ala-32 to Thr-38) therefore appeared important for the binding of 5B9 and KKO on PF4 modified by heparin. Conclusions 5B9 is the first anti-PF4/H monoclonal antibody with a human Fc fragment, which induces similar cellular activation as HIT antibodies. Moreover, 5B9 binds epitopes within PF4 that are likely to be critical for the pathogenicity of HIT antibodies.

Different fermentation processes produced variants of an anti-CD52 monoclonal antibody that have divergent in vitro and in vivo characteristics.

The anti-CD52 antibody has already been approved for the treatment of patients with resistant chronic lymphocytic leukemia, relapsing-remitting multiple sclerosis, and has demonstrable efficacy against stem cell transplantation rejection. A CHO cell line expressing a humanized anti-CD52 monoclonal antibody (mAb-TH) was cultivated in both fed-batch and perfusion modes, and then purified. The critical quality attributes of these mAb variants were characterized and the pharmacokinetics (PK) properties were investigated. Results showed that the perfusion culture achieved higher productivity, whereas the fed-batch culture produced more aggregates and acid components. Additionally, the perfusion culture produced similar fucose, more galactose and a higher proportion of sialic acid on the anti-CD52 mAb compared to the fed-batch culture. Furthermore, the perfusion process produced anti-CD52 mAb had higher complement-dependent cytotoxicity (CDC) efficacy than that produced by the fed-batch culture, a result probably linked to its higher galactose content. However, antibody produced by fed-batch and perfusion cultures showed similar PK profiles in vivo. In conclusion, perfusion is a more efficient method than fed-batch process in the production of functional anti-CD52 monoclonal antibody. Product quality variants of anti-CD52 mAb were found in different cell culture processes, which demonstrated different physiochemical and biological activities, but comparable PK properties. Whether these observations apply to all mAbs await further investigation.

Improving Pertuzumab production by gene optimization and proper signal peptide selection.

Using proper signal peptide and codon optimization are important factors that must be considered when designing the vector to increase protein expression in Chinese Hamster Ovary (CHO) cells. The aim of the present study is to investigate how to enhance Pertuzumab production through heavy and light chain coding gene optimization and proper signal peptide selection. First, CHO-K1 cells were transiently transfected with whole-antibody-gene-optimized, variable-regions-optimized and non-optimized constructs and then we employed five different signal peptides to improve the secretion efficiency of Pertuzumab. Compared to the native antibody gene, a 3.8 fold increase in Pertuzumab production rate was achieved with the whole heavy and light chain sequence optimization. Although an overall two fold increase in monoclonal antibody production was achieved by human albumin signal peptide compared to the control signal peptide, this overproduction was not statistically significant. Selected signal peptides had no effect on the binding of Pertuzumab to the ErbB2 antigen. The combined data indicate that human albumin signal peptide along with whole antibody sequence optimization can be used to improve Pertuzumab production rates. This sequence was used to produce Pertuzumab producing CHO-K1 stably transfected cells. This result is useful for producing Pertuzumab as a biosimilar drug.