Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb² format where a set of mutated amino acid residues in the C_H3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab C_H1/C_L domain pair with a pair of antigen-binding C_H3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding C_H3 domains. Such bispecific molecules were produced in a “Fab-like” format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding C_H3 domains offer an alternative solution in positioning and valency of antigen binding sites.     

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the C_H1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass. HPLC-MS results indicated that cation-exchange chromatography acidic variant populations were enriched in antibody with a second glycosylation site, in addition to the well documented canonical glycosylation site located in the C_H2 domain. Subsequent tryptic and chymotryptic peptide map data indicated that the second glycosylation site was associated with the amino acid sequence TVSWN¹⁶²SGAL in the C_H1 domain of the antibody. This highly atypical modification is present at levels of 0.5–2.0% on most of the recombinant antibodies that have been tested and has also been observed in IgG1 antibodies derived from human donors. Site-directed mutagenesis of the C_H1 domain sequence in a recombinant-human IgG1 antibody resulted in an increase in non-consensus glycosylation to 3.15%, a greater than 4-fold increase over the level observed in the wild type, by changing the −1 and +1 amino acids relative to the asparagine residue at position 162. We believe that further understanding of the phenomenon of non-consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery.     

With or Without Sugar? (A)glycosylation of Therapeutic Antibodies

Antibodies and antibody-based drugs are currently the fastest-growing class of therapeutics. Over the last three decades, more than 30 therapeutic monoclonal antibodies and derivatives thereof have been approved for and successfully applied in diverse indication areas including cancer, organ transplants, autoimmune/inflammatory disorders, and cardiovascular disease. The isotype of choice for antibody therapeutics is human IgG, whose Fc region contains a ubiquitous asparagine residue (N₂₉₇) that acts as an acceptor site for N-linked glycans. The nature of these glycans can decisively influence the therapeutic performance of a recombinant antibody, and their absence or modification can lead to the loss of Fc effector functions, greater immunogenicity, and unfavorable pharmacokinetic profiles. However, recent studies have shown that aglycosylated antibodies can be genetically engineered to display novel or enhanced effector functions and that favorable pharmacokinetic properties can be preserved. Furthermore, the ability to produce aglycosylated antibodies in lower eukaryotes and bacteria offers the potential to broaden and simplify the production platforms and avoid the problem of antibody heterogeneity, which occurs when mammalian cells are used for production. In this review, we discuss the importance of Fc glycosylation focusing on the use of aglycosylated and glyco-engineered antibodies as therapeutic proteins.     

Mathematical model of clonal selection and antibody production. II

Several modifications are proposed to a recent mathematical model (Bell, 1970) of the clonal selection and antibody production which take place when an adult animal is injected with an antigen. In the original model, antigen molecules were assumed univalent, i.e. to have only one combining site per molecule, and the first modification is an allowance for multivalent antigens by permitting an antigen molecule to interact with only one cell at a time. It is found that, for antigens with few (? 10) sites per molecule this modification is not important while for many sites per molecule, the modification will reduce the response as compared to that from the same number of independent antigenic sites. In section 3, it is seen that by restricting the number of cells which can arise in an immune response, much more realistic responses to high antigen doses are obtained. Moreover, the response is made more predictable by assuming that both target and proliferating cells are stimulated by antigen when a fraction of their receptors, between f_min and f_max, is bound to antigen. The parameter f_min is shown to determine the extent to which a molecule which can be recognized by antibodies will also serve as an immunogen giving rise to cellular proliferation and antibody production. In section 4, it is found that if more plasma cells than memory cells result from antigen stimulation, then weak stimulation will lead to a depletion of target plus memory cells and to at least partial immunological paralysis. Optimum antigen doses and times for the induction of such paralysis are examined. In section 5, precipitation of antigen-antibody mixtures is considered and it is shown that the number of doubly bound antibody molecules per antigen site determines whether precipitation will occur. This number is easily computed for heterogeneous bivalent antibodies.     

Monoclonal antibody covalently coupled to liposomes: Specific targeting to cells

We have evaluated optimal conditions for coupling monoclonal antibody to small unilamellar lipisomes. Coupling of an IgG_2a monoclonal anti-β₂-microglobulin antibody, which reacts with human cells, was examined in detail. Liposomes were composed of dipalmitoyl lecithin and cholesterol, and variable quantities of phosphatidylethanolamine substituted with the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP). They were reacted with antibody derivatized with the same reagent at a 5- to 20-fold molar excess, and activated by mild reduction. This degree of SPDP modification had no effect on the capacity of the antibody to bind to its target antigen. More than 40% of antibody could be reproducibly bound to liposomes, resulting in the coupling of from 1 to 10 antibody molecules per liposome (mean diameter.580 Å). The coupling reaction did not lead to loss of carboxyfluorescein encapsulated within liposomes. At least 80% of liposomes carried nondenatured antibody, as confirmed by precipitation of liposomes and encapsulated carboxyfluorescein by Staphylococcus aureus, strain Cowan I. The liposome-coupled antibody retained its immunological specificity: only cells expressing human β₂-microglobulin bound liposomes in vitro, and the binding was inhibited by the free antibody in solution. Results with antibodies of different antigenic specificity confirm that the technique can be generally applied.     

The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS

It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS™ protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V_λ and 13/35 V_κ light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V_H CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC₅₀ values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.     

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_H3 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.     

Molecular dissection of the human antibody response to the structural repeat epitope of Plasmodium falciparum sporozoite from a protected donor

Background

The circumsporozoite surface protein is the primary target of human antibodies against Plasmodium falciparum sporozoites, these antibodies are predominantly directed to the major repetitive epitope (Asn-Pro-Asn-Ala)_n, (NPNA)_n. In individuals immunized by the bites of irradiated Anopheles mosquitoes carrying P. falciparum sporozoites in their salivary glands, the anti-repeat response dominates and is thought by many to play a role in protective immunity.

Methods

The antibody repertoire from a protected individual immunized by the bites of irradiated P. falciparum infected Anopheles stephensi was recapitulated in a phage display library. Following affinity based selection against (NPNA)₃ antibody fragments that recognized the PfCSP repeat epitope were rescued.

Results

Analysis of selected antibody fragments implied the response was restricted to a single antibody fragment consisting of V_H3 and V_κI families for heavy and light chain respectively with moderate affinity for the ligand.

Conclusion

The dissection of the protective antibody response against the repeat epitope revealed that the response was apparently restricted to a single V_H/V_L pairing (PfNPNA-1). The affinity for the ligand was in the μM range. If anti-repeat antibodies are involved in the protective immunity elicited by exposure to radiation attenuated P. falciparum sporozoites, then high circulating levels of antibodies against the repeat region may be more important than intrinsic high affinity for protection. The ability to attain and sustain high levels of anti-(NPNA)_n will be one of the key determinants of efficacy for a vaccine that relies upon anti-PfCSP repeat antibodies as the primary mechanism of protective immunity against P. falciparum.     

Expression of catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of the key problems in immuno-biotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively performed in yeast cells. We obtained expression strains of the methylotrophic yeast Pichia pastoris producing single-chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab), and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant K _d and the first order constant (k ₂) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and to the single-chain antibody A.17 expressed in CHO and E. coli cells, respectively.     

Identification of a highly specific and versatile vitamin D receptor antibody

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is critical for regulation of serum calcium and phosphorus levels and for proper maintenance of bone mineralization and neuromuscular function. Biological effects of 1,25(OH)₂D₃ are mediated through a nuclear steroid hormone receptor, known as the vitamin D receptor (VDR). The discovery of VDR in a number of different cell and tissue types, suggests that the physiological role of vitamin D may extend beyond the regulation of calcium homeostasis and bone function. Unfortunately, identification of tissues expressing VDR has been controversial due to low abundance of the receptor and quality of the antibodies used. Therefore, we elected to characterize a panel of commercially available VDR antibodies in order to identify antibodies with high specificity and sensitivity. To address these objectives, we have used multiple immunoassays to determine VDR expression in tissues from several organs from multiple species employing tissues from VDR knockout mice as critical negative controls. Many of the antibodies tested showed nonspecific binding that can account for divergent reports. However, one antibody, identified as D-6, is highly specific and extremely sensitive. The specificity, sensitivity, and versatility of this antibody make it the preferred antibody for identifying VDR expression in target tissues using immunological methods.     

Genetic control of the immune response

(1) Inbred strains of mice when immunized withp-aminobenzoic acid and sulphanilic acid bound by diazo-linkage to the same protein carrier molecule (bovine gamma globulin) differ in their ability to respond by antibody formation. The strains A and CBA/J form only low levels of antibodies to the haptens after immunization; in strains ScSN and B10.LP the same high titers of antibodies to both haptens were found under these conditions. The strain B10.D₂ forms antibodies well to sulphanilic acid, antip-aminobenzoic acid antibodies are formed only in very low quantity. (2) Individual mice of an inbred strain form a homogeneous population in respect of their capability or inability to form a particular antihapten antibody. The individual titers in a given inbred strain vary only slightly. On the contrary the noninbred strain H shows great variability both in quantity and quality of the immune response to the haptens. (3) The crossing of good and poor anti-hapten antibody producing strains shows in F₁; F₂ and B₁ generation, that the ability to produce antibodies againstp-aminobenzoic and sulphanilic acid depends on the genotype of a given individual. The ability to respond is transmitted to the offspring as a dominant trait. (4) There is no difference in the response to the haptens between males and females of the same strain. (5) The antibodies to the haptens in different strains of mice differ in the ratio of 2-mercaptoethanol sensitive and 2-mercaptoethanol resistant antibody. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

The impact of glycosylation on monoclonal antibody conformation and stability

Antibody glycosylation is a common post-translational modification and has a critical role in antibody effector function. The use of glycoengineering to produce antibodies with specific glycoforms may be required to achieve the desired therapeutic efficacy. However, the modified molecule could have unusual behavior during development due to the alteration of its intrinsic properties and stability. In this study, we focused on the differences between glycosylated and deglycosylated antibodies, as aglycosyl antibodies are often chosen when effector function is not desired or unimportant. We selected three human IgG1 antibodies and used PNGase F to remove their oligosaccharide chains. Although there were no detected secondary or tertiary structural changes after deglycosylation, other intrinsic properties of the antibody were altered with the removal of oligosaccharide chains in the Fc region. The apparent molecular hydrodynamic radius increased after deglycosylation based on size-exclusion chromatography analysis. Deglycosylated antibodies exhibited less thermal stability for the CH₂ domain and less resistance to GdnHCl induced unfolding. Susceptibility to proteolytic cleavage demonstrated that the deglycosylated version was more susceptible to papain. An accelerated stability study revealed that deglycosylated antibodies had higher aggregation rates. These changes may impact the development of aglycosyl antibody biotherapeutics.Key words: monoclonal antibody, glycosylation, stability, liquid chromatography-mass spectroscopy, Fourier transform infrared, fluorescence spectroscopy, size-exclusion chromatography, differential scanning calorimetry     

Characterization and cancer cell targeted imaging properties of human antivascular endothelial growth factor monoclonal antibody conjugated CdTe/ZnS quantum dots

High luminescence quantum yield water‐soluble CdTe/ZnS core/shell quantum dots (QDs) stabilized with thioglycolic acid were synthesized. QDs were chemically coupled to fully humanized antivascular endothelial growth factor₁₆₅ monoclonal antibodies to produce fluorescent probes. These probes can be used to assay the biological affinity of the antibody. The properties of QDs conjugated to an antibody were characterized by ultraviolet and visible spectrophotometry, fluorescent spectrophotometry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transmission electron microscopy and fluorescence microscopy. Cell‐targeted imaging was performed in human breast cancer cell lines. The cytotoxicity of bare QDs and fluorescent probes was evaluated in the MCF‐7 cells with an MTT viability assay. The results proved that CdTe/ZnS QD–monoclonal antibody nanoprobes had been successfully prepared with excellent spectral properties in target detections. Surface modification by ZnS shell could mitigate the cytotoxicity of cadmium‐based QDs. The therapeutic effects of antivascular endothelial growth factor antibodies towards cultured human cancer cells were confirmed by MTT assay. Copyright © 2014 John Wiley & Sons, Ltd.     

Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three V_H gene segments. Panels of somatically mutated antibodies encoded by a common V_H gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the V_H gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.     

Naturally acquired antibodies to Plasmodium vivax blood-stage vaccine candidates (PvMSP-1₁₉ and PvMSP-3α₃₅₉₋₇₉₈ and their relationship with hematological features in malaria patients from the Brazilian Amazon

An important step when designing a vaccine is identifying the antigens that function as targets of naturally acquired antibodies. We investigated specific antibody responses against two Plasmodium vivax vaccine candidates, PvMSP-1₁₉ and PvMSP-3α_359?798. Moreover, we assessed the relationship between these antibodies and morbidity parameters. PvMSP-1₁₉ was the most immunogenic antigen and the frequency of responders to this protein tended to increase in P. vivax patients with higher parasitemia. For both antigens, IgG antibody responses tended to be lower in patients who had experienced their first bout of malaria. Furthermore, anemic patients presented higher IgG antibody responses to PvMSP-3α_359?798. Since the humoral response involves a number of antibodies acting simultaneously on different targets, we performed a Principal Component Analysis (PCA). Anemic patients had, on average, higher first principal component scores (IgG1/IgG2/IgG3/IgG4 anti-MSP3α), which were negatively correlated with hemoglobin levels. Since antibodies against PfMSP-3 have been strongly associated with clinical protection, we cannot exclude the possibility of a dual role of PvMSP-3 specific antibodies in both immunity and pathogenesis of vivax malaria. Our results confirm the high immunogenicity of the conserved C terminus of PvMSP-1 and points to the considerable immunogenicity of polymorphic PvMSP-3α_359?798 during natural infection.     

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding V_H CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long V_H CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long V_H CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.     

Low pKa of Lys promotes glycation at one complementarity-determining region of a bispecific antibody

Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. Cation exchange chromatography and liquid chromatography-mass spectrometry were used to characterize glycation at a lysine residue within a heavy chain (HC) CDR (HC-CDR3-Lys98) of a bispecific antibody. Thermodynamic analysis revealed that this reaction is reversible and can occur under physiological conditions with an apparent affinity of 8–10 mM for a glucose binding to HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5°C–45°C and can be well predicted in vitro and in a non-human primate. In addition, this glycation reaction was found to be driven by an unusually low pK_a on the ε-amino group of HC-CDR3-Lys98. Van't Hoff analysis and homology modeling suggested that this reaction is enthalpically driven, with this lysine residue surrounded by a microenvironment with low polarity. This study provides, to our knowledge, new insights toward a mechanistic understanding of protein glycation and strategies to mitigate the impact of protein glycation during pharmaceutical development.     

Affinity maturation of a novel antagonistic human monoclonal antibody with a long VH CDR3 targeting the Class A GPCR formyl-peptide receptor 1

Therapeutic monoclonal antibodies targeting G-protein-coupled receptors (GPCRs) are desirable for intervention in a wide range of disease processes. The discovery of such antibodies is challenging due to a lack of stability of many GPCRs as purified proteins. We describe here the generation of Fpro0165, a human anti-formyl peptide receptor 1 (FPR1) antibody generated by variable domain engineering of an antibody derived by immunization of transgenic mice expressing human variable region genes. Antibody isolation and subsequent engineering of affinity, potency and species cross-reactivity using phage display were achieved using FPR1 expressed on HEK cells for immunization and selection, along with calcium release cellular assays for antibody screening. Fpro0165 shows full neutralization of formyl peptide-mediated activation of primary human neutrophils. A crystal structure of the Fpro0165 Fab shows a long, protruding V_H CDR3 of 24 amino acids and in silico docking with a homology model of FPR1 suggests that this long V_H CDR3 is critical to the predicted binding mode of the antibody. Antibody mutation studies identify the apex of the long V_H CDR3 as key to mediating the species cross-reactivity profile of the antibody. This study illustrates an approach for antibody discovery and affinity engineering to typically intractable membrane proteins such as GPCRs.     

Kinetic measurements of molecular interactions by spectrofluorometry

The kinetics of antibody–antigen interactions are reviewed in terms of general trends observed in both polyclonal and monoclonal antibody populations. Anti-fluorescein antibodies are featured in the review as model proteins to explore fluorescence-based kinetic measurements. Since the fluorescence of the fluorescein ligand is significantly quenched upon interaction with both polyclonal and monoclonal anti-fluorescein antibodies, the quenching parameter can be advantageously employed in measuring the rates of association (k₁) and dissociation (k₂). The near diffusion-limited k₁ rates and the prolonged k₂ rates are discussed in terms of antibody affinity and mechanisms involved in ligand binding. Specific prolongation effects of reagents, such as anti-metatype antibodies, on the dissociation rate are discussed in terms of antibody dynamics and conformational substates.     

Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies

BackgroundBispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing.MethodsWe have designed a solution in which the C_H1 and C_L domain pair in one of the Fab fragments is replaced with a C_H3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII).ResultsBispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains.ConclusionIncorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties.General significanceOur results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with C_H3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing.