Production Of BsAb Research Articles

Large scale controlled Fab exchange GMP process to prepare bispecific antibodies.

Objective: Bispecific antibodies (BsAbs) have demonstrated significant therapeutic impacts for the treatment of a broad spectrum of diseases that include oncology, auto-immune, and infectious diseases. However, the large-scale production of clinical batches of bispecific antibodies still has many challenges that include having low yield, poor stability, and laborious downstream purification processes. To address such challenges, we describe the optimization of the controlled Fab arm exchange (cFAE) process for the efficient generation of BsAbs. Methods: The process optimization of a large-scale good manufacturing practice (GMP) cFAE strategy to prepare BsAbs was based on screening the parameters of temperature, reduction, oxidation, and buffer exchange. We include critical quality standards for the reducing agent cysteamine hydrochloride. Results: This large-scale production protocol enabled the generation of bispecific antibodies with >90% exchange yield and at >95% purity. The subsequent downstream processing could use typical mAb procedures. Furthermore, we demonstrated that the bispecific generation protocol can be scaled up to ∼60L reaction scale using parental monoclonal antibodies that were expressed in a 200L bioreactor. Conclusion: We presented a robust development strategy for the cFAE process that can be used for a larger scale GMP BsAb production.

Rapid Generation of Murine Bispecific Antibodies Using FAST-IgTM for Preclinical Screening of HER2/CD3 T-Cell Engagers.

Bispecific antibodies (BsAbs) can bind to two different antigens, enabling therapeutic concepts that cannot be achieved with monoclonal antibodies. Immuno-competent mice are essential for validating drug discovery concepts, necessitating the development of surrogate mouse BsAbs. In this study, we explored the potential of FAST-IgTM, a previously reported BsAb technology, for mouse BsAb production. We investigated charge-based orthogonal Fab mutations to facilitate the correct assembly of heavy and light chains of mouse antibodies and employed knobs-into-holes mutations to facilitate the heterodimerization of heavy chains. We combined five anti-CD3 and two anti-HER2 antibodies in mouse IgG1 and IgG2a subclasses. These 20 BsAbs were analyzed using mass spectrometry or ion exchange chromatography to calculate the percentages of BsAbs with correct chain pairing (BsAb yields). Using FAST-Ig, 19 out of the 20 BsAbs demonstrated BsAb yields of 90% or higher after simple protein A purification from transiently expressed antibodies in Expi293F cells. Importantly, the mouse BsAbs maintained their fundamental physicochemical properties and affinity against each antigen. A Jurkat NFAT-luciferase reporter cell assay demonstrated the combined effects of epitope, affinity, and subclasses. Our findings highlight the potential of FAST-Ig technology for efficiently generating mouse BsAbs for preclinical studies.

Construction of IgG–Fab2 bispecific antibody via intein-mediated protein trans-splicing reaction

A bispecific antibody (bsAb) is a class of engineered antibody molecules that simultaneously binds to two different antigens by having two kinds of antigen-binding domains. One of the major obstacles for the bsAb production is the incorrect chain-pairing problem, wherein each heavy and light chain should form pairings with the correct counterpart’s chains, but the structural similarity of the incorrect partners also forms the incorrect pairings. This study aimed to demonstrate a bsAb construction method using intein-mediated protein trans-splicing to create IgG–Fab2–type bsAbs, which is a modified antibody with a structure in which two additional Fabs are linked to the N-terminus of the heavy chain of an IgG molecule. The chain-paring problem between a heavy chain and a light chain is circumvented by separate expression and purification of the IgG part and the Fab part. We found that the deletion of a possible glycosylation residue improved the reaction yield and side-reaction cleavage in the protein ligation step. The resulting bsAb, IgG–Fab2 (Her2/CD3), demonstrated target binding activity and cytotoxicity mediated by activated T cells. These results indicate that the use of the protein ligation to produce the IgG–Fab2 type bsAb will expand the bsAb production method.

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO–K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

Epsilon-caprolactone-modified polyethylenimine as a genetic vehicle for stem cell-based bispecific antibody and exosome synergistic therapy.

Bispecific antibodies (BsAb) have gained significant momentum in clinical application. However, the rapid enzymolysis and metabolism of protein drugs usually induce short circulation in vivo, and developing an efficient protein delivery system still is a bottleneck. Mesenchymal stem cells (MSCs) have become an attractive therapeutic carrier for cancers. Genetic modification enables MSCs to express and secrete specific proteins, which is essential for therapeutic efficacy. However, efficient gene transfer into MSCs is still a challenge. In this study, we applied epsilon-caprolactone-modified polyethylenimine (PEI-CL) as an efficacy carrier for plasmid transfection into MSC that served as in situ 'cell factory' for anti-CD3/CD20 BsAb preparation. Herein, the PEI-CL encapsulates the minicircle plasmid and mediates cell transfection efficiently. Thus, the anti-CD3/CD20 BsAb is secreted from MSC and recruited T cell, resulting in highly sensitive cytotoxicity in the human B-cell lymphoma. Furthermore, these stem cells produce exosomes bearing MiR-15a/MiR-16, which could negatively regulate cancer's oncogenes BCL-2 for adjuvant therapy. Meanwhile, high immunologic factors like tumor necrosis factor-α and interferon-γ are generated and enhance immunotherapy efficacy. The engineered MSCs are demonstrated as an efficient route for BsAb production, and these bioactive components contribute to synergistic therapy, which would be an innovative treatment.

Vector design for enhancing expression level and assembly of knob-into-hole based FabscFv-Fc bispecific antibodies in CHO cells

Two-armed FabscFv-Fc is a favoured bispecific antibody (BsAb) format due to its advantages of the conventional IgG structure. Production of FabscFv-Fc requires expression of three polypeptide chains, one light chain (LC), one heavy chain (HC) and a scFv fused to the Fc (scFvFc) at optimal ratios. We designed a set of internal ribosome entry site (IRES)-mediated multi-cistronic vectors tailoring to various expression ratios of the three polypeptides to study how the chain ratios affect the FabscFv-Fc production. Expression of HC and scFvFc chains at 1:1 ratio and excess LC gave the highest yield of correctly assembled product. Compared to the use of IRES and multiple promoters, using 2A peptides for co-expression of the three polypeptides gave the highest titre and correctly assembled product. The results obtained in this work provide insights to the impacts of hetero-chain ratios on the BsAb production.

Role of harvest depth filtration in controlling product-related impurities for a bispecific antibody.

BackgroundBispecific antibodies (BsAb) belong to a novel antibody category with advantages over traditional mono-specific therapeutic antibodies. However, product variants are also commonly seen during the production of BsAb, which poses significant challenges to downstream processing. In this study, the adsorptive characteristics of a BsAb product and its variants were investigated for a set of depth filters during primary recovery of the cell culture fluid.MethodsThe retention of the BsAb product and its variants on a set of Millistak+® D0HC and X0HC depth filters were first investigated, followed by studying the mechanism of their adsorption on the depth filters. The chemical and structural properties of depth filters along with the molecular properties of the product and its variants were studied subsequently.ResultsThe X0HC filter was found to be able to retain a significant amount of low molecular weight (LMW) variants along with a low amount of main product retained. Different levels of retention, observed for these variants, were correlated to their different hydrophobic and charge characteristics in relation with the adsorptive properties of the depth filters used. Electrostatic, hydrophobic, and hydrogen bonding interactions were found to be the key forces to keep product variants retained on the depth filter where the higher hydrophobicity of the LMW variants may cause them to be preferentially retained.ConclusionHarvest depth filters potentially can be utilized for retaining the BsAb variants, which depends on relative molecular properties of the product and its variants and adsorptive properties of the depth filters used.

Rapid Production of Bispecific Antibodies from Off-the-Shelf IgGs with High Yield and Purity.

Bispecific antibodies (BsAb) refer to a class of biomacromolecules that are capable of binding two antigens or epitopes simultaneously. This can elicit unique biological effects that cannot be achieved with either individual antibody or two unlinked antibodies. Bispecific antibodies have been used for targeting effector cells to tumor cells, preferential targeting of cells expressing two target biomarkers over cells expressing either target biomarker individually, or to couple two molecular targets on the same cell surface to trigger unique intracellular signaling pathways. Here, we present two related methods that enable direct, rapid assembly of bispecific antibodies from any two "off-the-shelf" Immunoglobulin G (IgG) antibodies, in as little as 1 day. Both workflows can be summarized into two steps: (1) attach a small photoreactive antibody binding domain (pAbBD) fused to SpyCatcher or SpyTag (peptide-protein partners derived from the S. pyogenes fibronectin-binding protein FbaB) to each component IgG, respectively; (2) assemble the BsAb through the spontaneous isopeptide bond formation that occurs between SpyTag and SpyCatcher. These approaches enable production of BsAbs from any two IgG molecules without the need to elucidate their amino acid sequences or genetically alter their structure. Binding assays and T cell-mediated cytolysis assays were performed to validate the binding and functional properties of Trastuzumab × Cetuximab BsAb and Cetuximab × OKT3 BsAb, respectively. This approach enables rapid, low-cost production of highly homogeneous tetravalent BsAbs in a modular fashion, presenting an opportunity to quickly evaluate antibody pairs in a BsAb format for unique or synergistic functionalities.

A cell line development strategy to improve a bispecific antibody expression purity in CHO cells

• A cell line development strategy to improve BsAb expression purity was developed. • The strategy increased the expression purity of a BsAb to from 44.5%–88.6%. • Enhanced ScFv-Fc expression was responsible for the increased purity. • Large-scale manufacturing batches were completed with the established clone. Among antibody-based therapies, bispecific antibodies (BsAbs) have gained momentum in preclinical and clinical investigations following the regulatory approvals of the T-cell engaging BsAb blinatunomab and the FVIII cofactor mimicking BsAb emicizumab. Production of BsAbs with high purity is one of the challenges in commercializing BsAb therapeutics. Therefore, several protein engineering approaches have been developed to increase the correct assembly of the desired target product. However, few reports investigated the cell line development strategies in further improving the expression purity of BsAbs. In this paper, we investigated the plasmid transfection ratio and pool selection approach to enhance the purity of an IgG-like BsAb containing a heavy chain (H), a light chain (L), and a ScFv-Fc polypeptide (ScFv-Fc). Two plasmids were used to express HL and ScFv-Fc, respectively. A two-stage pool selection method to favor ScFv-Fc expression increased the expression purity of the BsAb from 44.5%–88.6%. The single clones isolated from the best pool after the second stage of pool selection could produce the BsAb with tripled titer and similar purity to its parental pool.

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.

Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product‐related impurities in CHO cells

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAbit can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.

Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies.

The use of bispecific antibodies (BsAbs) to treat human diseases is on the rise. Increasingly complex and powerful therapeutic mechanisms made possible by BsAbs are spurring innovation of novel BsAb formats and methods for their production. The long-lived in vivo pharmacokinetics, optimal biophysical properties and potential effector functions of natural IgG monoclonal (and monospecific) antibodies has resulted in a push to generate fully IgG BsAb formats with the same quaternary structure as monoclonal IgGs. The production of fully IgG BsAbs is challenging because of the highly heterogeneous pairing of heavy chains (HCs) and light chains (LCs) when produced in mammalian cells with two IgG HCs and two LCs. A solution to the HC heterodimerization aspect of IgG BsAb production was first discovered two decades ago; however, addressing the LC mispairing issue has remained intractable until recently. Here, we use computational and rational engineering to develop novel designs to the HC/LC pairing issue, and particularly for κ LCs. Crystal structures of these designs highlight the interactions that provide HC/LC specificity. We produce and characterize multiple fully IgG BsAbs using these novel designs. We demonstrate the importance of specificity engineering in both the variable and constant domains to achieve robust HC/LC specificity within all the BsAbs. These solutions facilitate the production of fully IgG BsAbs for clinical use.

Cryogels to establish immunotherapeutic organoids

Event Abstract Back to Event Cryogels to establish immunotherapeutic organoids Roberta Aliperta1, Petra Welzel2, 3, Marcello Stanzione4, Marc Cartellieri5, Uwe Freudenberg2, 3, Claudia Arndt1, Carsten Werner2, 3, Martin Bornhäuser6, Gerhard Ehninger6 and Michael Bachmann1, 3, 7 1 Helmholtz-Zentrum Dresden-Rossendorf, Department of Radioimmunology, Institute of Radiopharmaceutical Cancer Research, Germany 2 Leibniz Institute of Polymer Research Dresden, Max Bergmann Center of Biomaterials Dresden, Germany 3 Technische Universität Dresden, Center for Regenerative Therapies Dresden, Germany 4 Technische Universität Dresden, Institute of Physiological Chemistry, Germany 5 Cellex Patient Treatment GmbH, Germany 6 University Hospital 'Carl Gustav Carus', Technische Universität Dresden, Medical Clinic and Polyclinic I, Germany 7 Technische Universität Dresden, University Cancer Center (UCC), Tumorimmunology, Germany Introduction: T-cell based immunotherapy of tumors has gained much prominence in the last years. In this context treatments relying on the administration of recombinant bispecific antibodies (bsAbs) for retargeting effector T-lymphocytes towards cancer cells are of particular interest[1],[2]. However, several challenges with respect to pharmacokinetics, efficiency and safety need to be overcome. Our aim is to develop, test and apply a transplantable cell/biomaterial-based bsAb production platform (immunotherapeutic organoid) ensuring customized sustained release and effective and persistent levels of these T-cell stimulating therapeutic proteins over time with low risk of side effects. Materials and Methods: Macroporous biohybrid hydrogel matrices (cryogels) were prepared by combining hydrogel network formation via chemical cross-linking of four-arm poly(ethylene glycol) (starPEG) and heparin with the cryogelation technology[3]. The capability of these starPEG-heparin cryogel materials to support the ingrowth, attachment, distribution and proliferation of gene-modified mesenchymal stem cells (MSCs) in vitro was tested by immunostaining/fluorescence microscopy and by using metabolic activity assays. Cell migration experiments were used to verify the attachment between the MSCs and the functionalized biomaterial under various experimental conditions. The production of therapeutic antibodies by the cell/cryogel machinery and their potential to kill tumor cells was quantified by enzyme-linked immunosorbent assays (ELISAs) at different time points and standard T-cell mediated cytotoxicity assays. Results and Discussion: The unique sponge-like structure of the starPEG-heparin cryogel material with interconnected macropores provides a high surface-to-volume ratio and enabled ingrowth, proliferation and viability of gene-modified bsAb-releasing-MSCs and effective transport of nutrients, metabolites and therapeutic antibodies. Functionalization of the gels with peptide sequences allowed for integrin-mediated adhesion of the modified MSCs. The established cell/cryogel machinery was proved to be feasible for maintaining MSCs in a supporting microenvironment, and for producing recombinant bsAbs for prolonged time spans resulting in specific T-cell mediated tumor cell killing. Conclusion: The proposed cell/cryogel system is a suitable candidate for developing transplantable immunotherapeutic organoids for future in vivo applications. It ensures the constant delivery of bsAbs to promote customized T-cell activation and efficient killing of tumor cells and paves the way to a safer and more effective tumor-specific strategy for T-cell mediated cancer treatment.

Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and “Bispecific” Monoclonal Antibody Formation

Monoclonal antibodies (mAbs) and derivatives such as antibody-drug conjugates (ADC) and bispecific antibodies (bsAb), are the fastest growing class of human therapeutics. Most of the therapeutic antibodies currently on the market and in clinical trials are chimeric, humanized, and human immunoglobulin G1 (IgG1). An increasing number of IgG2s and IgG4s that have distinct structural and functional properties are also investigated to develop products that lack or have diminished antibody effector functions compared to IgG1. Importantly, wild type IgG4 has been shown to form half molecules (one heavy chain and one light chain) that lack interheavy chain disulfide bonds and form intrachain disulfide bonds. Moreover, IgG4 undergoes a process of Fab-arm exchange (FAE) in which the heavy chains of antibodies of different specificities can dissociate and recombine in bispecific antibodies both in vitro and in vivo. Here, native mass spectrometry (MS) and time-resolved traveling wave ion mobility MS (TWIM-MS) were used for the first time for online monitoring of FAE and bsAb formation using Hz6F4-2v3 and natalizumab, two humanized IgG4s which bind to human Junctional Adhesion Molecule-A (JAM-A) and alpha4 integrin, respectively. In addition, native MS analysis of bsAb/JAM-A immune complexes revealed that bsAb can bind up to two antigen molecules, confirming that the Hz6F4 family preferentially binds dimeric JAM-A. Our results illustrate how IM-MS can rapidly assess bsAb structural heterogeneity and be easily implemented into MS workflows for bsAb production follow up and bsAb/antigen complex characterization. Altogether, these results provide new MS-based methodologies for in-depth FAE and bsAb formation monitoring. Native MS and IM-MS will play an increasing role in next generation biopharmaceutical product characterization like bsAbs, antibody mixtures, and antibody-drug conjugates (ADC) as well as for biosimilar and biobetter antibodies.

Highly Enhanced Cytotoxicity of a Dimeric Bispecific Diabody, the hEx3 Tetrabody

We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy.

Single Variable Domain-IgG Fusion: A NOVEL RECOMBINANT APPROACH TO Fc DOMAIN-CONTAINING BISPECIFIC ANTIBODIES

Both laboratory and early clinical studies to date have demonstrated that bispecific antibodies (BsAb) may have potentially significant application in cancer therapy. The clinical development of BsAb as therapeutics has been hampered, however, by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods has been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. Here we describe a novel recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a single variable domain antibody to the N terminus of the light chain of a functional IgG antibody of different specificity. A model BsAb was constructed using a single variable domain antibody to mouse platelet-derived growth factor receptor alpha and a conventional IgG antibody to mouse vascular endothelial growth factor receptor 2. The BsAb was expressed in mammalian cells and purified to homogeneity by one-step protein A affinity chromatography. Furthermore, the BsAb retains the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. This design and expression of Fc domain-containing, IgG-like BsAb should be applicable to the construction of similar BsAb from antibodies recognizing any pair of antigens.

The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody

In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats. Bispecific diabody (bDAb), a 55–60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production. Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks. A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V H) and variable light (V L) chain domains but arranged in different orientations. V H/V L orientation, i.e., V H–linker–V L or V L–linker–V H, showed significant effects on the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12. Further, only 2 out of the 4 possible V H/V L orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR. Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 °C in mouse serum for up to 7 days, indicating excellent stability of the constructs. Taken together, our results underscore the importance of identifying/selecting optimal V H/V L orientation/arrangement for efficient production of active bDAb.

Di-diabody: a novel tetravalent bispecific antibody molecule by design

The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens. Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH 3 domain of an IgG. The novel BsAb, which we termed “di-diabody”, represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH 3 domains. A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography. Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors. The di-diabody retained good antigen-binding activity after incubation at 37 °C in mouse serum for 72 h, demonstrating good product stability. Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity. This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.

Specific targeting immunotherapy of cancer with bispecific antibodies.

In order to enhance cell mediated cytotoxicity, bispecific antibodies (BsAbs), molecules combining two or more antibodies with different antigenic specificities, have been developed as new agents for immunotherapy. Our recent studies revealed that simultaneous administration of two kinds of BsAbs (anti-tumor x anti-CD3 plus anti-tumor x anti-CD28) together with lymphokine activated killer cells with a T cell phenotype (T-LAK cells) inhibited growth of human xenotransplanted tumors in severe combined immunodeficient (SCID) mice, while single BsAb was without effect. Three kinds of BsAbs (anti-tumor x anti-CD3, anti-tumor x anti-CD28, anti-tumor x anti-CD2) showed the highest cytotoxicity against tumor cells when given simultaneously with T-LAK cells or peripheral blood mononuclear cells in vitro and in vivo. BsAbs can be preserved for immediate application, while cytotoxic T lymphocytes (CTLs) must be made-to-order, and are time-consuming to prepare. Tumor associated antigens, such as MAGE antigens, SART antigens, MUC1 antigen, c-erbB 2 antigen or cancer/testis antigens can be served to target antigens for BsAb production. By conjugation with antibodies to effector cells (anti-CD3, anti-CD28, anti-CD16, anti-CD64, anti-CD89 or anti-CD2), many kinds of BsAbs can be produced to cover most types of cancers from different organs. Therefore this strategy might be ubiquitously applicable to most malignancies.