Production Of Bispecific Antibodies Research Articles

New structural formats of therapeutic antibodies for rheumatology

Pharmaceutical companies strive continuously to develop better medications in order to remain competitive. In the arena of monoclonal antibodies and related biologics (fusion proteins containing an IgG Fc fragment), the thrust is not only toward identifying new targets, but also toward developing new molecular formats. Here, new-generation antibodies used to treat rheumatic diseases are discussed, with emphasis on relations linking structure to pharmacological effects and on the improvements expected from the new formats. Isotypic and allotypic antibody diversity has pharmacological implications and is already exploited in commercially available antibodies. Efforts to engineer the Fc fragment of the various immunoglobulin G subclasses are reviewed with reference to abatacept, ixekizumab, other mutated IgG4 antibodies currently in development, sapelizumab, anifrolumab, and tanezumab. Bispecific antibodies are a focus of increasing interest (particularly those binding to both IL-17 and TNFα) and may earn a place in the therapeutic armamentarium as a means of avoiding the use of antibody combinations. However, the construction and production of bispecific antibodies continues to raise major technological challenges. Other molecular formats involve the fusion of antibodies to cytokines or the use of nanobodies and peptibodies. These new formats are at the very early stages of development, and their clinical relevance remains unclear.

Effector-attenuating Substitutions That Maintain Antibody Stability and Reduce Toxicity in Mice

The antibody Fc region regulates antibody cytotoxic activities and serum half-life. In a therapeutic context, however, the cytotoxic effector function of an antibody is often not desirable and can create safety liabilities by activating native host immune defenses against cells expressing the receptor antigens. Several amino acid changes in the Fc region have been reported to silence or reduce the effector function of antibodies. These earlier studies focused primarily on the interaction of human antibodies with human Fc-γ receptors, and it remains largely unknown how such changes to Fc might translate to the context of a murine antibody. We demonstrate that the commonly used N297G (NG) and D265A, N297G (DANG) variants that are efficacious in attenuating effector function in primates retain potent complement activation capacity in mice, leading to safety liabilities in murine studies. In contrast, we found an L234A, L235A, P329G (LALA-PG) variant that eliminates complement binding and fixation as well as Fc-γ-dependent, antibody-dependent, cell-mediated cytotoxity in both murine IgG2a and human IgG1. These LALA-PG substitutions allow a more accurate translation of results generated with an "effectorless" antibody between mice and primates. Further, we show that both human and murine antibodies containing the LALA-PG variant have typical pharmacokinetics in rodents and retain thermostability, enabling efficient knobs-into-holes bispecific antibody production and a robust path to generating highly effector-attenuated bispecific antibodies for preclinical studies.

An Alternative Chemical Redox Method for the Production of Bispecific Antibodies: Implication in Rapid Detection of Food Borne Pathogens

Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC) and the food borne pathogen Listeria monocytogenes (L. monocytogenes) were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches.

Fast identification of reliable hosts for targeted cell line development from a limited‐genome screening using combined φC31 integrase and CRE‐Lox technologies

The use of targeted integration (TI) in cell line development (CLD) usually introduces one copy of a recombinant gene into a predetermined transcriptionally active locus. This reduces the heterogeneity typically associated with traditional random integration (RI) CLD with regards to varied productivity and instability, resulting from diverse chromosomal influences, varied copy numbers, and repeat-induced rearrangement. As such, TI CLD offers the hope of a predictable and consistent CLD process for establishing stable clones. However, given the low copy number, cell lines established from a TI CLD process tend to exhibit low productivity. Here, we describe our nonviral-based approach for quickly establishing and identifying TI hosts from a limited genome screening. Importantly, the TI hosts identified are consistent and reliable in supporting the production of diverse antibodies regardless of antibody subclass (IgG1 vs. IgG4) or prior traditional CLD performance (relatively easy vs. difficult to express antibodies). Moreover, an approximately twofold increase in titer can be achieved by using a CRE recombinase-mediated cassette exchange (RMCE) strategy with an exchange vector carrying two units of the antibody gene. Two RMCE hosts that were established were able to produce up to ∼ 1.7 and 2 g/L of antibodies in nonoptimized fed-batch shake flask production cultures with chemically defined media. Potentially, this strategy may be applied to the production of bispecific antibodies with a fast turnaround time.

LUZ-Y, a Novel Platform for the Mammalian Cell Production of Full-length IgG-bispecific Antibodies

The ability of bispecific antibodies to simultaneously bind two unique antigens has great clinical potential. However, most approaches utilized to generate bispecific antibodies yield antibody-like structures that diverge significantly from the structure of archetype human IgG, and those that do approach structural similarity to native antibodies are often challenging to engineer and manufacture. Here, we present a novel platform for the mammalian cell production of bispecific antibodies that differ from their parental mAbs by only a single point mutation per heavy chain. Central to this platform is the addition of a leucine zipper to the C terminus of the C(H)3 domain of the antibody that is sufficient to drive the heterodimeric assembly of antibody heavy chains and can be readily removed post-purification. Using this approach, we developed various antibody constructs including one-armed Abs, bispecific antibodies that utilize a common light chain, and bispecific antibodies that pair light chains to their cognate heavy chains via peptide tethers. We have applied this technology to various antibody pairings and will demonstrate the engineering, purification, and biological activity of these antibodies herein.

A Novel Redox Method for Rapid Production of Functional Bi-Specific Antibodies For Use in Early Pilot Studies

We demonstrate here a rapid alternative method for the production of functional bi-specific antibodies using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA). Following reduction of a mixture of two monoclonal antibodies with MESNA to break inter heavy chain bonds, this solution is dialysed under oxidising conditions and antibodies are allowed to reform. During this reaction a mixture of antibodies is formed, including parental antibodies and bi-specific antibody. Bi-specific antibodies are purified over two sequential affinity columns. Following purification, bi-specificity of antibodies is determined in enzyme-linked immunosorbent assays and by flow cytometry. Using this redox method we have been successful in producing hybrid and same-species bi-specific antibodies in a time frame of 6–10 working days, making this production method a time saving alternative to the time-consuming traditional heterohybridoma technology for the production of bi-specific antibodies for use in early pilot studies. The use of both rat and mouse IgG antibodies forming a rat/mouse bi-specific antibody as well as producing a pure mouse bi-specific antibody and a pure rat bi-specific antibody demonstrates the flexibility of this production method.

Production of a Recombinant Anti-Human CD4 Single-Chain Variable-Fragment Antibody Using Phage Display Technology and Its Expression in Escherichia coli

Single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulin, connected with a short linker peptide of 10 to about 20 amino acids. In this study, the scFv of a monoclonal antibody against the third domain of human CD4 was cloned from OKT4 hybridoma cells using the phage display technique and produced in E. coli. The expression, production, and purification of anti-CD4 scFv were tested using SDS-PAGE and Western blot, and the specificity of anti-CD4 scFv was examined using ELISA. A 31 kDa recombinant anti-CD4 scFv was expressed and produced in bacteria, which was confirmed by SDS-PAGE and Western blot assays. Sequence analysis proved the ScFv structure of the construct. It was able to bind to CD4 in quality ELISA assay. The canonical structure of anti-CD4 scFv antibody was obtained using the SWISS_MODEL bioinformatics tool for comparing with the scFv general structure. To the best of our knowledge, this is the first report for generating scFv against human CD4 antigen. Engineered anti-CD4 scFv could be used in immunological studies, including fluorochrome conjugation, bispecific antibody production, bifunctional protein synthesis, and other genetic engineering manipulations. Since the binding site of our product is domain 3 (D3) of the CD4 molecule and different from the CD4 immunological main domain, including D1 and D2, further studies are needed to evaluate the anti-CD4 scFv potential for diagnostic and therapeutic applications.

Development and prospects for bispecific antibody-based therapeutics in cancer and other applications

Background: Progress in molecular biology for the production of bispecific antibodies (BiAbs) and the expanding knowledge on receptors on malignant and normal target cells provide the basis for developing new strategies using antibody- and/or receptor-based platform technology for the treatment of cancer and other diseases. Objective: This review provides a preclinical and clinical perspective on application of bispecific antibodies for the treatment of solid tumors, hematologic malignancies and other diseases. Methods: This review focuses on BiAb-based immunotherapy, clinical trials, alternative strategies, the challenges of technology and future applications. Results/conclusion: The successful application of a particular BiAb will depend on a thorough evaluation of the expected functional application and thoughtful engineering of structure, affinity and number of binding sites based on the desired function.

Bispecific antibodies as emerging pharmaceuticals for tumor immunotherapy

Bispecific antibodies consist of a targeting arm specific to the tumor antigen and a functional arm triggering an antitumor response through cytotoxic T-lymphocytes (CTLs), natural killer (NK) cells, macrophages, etc. Bispecific antibodies prepared by conventional hybrid hybridoma or chemical conjugation methods have been tested in both the preclinical and clinical setting for the treatment of various cancers. The results from these studies revealed several major limitations of these molecules, such as difficulty in large-scale production of bispecific antibodies, immunogenicity due to murine components of the antibodies and severe side effects caused by the mass release of inflammatory cytokines. Rapid progress in the field of antibody engineering has made recombinant bispecific antibodies a paradigm for overcoming these obstacles. Some recombinant bispecific antibodies have already shown promising results and are expected to enter clinical trials over the next few years. This review focuses on the recent progress in the preclinical and clinical evaluation of recombinant bispecific antibodies.

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.

Chemical production of bispecific antibodies.

This chapter discusses two related methods for creating Fab' x Fab' chemically linked BsAb. Both methods require the generation of purified F(ab')2 fragments of each antibody and use reagents that react with the free thiols generated upon reduction of interheavy chain disulfide bonds of the F(ab')2 fragments. Upon reduction, the resulting Fabs are then recombined to form a Fab' x Fab' BsAb. 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) acts to regenerate disulfide bonds between the two Fabs, whereas o-phenylenedimaleimide (o-PDM) acts to form a thioether bond between the two Fabs. After coupling, the bispecific antibody is purified from the uncoupled Fabs by size-exclusion chromatography. The advantages and disadvantages of each conjugation method are discussed.

Construction and characterization of minibodies for imaging and therapy of colorectal carcinomas.

Engineering of antibodies specific for tumor-associated antigens provides great flexibility in improving their properties for eventual use in the clinic, for the detection of cancer, or as a therapeutic. In some instances, murine monoclonal antibodies (MAbs) have been identified with innate biological anti-tumor activity. Production of chimeric or humanized (CDR-grafted) antibodies lowers the immunogenicity of murine MAbs, a necessary modification if repeat administration is desired (1,2). Chimerization or humanization can also increase the ability of murine MAbs to interact with the human immune system. The anti-lymphoma and anti-breast cancer antibodies RituxanTM (Rituximab) and HerceptinTM (Trastuzumab) are robust examples of this path to a clinically useful reagent (3,4). Production of chimeric or humanized antibodies with Fc regions of a suitable subclass allows engagement of human host immune responses (complement activation, antibody-dependent cellular cytotoxity). Many anti-tumor MAbs, however, must be further “armed” in some fashion in order to be useful as therapeutics. Alternate approaches, described in detail elsewhere in this volume, include fusion of antibodies to toxins or cytotoxic proteins such as ribonuclease, production of bispecific antibodies for recruitment of effector T cells, or direct activation of T cells through chimeric T-cell receptors. One of the classic approaches toward arming antibodies has been radiolabeling. Antibodies can be tagged with radionuclides such as 99mTc, 111In, 123I, or many other isotopes that emit gamma rays, and be used for imaging purposes. The approach can be extended to radiolabeling with positron-producing radionuclides such as 18F, 64Cu, 124I, and so on, for positron emission tomography (PET), affording higher resolution and better quantitation than single-photon approaches. By switching to radionuclides

Tolerance is overcome in beef insulin‐transgenic mice by activation of low‐affinity autoreactive T cells

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self-reactive BI-specific T cell in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1-14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.

Production of bispecific antibodies.

Bispecific antibodies (bsAbs) contain two different binding specificities within a single molecule and can specifically bind two different molecules together. BsAbs can be produced by chemically cross-linking purified antibodies or Fab fragments with reducible disulfide bonds or nonreducible thioether bonds, both of which are described in this unit. Protocols are also presented for producing BsAbs by fusing two antibody-producing hybridomas that can be selected for based on drug resistance, or by double labeling with fluorochromes and FACS. Support protocols describe screening and purification of bsAbs.

Production and characterisation of a set of monoclonal antibodies against tissue-type plasminogen activator (t-PA)

To generate bispecific monoclonal antibodies, reactive to both fibrin and tissue-type plasminogen activator (t-PA), we planned to generate anti-t-PA monoclonal antibodies (mAb) which eliminate negative aspects of t-PA such as the inhibition by plasminogen activator inhibitor-type 1 (PAI-1) and the rapid clearance of t-PA. Here we report on the isolation and characterisation of a set of 13 mAb against t-PA, some of which meet the above requirements. Apart from their potential in the production of bispecific antibodies, these and the other mAb can be useful in structure-function analysis and a variety of other applications. Experiments involving PAI-1 showed that one mAB (12-5-3) reacts only with free t-PA, and prevents the subsequent binding of PAI-1 to mAb-bound t-PA. In vitro studies on the receptor mediated uptake of t-PA by hepatic cells, showed that one mAb (1-3-1) specifically inhibited the association of t-PA with liver endothelial cells. Other tests showed that mAb 7-8-4 and 12-5-3, but not 1-3-1, inhibited in vitro the enzymatic activity of t-PA. On the basis of these and other observations, we conclude that especially mAb 1-3-1, and in vivo possibly 7-8-4 and 12-5-3 may be good candidates for incorporation in bispecific monoclonal antibodies.

Absence of preferential homologous H/L chain association in hybrid hybridomas.

Bispecific mAb contain two Ag-combining sites each composed of a different combination of H and L chains. The resulting ability to react with and cross-link two different Ag makes these molecules a novel tool for application in biology and medicine. Intact bispecific mAb can be made only by biologic means, e.g., by fusion of two established hybridomas. Appropriate assembly of bispecific mAb by these hybrid cells depends on H = L chain behavior: strong preferential homologous H-L pairing would benefit the yield of bispecific antibodies. We have analyzed the Ig species produced by eight hybrid hybridomas (quadromas). Quadroma-produced IgG was fractionated and characterized for H and L chain content. The Ag reactivities were verified by using ELISA and immunofluorescence. Preferential homologous pairing was seen only with a minority of H-L chain pairs; L chains associated on average in a random fashion with H chains. This indicates that in the B cells from which the parental hybridomas were obtained, no strong selection had occurred on H-L recombination. Our results extend recent biochemical competitive H-L reassociation experiments, where on average an at random association of L chains with H chains was found; evidently this random association occurs in our biologic system as well. For the biologic production of bispecific antibodies this means that only in a small number of cases the "ideal" producer will be met. From the viewpoint of generation of antibody diversity, our results favor a large freedom for combinatorial binding of H and L chains during B cell ontogeny.

The award of medals by the President, Sir George Porter, at the Anniversary Meeting, 30 November 1989

The Copley Medal is awarded to Dr C. Milstein, F. R. S., in recognition of his outstanding contributions to immunology, in particular to the discovery of monoclonal antibodies and to the understanding of the role of somatic mutations in the maturation of the immune response. The thrust of Dr Milstein’s work has been to understand, at a molecular level, how the diversity of antibodies arises. With G. Brownlee he first proved that the variable and constant regions of antibody light chains are encoded in one messenger RNA molecule. His studies with G. Kohler on certain cell hybrids led to the immortalization of antibody secreting cells, and hence monoclonal antibodies, for which discovery they shared the Nobel Prize for Physiology and Medicine. He has applied these methods to the production of diagnostic reagents and the isolation of cell surface antigens, and has extended the technology to the production of bispecific antibodies, the first example of antibody engineering. Most recently he has developed an approach to studying steps in the antibody response, and has demonstrated the critical role of hypermutation in the maturation process. In all his work, Dr Milstein has shown an unusual gift in making major discoveries by unconventional routes, a gift that he continues to apply in his current work in immunology.

The award of medals by the President, Sir George Porter, at the Anniversary Meeting, 30 November 1989

The Copley Medal is awarded to Dr C. Milstein, F. R. S., in recognition of his outstanding contributions to immunology, in particular to the discovery of monoclonal antibodies and to the understanding of the role of somatic mutations in the maturation of the immune response. The thrust of Dr Milstein’s work has been to understand, at a molecular level, how the diversity of antibodies arises. With G. Brownlee he first proved that then variable and constant regions of antibody light chains are encoded in one messenger RNA molecule. His studies with G. Kohler on certain cell hybrids led to the immortalization of antibody secreting cells, and hence monoclonal antibodies, for which discovery they shared the Nobel Prize for Physiology and Medicine. He has applied these methods to the production of diagnostic reagents and the isolation of cell surface antigens, and has extended the technology to the production of bispecific antibodies, the first example of antibody engineering. Most recently he has developed an approach to studying steps in the antibody response, and has demonstrated the critical role of hypermutation in the maturation process. In all his work, Dr Milstein has shown an unusual gift in making major discoveries by unconventional routes, a gift that he continues to apply in his current work in immunology.