Expression of Multispecific Antibodies.

Bi- and multispecific antibody formats allow the development of new therapeutic strategies to address previously unmet medical needs. However, due to the increased complexity (e.g., the interface design and the presence of multiple binders), such molecules are generally more challenging to express and purify compared to standard monoclonal antibodies (mAbs). We describe here an optimized methodology to express and purify basic bispecific antibodies using the BEAT® interface. This interface allows to generate antibodies with very high levels of heterodimer product (reported titers exceed 10 g/L) and comes with a built-in purification strategy allowing removal of residual levels of undesired product-related impurities (e.g., homodimers and half molecules).

CaptureSelect FcXP affinity medium exhibits strong aggregate separation capability.

Protein A affinity chromatography has been widely used for initial product capture in recombinant antibody/Fc-fusion purification. However, in general Protein A lacks the capability of separating aggregates (unless the aggregates are too large to enter the pores of resin beads or have their Protein A binding sites buried, in which case the aggregates do not bind). In the current work, we demonstrated that CaptureSelect FcXP affinity medium exhibited strong aggregate separation capability and effectively removed aggregates under pH or conductivity gradient elution in two bispecific antibody (bsAb) cases. For these two cases, aggregate contents were reduced from >16% and >22% (in the feed) to <1% and <5% (in the eluate) for the first and second bsAbs, respectively. While more case studies are required to further demonstrate FcXP's superiority in aggregate removal, findings from the current study suggest that FcXP can potentially be a better alternative than Protein A for product capture in cases where aggregate content is high.

Systematic workflow for studying domain contributions of bispecific antibodies to selectivity in multimodal chromatography.

In this article, a systematic workflow was formulated and implemented to understand selectivity differences and preferred binding patches for bispecific monoclonal antibodies (mAbs) and their parental mAbs on three multimodal cation exchange resin systems. This workflow incorporates chromatographic screening of the parent mAbs and their fragments at various pH followed by surface property mapping and protein footprinting using covalent labeling followed by liquid chromatography-mass spectrometry analysis. The chromatography screens on multimodal resins with the intact mAbs indicated enhanced selectivity as compared to single-mode interaction systems. While the bispecific antibody (bsAb) eluted between the two parental mAbs on most of the resins, the retention of the bispecific transitioned from co-eluting with one parental mAb to the other parental mAb on Capto MMC. To investigate the contribution of different domains, mAb fragments were evaluated and the results indicated that the interactions were likely dominated by the Fab domain at higher pH. Protein surface property maps were then employed to hypothesize the potential preferred binding patches in the solvent-exposed regions of the parental Fabs. Finally, protein footprinting was carried out with the parental mAbs and the bsAb in the bound and unbound states at pH 7.5 to identify the preferred binding patches. Results with the intact mAb analysis supported the hypothesis that interactions with the resins were primarily driven by the residues in the Fab fragments and not the Fc. Furthermore, peptide mapping data indicated that the light chain may be playing a more important role in the higher binding of Parent A as compared with Parent B in these resin systems. Finally, results with the bsAb indicated that both halves of the molecule contributed to binding with the resins, albeit with subtle differences as compared to the parental mAbs. The workflow presented in this paper lays the foundation to systematically study the chromatographic selectivity of large multidomain molecules which can provide insights into improved biomanufacturability and expedited downstream bioprocess development.

Production of IgG1-based bispecific antibody without extra cysteine residue via intein-mediated protein trans-splicing.

A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

Immunoglobulin-binding protein-based affinity chromatography in bispecific antibody purification: Functions beyond product capture.

In general, purification of bispecific antibody (bsAb) is more challenging than that of monospecific antibody due to the increased complexity in byproduct profile. Like in the case of monospecific antibody purification, immunoglobulin-binding protein-based affinity chromatography is an indispensable tool for bsAb purification. For example, Protein A affinity chromatography has been widely used to capture Fc-containing bsAbs whereas other affinity media such as Protein L and KappaSelect, which bind kappa light chain, are used to capture bsAbs that do not contain a Protein A-binding site. In fact, affinity chromatography also possesses the capability of removing certain product-related impurities in bsAb purification when it is conducted with suitable medium and under appropriate conditions. Fully exploring the potential of affinity chromatography in bsAb purification to achieve both product capture and byproduct removal is highly desirable, as this can greatly alleviate the purification burden on subsequent polishing steps and hence improves the overall robustness of the downstream process. This article briefly reviews the byproduct clearance potential of several commonly used affinity media under relevant bsAb purification scenarios.

IgG-like bispecific antibody platforms with built-in purification-facilitating elements.

Assembly of IgG-like asymmetric bispecific antibodies (bsAbs) requires heavy chain heterodimerization and cognate heavy-light chain pairings. Multiple strategies have been developed to solve these chain association issues. While these strategies greatly promote correct chain pairing, they normally cannot prevent low amount of chain mispaired byproducts from being generated. Besides, byproducts can also be generated as a result of discordant chain expression. The existence of various byproducts poses considerable challenges to downstream processing during the production of recombinant IgG-like bsAbs. In many cases, yield is greatly compromised for purity improvement. This mini review introduces eight IgG-like bsAb platforms, which share a common feature: they all contain built-in purification-facilitating elements in addition to chain pairing control designs. These platforms, by simultaneously providing solutions to the two issues associated with bsAb production (i.e., correct chain pairing and efficient purification), improve both efficiency and robustness of bsAb production.

Comparing the relative robustness of anion exchange and the corresponding mixed-mode chromatography on removing a weakly-bound byproduct: A case study of bispecific antibody purification.

A certain impurity may bind weaker or tighter to a particular type of chromatography column (e.g., ion exchange, hydrophobic interaction or mixed-mode) than the target protein, and this forms the basis for separation. For impurities that bind weaker, they can be removed by an appropriate pre-elution wash. However, we previously showed that wash-enabled impurity clearance is usually sensitive to loading density, causing poor robustness of the process. In this work, with a bispecific antibody case study, we compared the relative robustness of anion exchange and the corresponding mixed-mode chromatography, which mediates both anion exchange and hydrophobic interactions, on removing a weakly-bound byproduct by wash. It was learned that under a fixed appropriate wash condition, the latter achieves consistent byproduct clearance and good yield over a much wider range of loading density than the former. As wide loading density range is highly desirable for large-scale manufacturing, the above finding suggests that for a chromatography step that employs stepwise elution and relies on pre-elution wash for removing weakly-bound impurities, mixed-mode chromatography could be a better choice than the corresponding monomode ion exchange chromatography.

Evaluating a new mixed-mode resin Diamond MMC Mustang using Capto MMC ImpRes as a benchmark.

Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. In this work, we evaluated this resin using Cytiva's Capto MMC ImpRes, a well-established mixed-mode resin with similar properties, as a benchmark. The data suggest that in comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable binding capacity and resolution. In addition, the resin under evaluation shows good lot-to-lot consistency. The information provided in this study allows users to have additional options when selecting mixed-mode resin for intermediate purification or final polishing, which is favourable especially at the present time when the supply chains of many manufacturers are negatively impacted by the coronavirus pandemic.

Removing a light-chain missing byproduct by MMC ImpRes mixed-mode chromatography under weak partitioning mode in purifying a WuXiBody-based bispecific antibody.

WuXiBody is a bispecific antibody (bsAb) platform developed by WuXi Biologics. Its key feature is the replacement of one parental antibody's CH1/CL region with the T cell receptor (TCR) constant domain, a design that promotes cognate heavy chain (HC)-light chain (LC) pairing. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. For purifying a WuXiBody-based symmetric bsAb, we identified a LC-missing species as a major byproduct. While for bsAbs based on other platforms removal of such byproduct can pose considerable challenge to the downstream team, in this case WuXiBody's unique design makes separation relatively straightforward. We previously showed that Capto MMC ImpRes mixed-mode chromatography under bind-elute mode can effectively remove this LC-missing species. However, the dynamic binding capacity (DBC) of Capto MMC ImpRes is relatively low under the selected condition, making the process less desirable for large-scale manufacturing. In this study, we demonstrated that when Capto MMC ImpRes chromatography is conducted under weak partitioning mode, high throughput, good yield, and effective byproduct removal are simultaneously achieved.

Sodium caprylate wash during Protein A chromatography as an effective means for removing protease(s) responsible for target antibody fragmentation.

For recombinant proteins produced in Chinese hamster ovary (CHO) cells, fragmentation is a common phenomenon that results in generation of product-related low-molecular-weight (LMW) species. Recently while purifying a bispecific antibody (bsAb), we observed that the target protein experienced cleavage at a couple of potential sites, leading to truncated products. Further studies suggest that the cleavage can likely be attributed to residual CHO cell protease activity. In order to maximally remove potential protease(s) that contribute fragmentation, we optimized Protein A chromatography by adding sodium caprylate (SC) to the wash buffer. Upon optimization, fragmentation of Protein A eluate happened to a much lesser degree as compared to that of eluate from unoptimized process, and the increased sample stability is in accordance with significantly reduced host cell protein (HCP) level. Taken together, the data suggest that SC wash during Protein A chromatography is an effective means for removing HCPs including endogenous protease(s) that are responsible for target antibody fragmentation.

Bispecific Antibody PD-L1 x CD3 Boosts the Anti-Tumor Potency of the Expanded Vγ2Vδ2 T Cells.

Vγ2Vδ2 T cell-based immunotherapy has benefited some patients in clinical trials, but the overall efficacy is low for solid tumor patients. In this study, a bispecific antibody against both PD-L1 and CD3 (PD-L1 x CD3), Y111, could efficiently bridge T cells and PD-L1 expressing tumor cells. The Y111 prompted fresh CD8+ T cell-mediated lysis of H358 cells, but spared this effect on the fresh Vδ2+ T cells enriched from the same donors, which suggested that Y111 could bypass the anti-tumor capacity of the fresh Vγ2Vδ2 T cells. As the adoptive transfer of the expanded Vγ2Vδ2 T cells was approved to be safe and well-tolerated in clinical trials, we hypothesized that the combination of the expanded Vγ2Vδ2 T cells with the Y111 would provide an alternative approach of immunotherapy. Y111 induced the activation of the expanded Vγ2Vδ2 T cells in a dose-dependent fashion in the presence of PD-L1 positive tumor cells. Moreover, Y111 increased the cytotoxicity of the expanded Vγ2Vδ2 T cells against various NSCLC-derived tumor cell lines with the releases of granzyme B, IFNγ, and TNFα in vitro. Meanwhile, the adoptive transferred Vγ2Vδ2 T cells together with the Y111 inhibited the growth of the established xenografts in NPG mice. Taken together, our data suggested a clinical potential for the adoptive transferring the Vγ2Vδ2 T cells with the Y111 to treat PD-L1 positive solid tumors.

A human bispecific neutralization antibody against four serotypes of dengue virus.

In tropical and subtropical countries, dengue virus (DENV) infections have been increasing; however, we still lack effective therapy. In the present study, we aimed to engineer a bispecific antibody (subsequently named LUZ-8F2-6B1), based on monoclonal antibody 6B1, which has anti DENV-1, 2, and 3 activity, and 8F2, which has anti DENV-4 activity. LUZ-8F2-6B1 displayed potent neutralization activity against four serotypes of DENV by binding to the envelop protein. In vivo, we demonstrated that LUZ-8F2-6B1 could provide protection against infection by four serotypes of DENV in a mouse model. In addition, the deletion of nine amino acids in the Fc region (LUZ-8F2-6B1-9del) completely abolished the antibody-dependent enhancement observed at lower doses of the antibody. Thus, LUZ-8F2-6B1 is a promising, safe, and effective agent for the prophylaxis and treatment of DENV infection.

A bispecific nanobody targeting the dimerization interface of epidermal growth factor receptor: Evidence for tumor suppressive actions in vitro and in vivo.

Targeting the dimer interface for the epidermal growth factor receptor (EGFR) that is highly conserved in the structure and directly involved in dimerization may solve the resistance problem that plagues anti-EGFR therapy. Heavy chain single domain antibodies have promising prospects as therapeutic antibodies. A bispecific nanobody was constructed based on previously screened humanized nanobodies that target the ß-loop at the EGFR dimer interface, an anti-FcγRIIIa (CD16) of natural killer cells (NK) nanobodies and anti-human serum albumin (HSA) nanobodies. The target gene was effectively expressed and secreted while controlled by promoter GAP in Pichia pastoris X33, and the expressed product was purified with a cation exchange and nickel chelation chromatography. The bispecific nanobody specifically bound to the surfaces of EGFR-overexpressed human epidermal carcinoma A431 cells and effectively inhibited tumor cell growth both in vitro and in vivo. In the A431 cell nude mouse xenograft model, the growth inhibition effect from the bispecific nanobody was significantly increased with the assistance of peripheral blood mononuclear cells (PBMCs), which was consistent with the results obtained in vitro, suggesting that there was an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. In addition, the intraperitoneal administration of bispecific nanobodies effectively reached tumor tissues in the shoulder dorsal region, but in significantly less distributed quantities than EGFR Dimer Nb77. To conclude, a bispecific nanobody targeting the EGFR dimer interface with ADCC effect was successfully constructed.

Removing a single-arm species by Fibro PrismA in purifying an asymmetric IgG-like bispecific antibody.

MabSelect PrismA is an affinity resin whose ligand is derived from the B-domain of Protein A. Compared to its predecessor resins (i.e., MabSelect SuRe LX and MabSelect SuRe), the PrismA ligand has enhanced affinity for the VH3 chain. Consistently, when being used for the purification of an asymmetric IgG-like bispecific antibody (bsAb), MabSelect PrismA resin shows certain resolution between the intact product and a single-arm byproduct, which contain the same Fc region but different numbers of VH3 domain. Fibro PrismA is a newly launched product which has the same PrismA Protein A ligand as MabSelect PrismA but uses cellulose fiber instead of resin as its matrix. It was learned that Fibro PrismA, in comparison to PrismA resin, exhibits further improved resolution, allowing efficient clearance of the single-arm byproduct as well as good recovery of the target bsAb. This finding suggests that Fibro PrismA provides a potential solution for separating antibody species containing the same Fc region but different numbers of VH3 domain, which can otherwise be difficult to separate.

Bispecific antibody target pair discovery by high-throughput phenotypic screening using in vitro combinatorial Fab libraries.

Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, high-content imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins ß1 and ß6 or αV and ß1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.

Application of pH-salt dual gradient elution in purifying a WuXiBody-based bispecific antibody by MMC ImpRes mixed-mode chromatography.

WuXiBody is a novel bispecific antibody (bsAb) platform developed by WuXi Biologics. BsAbs based on WuXiBody can adopt either asymmetric or symmetric format. In general, recombinant production of asymmetric bsAbs has a more complex impurity profile than that of symmetric ones, as assembly of the former usually involves an increased number of distinct chains. Consequently, purification of asymmetric bsAb typically poses greater challenges to the downstream team. Recently while purifying a WuXiBody-based bsAb in asymmetric format, we learned that MMC ImpRes mixed-mode chromatography under linear pH or salt gradient elution can effectively remove two out of the three low-molecular-weight byproducts post Protein A chromatography but not the third one, which was eventually removed under pH-salt dual gradient elution. This finding suggests that MMC ImpRes chromatography performed under pH-salt dual gradient elution provides better resolution than that performed under pH/salt mono gradient elution.

Sheathless CE-MS as a tool for monitoring exchange efficiency and stability of bispecific antibodies.

Bispecific monoclonal antibodies (BsAbs) are receiving great attention due to their extensive benefits as biopharmaceuticals and their involvement in IgG4 mediated autoimmune diseases. While the production of BsAbs is getting more accessible, their analytical characterization remains challenging. We explored the potential of sheathless CE-MS for monitoring exchange efficiency and stability of in-house produced bispecific antibodies. Two IgG4 bispecific antibodies with different molecular characteristics were prepared using controlled Fragment antigen binding (Fab)-arm exchange. Separation of BsAbs from their parent monospecific antibodies was achieved using a polyethyleniimine (PEI)-coated capillary and acidic background electrolytes permitting reliable assessment of the exchange efficiency. This was especially valuable for a Fab-glycosylated BsAb where the high glycan heterogeneity resulted in an overlap of masses with the monospecific parent antibody, hindering their discrimination by MS only. The method showed also good capabilities to monitor the stability of the generated BsAbs under different storage conditions. The levels of degradation products were different for the studied antibodies indicating pronounced differences in stability. Overall, the proposed method represents a useful analytical tool for exchange efficiency and stability studies of bispecific antibodies.

Comparing the relative robustness of byproduct removal by wash and by elution in column chromatography for the purification of a bispecific antibody.

For recombinant antibody purification, removal of product-related impurities usually relies on the two polishing steps post Protein A chromatography. A certain impurity may bind weaker or tighter to a particular type of column than the target antibody, and this forms the basis for separation. For impurities that bind weaker, they can be removed by pre-elution wash under appropriate conditions. For impurities that bind stronger, they can be separated by using a suitable condition that selectively elutes the product. In this study, with a bispecific antibody case, we compared the relative robustness of byproduct removal by wash and by elution using two different types of chromatography. The data suggest that elution-enabled byproduct clearance is more robust than wash-enabled clearance, and the former approach provides consistent impurity clearance over a relatively wide range of loading density.

Production of a novel bispecific protein ULBP1×CD19-scFv targeting the NKG2D receptor and CD19 to promote the activation of NK cells.

Natural killer (NK) cells are potent cytotoxic effector cells of the innate immune system and play an important role in tumor immunosurveillance and control. NKG2D is an activating receptor of NK cells. The NKG2D receptor-ligand system has contributed to immune cells recognizing tumor cells and the tumor microenvironment. In order to stretch the application of NK cells on adoptive immunotherapy for B-cell malignancies, we designed and produced a novel bispecific ULBP1×CD19-scFv fusion protein, in which the extracellular domain of NKG2D ligand ULBP1 was fused to a single chain variable fragment (scFv) of anti-CD19. The vector expressing ULBP1×CD19-scFv protein was constructed and expressed in Pichia pastoris. Effects of medium composition, concentration of methanol as the inducer, induction time and broth content in shake flask on the expression of the recombinant protein were investigated. The results showed that the optimized conditions for ULBP1×CD19-scFv expression were 1% methanol induction for 96 h with 15% broth content. The secreted recombinant protein was purified using ammonium sulfate fractionation and Ni-NTA affinity chromatography and the purity is about 93%. The cytotoxicity of NK92-MI cells against CD19+ Raji cells was enhanced in the presence of purified ULBP1×CD19-scFv protein. These results indicated that ULBP1 could be used as an activating element of bispecific killer engagers (BiKEs) and Pichia pastoris yeast might be an alternative expression host for BiKEs production.

Format chain exchange (FORCE) for high-throughput generation of bispecific antibodies in combinatorial binder-format matrices.

Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.