双特异抗体的研究进展

双特异抗体是指可以同时结合两个不同抗原或一个抗原不同表位的特殊抗体,目前已有3个双特异抗体批准上市,还有很多个双特异抗体处于临床或临床前研究阶段。文中就双特异抗体的发现、制备方法、结构类型和设计策略、作用机制以及目前研究现状进行综述。     

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

ABSTRACT

Bispecific antibody therapeutics can expand the functionality of a conventional monoclonal antibody drug because they can bind multiple antigens. However, their great potential is counterbalanced by the challenges faced in their production. The classic asymmetric bispecific containing an Fc requires the expression of four unique chains – two light chains and two heavy chains; each light chain must pair with its correct heavy chain, which then must heterodimerize to form the full bispecific. The light-chain pairing problem has several solutions, some of which require engineering and optimization for each bispecific pair. Here, we introduce a technology called EFab Domain Substitution, which replaces the Cε2 of IgE for one of the CL/CH1 domains into one arm of an asymmetric bispecific to encourage the correct pairing of the light chains. EFab Domain Substitution provides very robust correct pairing while maintaining antibody function and is effective for many variable domains. We report its effect on the biophysical properties of an antibody and the crystal structure of the EFab domain substituted into the adalimumab Fab (PDB ID 6CR1).     

Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

There is strong interest in the design of bispecific monoclonal antibodies (bsAbs) that can simultaneously bind 2 distinct targets or epitopes to achieve novel mechanisms of action and efficacy. Multiple bispecific formats have been proposed and are currently under development. Regeneron's bispecific technology is based upon a standard fully human IgG antibody in order to minimize immunogenicity and improve the pharmacokinetic profile. A single common light chain and 2 distinct heavy chains combine to form the bispecific molecule. One of the heavy chains contains a chimeric Fc sequence form (called Fc*) that ablates binding to Protein A via the constant region. As a result of co-expression of the 2 heavy chains and the common light chain, 3 products are created, 2 of which are homodimeric for the heavy chains and one that is the desired heterodimeric bispecific product. The Fc* sequence allows selective purification of the FcFc* bispecific product on commercially available affinity columns, due to intermediate binding affinity for Protein A compared to the high avidity FcFc heavy chain homodimer, or the weakly binding Fc*Fc* homodimer. This platform requires the use of Protein A chromatography in both a capture and polishing modality. Several challenges, including variable region Protein A binding, resin selection, selective elution optimization, and impacts upon subsequent non-affinity downstream unit operations, were addressed to create a robust and selective manufacturing process.     

Optimizing assembly and production of native bispecific antibodies by codon de-optimization

When production of bispecific antibodies requires the co-expression and assembly of three or four polypeptide chains, low expression of one chain can significantly limit assembly and yield. κλ bodies, fully human bispecific antibodies with native IgG structure, are composed of a common heavy chain and two different light chains, one kappa and one lambda. No engineering is applied to force pairing of the chains, thus both monospecific and bispecific antibodies are secreted in the supernatant. In this context, stoichiometric expression of the two light chains allows for maximal assembly of the bispecific antibody. In this study, we selected a κλ body with suboptimal characteristics due to low kappa chain expression. Codon optimization to increase expression of the kappa chain did not improve bispecific yield. Surprisingly, progressive introduction of non-optimal codons into the sequence of the lambda chain resulted in lowering its expression for an optimal tuning of the relative distribution of monospecific and bispecific antibodies. This codon de-optimization led to doubling of the κλ body yield. These results indicate that assembly of different proteins into a recombinant complex is an interconnected process and that reducing the expression of one polypeptide can actually increase the overall yield.     

Aberrant bispecific antibody pharmacokinetics linked to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys

Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgG-scFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.     

A practical strategy for using miniature chromatography columns in a standardized high‐throughput workflow for purification development of monoclonal antibodies

The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high‐throughput process development (HTPD) strategy implementing several high‐throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high‐throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high‐throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high‐throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:626–635, 2014     

Mobile phase modifier effects in multimodal cation exchange chromatography

This study examines protein adsorption behavior and the effects of mobile phase modifiers in multimodal chromatographic systems. Chromatography results with a diverse protein library indicate that multimodal and ion exchange resins have markedly different protein binding behavior and selectivity. NMR results corroborate the stronger binding observed for the multimodal system and provide insight into the structural basis for the observed binding behavior. Protein-binding affinity and selectivity in multimodal and ion exchange systems are then examined using a variety of mobile phase modifiers. Arginine and guanidine are found to have dramatic effects on protein adsorption, yielding changes in selectivity in both chromatographic systems. While sodium caprylate leads to slightly weaker chromatographic retention for most proteins, certain proteins exhibit significant losses in retention in both systems. The presence of a competitive binding mechanism between the multimodal ligand and sodium caprylate for binding to ubiquitin is confirmed using STD NMR. Polyol mobile phase modifiers are shown to result in increased retention for weakly bound proteins and decreased retention for strongly bound proteins, indicating that the overall retention behavior is determined by a balance between changes in electrostatic and hydrophobic interactions. This work provides an improved understanding of protein adsorption and mobile phase modifier effects in multimodal chromatographic systems and sets the stage for future work to develop more selective protein separation systems.     

Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography

Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.     

Weak partitioning chromatography for anion exchange purification of monoclonal antibodies

Weak partitioning chromatography (WPC) is an isocratic chromatographic protein separation method performed under mobile phase conditions where a significant amount of the product protein binds to the resin, well in excess of typical flowthrough operations. The more stringent load and wash conditions lead to improved removal of more tightly binding impurities, although at the cost of a reduction in step yield. The step yield can be restored by extending the column load and incorporating a short wash at the end of the load stage. The use of WPC with anion exchange resins enables a two-column cGMP purification platform to be used for many different mAbs. The operating window for WPC can be easily established using high throughput batch-binding screens. Under conditions that favor very strong product binding, competitive effects from product binding can give rise to a reduction in column loading capacity. Robust performance of WPC anion exchange chromatography has been demonstrated in multiple cGMP mAb purification processes. Excellent clearance of host cell proteins, leached Protein A, DNA, high molecular weight species, and model virus has been achieved.     

Resolution of heterogeneous charged antibody aggregates via multimodal chromatography: A comparison to conventional approaches

Clearance of aggregates during protein purification is increasingly paramount as protein aggregates represent one of the major impurities in biopharmaceutical products. Aggregates, especially dimer species, represent a significant challenge for purification processing since aggregate separation coupled with high purity protein recovery can be difficult to accomplish. Biochemical characterization of the aggregate species from the hydrophobic interaction and cation exchange chromatography elution peaks revealed two different charged populations, i.e. heterogeneous charged aggregates, which led to further challenges for chromatographic removal. This paper compares multimodal versus conventional cation exchange or hydrophobic chromatography methodologies to remove heterogeneous aggregates. A full, mixed level factorial design of experiment strategy together with high throughput experimentation was employed to rapidly evaluate chromatographic parameters such as pH, conductivity, and loading. A variety of operating conditions were identified for the multimodal chromatography step, which lead to effective removal of two different charged populations of aggregate species. This multimodal chromatography step was incorporated into a monoclonal antibody purification process and successfully implemented at commercial manufacturing scale. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:636–645, 2014     

Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing C_H1 with Cα and C_L with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the C_H1/C_L and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.     

双特异性抗体在抗病毒方面的研究进展

随着基因工程抗体的快速发展,双特异性抗体技术也日趋成熟。双特异性抗体能够同时结合两个以上不同的抗原表位,在免疫治疗中具有独特的优势。双特异性抗体己经广泛应用于癌症治疗如黑色素瘤、霍奇金淋巴瘤以及肝癌、胃癌等,以及炎症方面的治疗如类风湿性关节炎、牛皮癣与克罗恩病等。双特异性抗体在病毒免疫治疗方面则刚刚起步。文中对双特异性抗体用于病毒免疫治疗的研究进展进行了综述,特别是在体内外效果较好的产品,为用于病毒免疫治疗的双特异性抗体药物开发与研究提供一定的参考。     

Effect of guanidine and arginine on protein–ligand interactions in multimodal cation‐exchange chromatography

The addition of fluid phase modifiers provides significant opportunities for increasing the selectivity of multimodal chromatography. In order to optimize this selectivity, it is important to understand the fundamental interactions between proteins and these modifiers. To this end, molecular dynamics (MD) simulations were first performed to study the interactions of guanidine and arginine with three proteins. The simulation results showed that both guanidine and arginine interacted primarily with the negatively charged regions on the proteins and that these regions could be readily predicted using electrostatic potential maps. Protein surface characterization was then carried out using computationally efficient coarse‐grained techniques for a broader set of proteins which exhibited interesting chromatographic retention behavior upon the addition of these modifiers. It was shown that proteins exhibiting an increased retention in the presence of guanidine possessed hydrophobic regions adjacent to negatively charged regions on their surfaces. In contrast, proteins which exhibited a decreased binding in the presence of guanidine did not have hydrophobic regions adjacent to negatively charged patches. These results indicated that the effect of guanidine could be described as a combination of competitive binding, charge neutralization and increased hydrophobic interactions for certain proteins. In contrast, arginine resulted in a significant decrease in protein retention times primarily due to competition for the resin and steric effects, with minimal accompanying increase in hydrophobic interactions. The approach presented in this paper which employs MD simulations to guide the application of coarse‐grained approaches is expected to be extremely useful for methods development in downstream bioprocesses. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:435–447, 2017     

Protein design of IgG/TCR chimeras for the co-expression of Fab-like moieties within bispecific antibodies

Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/β TCR. Replacing C_H1 with Cα and C_L with Cβ, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cβ with a canonical β-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the C_H1/C_L and Cα/Cβ domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.     

Select host cell proteins coelute with monoclonal antibodies in protein A chromatography

The most significant factor contributing to the presence of host cell protein (HCP) impurities in Protein A chromatography eluates is their association with the product monoclonal antibodies (mAbs) has been reported previously, and it has been suggested that more efficacious column washes may be developed by targeting the disruption of the mAbs-HCP interaction. However, characterization of this interaction is not straight forward as it is likely to involve multiple proteins and/or types of interaction. This work is an attempt to begin to understand the contribution of HCP subpopulations and/or mAb interaction propensity to the variability in HCP levels in the Protein A eluate. We performed a flowthrough (FT) recycling study with product respiking using two antibody molecules of apparently different HCP interaction propensities. In each case, the ELISA assay showed depletion of select subpopulations of HCP in Protein A eluates in subsequent column runs, while the feedstock HCP in the FTs remained unchanged from its native harvested cell culture fluid (HCCF) levels. In a separate study, the final FT from each molecule's recycling study was cross-spiked with various mAbs. In this case, Protein A eluate levels remained low for all but two molecules which were known as having high apparent HCP interaction propensity. The results of these studies suggest that mAbs may preferentially bind to select subsets of HCPs, and the degree of interaction and/or identity of the associated HCPs may vary depending on the mAb.     

A new class of bispecific antibodies to redirect T cells for cancer immunotherapy

Various constructs of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise in both preclinical and clinical studies. The single-chain variable fragment (scFv)-based formats, including bispecific T-cell engager (BiTE) and dual-affinity re-targeting (DART), which provide monovalent binding to both CD3 on T cells and to the target antigen on tumor cells, can exhibit rapid blood clearance and neurological toxicity due to their small size (~55 kDa). Herein, we describe the generation, by the modular DOCK-AND-LOCK^TM (DNL^TM) method, of novel T-cell redirecting bispecific antibodies, each comprising a monovalent anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs. The potential advantages of this design include bivalent binding to tumor cells, a larger size (~130 kDa) to preclude renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. These prototypes were purified to near homogeneity, and representative constructs were shown to provoke the formation of immunological synapses between T cells and their target tumor cells in vitro, resulting in T-cell activation and proliferation, as well as potent T-cell mediated anti-tumor activity. In addition, in vivo studies in NOD/SCID mice bearing Raji Burkitt lymphoma or Capan-1 pancreatic carcinoma indicated statistically significant inhibition of tumor growth compared with untreated controls.     

A new class of bispecific antibodies to redirect T cells for cancer immunotherapy

Various constructs of bispecific antibodies (bsAbs) to redirect effector T cells for the targeted killing of tumor cells have shown considerable promise in both preclinical and clinical studies. The single-chain variable fragment (scFv)-based formats, including bispecific T-cell engager (BiTE) and dual-affinity re-targeting (DART), which provide monovalent binding to both CD3 on T cells and to the target antigen on tumor cells, can exhibit rapid blood clearance and neurological toxicity due to their small size (~55 kDa). Herein, we describe the generation, by the modular DOCK-AND-LOCK^TM (DNL^TM) method, of novel T-cell redirecting bispecific antibodies, each comprising a monovalent anti-CD3 scFv covalently conjugated to a stabilized dimer of different anti-tumor Fabs. The potential advantages of this design include bivalent binding to tumor cells, a larger size (~130 kDa) to preclude renal clearance and penetration of the blood-brain barrier, and potent T-cell mediated cytotoxicity. These prototypes were purified to near homogeneity, and representative constructs were shown to provoke the formation of immunological synapses between T cells and their target tumor cells in vitro, resulting in T-cell activation and proliferation, as well as potent T-cell mediated anti-tumor activity. In addition, in vivo studies in NOD/SCID mice bearing Raji Burkitt lymphoma or Capan-1 pancreatic carcinoma indicated statistically significant inhibition of tumor growth compared with untreated controls.     

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore “pandemic mAb” timelines need to be shortened. One acceleration tool is “deferred cloning” and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future “pandemic mAb.”