Evaluation of a biosensor immunoassay for simultaneous characterization of isotype and binding region of human anti-tocilizumab antibodies with control by surrogate standards

This article describes the simultaneous Biacore analysis of human anti-human antibodies (HAHAs) with respect to the binding region and the isotype by a combination of 11 single measurements per sample. The multiplexing single assay setup made efficient use of the four parallel flow cells on one biosensor chip by immobilization of full-length antibody and its constant (Fc) and antigen binding (Fab) fragments for differential binding analysis of anti-drug antibodies (ADAs). Thereby, a complete time-specific immunogenicity profile (intensity, isotype, specificity, and kinetics) of a patient could be obtained by assessing the response patterns of serially collected samples analyzed in a single measurement run. The use of functionally active standard conjugates allowed control of the assay performance throughout the whole procedure. The positive control standard conjugates mimicking polyclonal human ADAs of different isotypes were obtained by conjugating polyclonal rabbit antibodies against the therapeutic antibody to human immunoglobulin (Ig) M, IgG, or IgE. In this article, the qualification of the assay is demonstrated and the application of the methodology to six representative rheumatoid arthritis patients treated with the therapeutic humanized IgG1 antibody tocilizumab (anti-IL-6R) is shown to illustrate the versatility of the assay. The presented method allows one to differentiate specific ADAs from drug-unspecific responses (e.g., rheumatoid factors). In addition, the method can be used to discriminate between isotype responses of the IgG, IgM, and IgE types and, thereby, allows one to describe the time course of specific ADA formation and its disappearance on the single patient level.     

Altering drug tolerance of surface plasmon resonance assays for the detection of anti-drug antibodies

Anti-drug antibody (ADA) responses are a concern for both drug efficacy and safety, and high drug concentrations in patient samples may inhibit ADA assays. We evaluated strategies to improve drug tolerance of surface plasmon resonance (SPR) assays that detect ADAs against a bispecific Adnectin drug molecule that consists of an anti-VEGFR2 domain linked to an anti-IGF-1R domain (V-I-Adnectin). Samples containing ADAs against V-I-Adnectin and various drug concentrations were tested in the presence of 1 M guanidine hydrochloride (Gdn), at pH values ranging from 4.5 to 7.4 and temperatures of up to 37 °C. Temperature had a negligible effect in weakening the affinity of interaction of monoclonal antibodies with polyethylene glycol(PEG)–V-I-Adnectin and did not increase drug tolerance of the ADA assay. Low pH increased drug tolerance of the assay relative to pH 7.4 but caused nonspecific binding of the drug during competition experiments. The chaotropic agent Gdn lowered the affinity of interaction between an anti-V-Adnectin monoclonal antibody and the drug (from K_D = 0.93 nM to K_D = 348 nM). That decrease in the affinity of drug–ADA interaction correlated with an increase of assay drug tolerance. Conditions that lower drug–ADA interaction affinity could also be used to develop drug-tolerant SPR assays for other systems.     

人源中和性抗汉滩病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面表达技术,获得人源和中性抗滩滩病毒汉滩型Ｇ１基因工程单克隆抗体Ｆａｂ段基因及其表达,并同时获得抗汉滩病毒核蛋白的Ｆａｂ抗体。从能综合征出血热疫区恢复期病人抗凝血中分离到的外周淋巴细胞中,提取了部细胞ＲＮＡ。通过ＲＴ－ＰＣＲ方法,用一组人ＩｇＧ Ｆａｂ基因特异性引物,从合成了ｃＤＮＡ中经ＰＣＲ扩增了一组轻链和重链Ｆａｂ段基因,将轻链和重链先后插入噬菌体载体ｐＣｏｍｂ３,ｄｎａｌｆ ｖｆ     

Identification of chimpanzee Fab fragments by repertoire cloning and production of a full-length humanized immunoglobulin G1 antibody that is highly efficient for neutralization of dengue type 4 virus

A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V(H) and V(L) sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V(H) sequences but varied in their V(L) sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Fab 3E4 and Fab 7G4 competed with each other for binding to DENV-4 in an enzyme-linked immunosorbent assay, as did Fab 3C1 and Fab 5A7. Fab 5H2 recognized an epitope on DENV-4 that was separate from the epitope(s) recognized by other Fabs. Both Fab 5H2 and Fab 5D9 neutralized DENV-4 efficiently with a titer of 0.24 to 0.58 micro g/ml by plaque reduction neutralization test (PRNT), whereas DENV-4-neutralizing activity of other Fabs was low or not detected. Fab 5H2 was converted to full-length immunoglobulin G1 (IgG1) by combining it with human sequences. The humanized chimpanzee antibody IgG1 5H2 produced in CHO cells neutralized DENV-4 strains from different geographical origins at a similar 50% plaque reduction (PRNT(50)) titer of 0.03 to 0.05 micro g/ml. The DENV-4 binding affinities were 0.42 nM for Fab 5H2 and 0.24 nM for full-length IgG1 5H2. Monoclonal antibody IgG1 5H2 may prove valuable for passive immunoprophylaxis against dengue virus in humans.     

Development of phage biopanning strategies to identify affinity peptide ligands for kappa light chain Fab fragments

In this work, two phage biopanning strategies were developed to identify affinity peptides for a single Fab and multiple kappa Fabs. For the biopanning rounds, protein L beads were employed to bind Fab targets in a fixed orientation, and NHS functionalized magnetic beads were used to facilitate evaluation of low pH elution conditions. The resulting peptide sequences were synthesized and the binding to different Fabs was evaluated using fluorescence polarization. The first biopanning approach yielded a peptide with similar affinities for two forms of the Fab (recombinantly expressed and post papain-digestion) as well as the intact antibody. While moderate affinity was observed toward a murine variant of the Fab with the same complementarity determining regions (CDR) region but different framework, minimal binding occurred to a Fab with high sequence homology but containing different CDR loops. The second biopanning strategy yielded a peptide with affinity for all three kappa Fabs indicating that it may be a good lead for the development of more general affinity reagents for recombinant kappa Fabs. Finally, an affinity peptide column was developed, and its efficacy was demonstrated for Fab purification from a complex cell culture fluid mixture. The results presented in this article demonstrate that different peptide-based phage biopanning strategies can be effectively employed to identify affinity peptide leads for specific Fab and more general kappa Fab purifications.     

Antigen, antibody and immune complex detection in serum samples from rats experimentally infected with Strongyloides venezuelensis

In order to establish an antigen, antibody and immune complex detection by enzyme-linked immunosorbent assay (ELISA) in serum samples, normal or immunocompromised Wistar rats experimentally infected with Strongyloides venezuelensis were used. The microtitre plates were coated with IgG anti-S. venezuelensis for antigen and immune complex detection and with alkaline parasite extract for antibody detection. Analysis revealed at least 12.5 μg/mL of S. venezuelensis specific antigens in serum samples. Assay for antigen detection was not a good approach for evaluating infection in normal or immunocompromised rats. In normal rats IgG specific for S. venezuelensis was preferentially detected during the first 13 days post-infection (p.i.) and immune complex detection was significantly reduced in 21 p.i. day. On the other hand, in immunocompromised rats, IgG and immune complex were detected during the entire kinetic (5, 8, 13 and 21 p.i). These results suggest that immune complex screening seems to be an alternative for early strongyloidiasis diagnosis in immunocompromised individuals.     

Guided selection of human antibody light chains against TAG-72 using a phage display chain shuffling approach

To enhance therapeutic potential of murine monoclonal antibody, humanization by CDR grafting is usually used to reduce immunogenic mouse residues. Most humanized antibodies still have mouse residues critical for antigen binding, but the mouse residues may evoke immune responses in humans. Previously, we constructed a new humanized version (AKA) of mouse CC49 antibody specific for tumor-associated glycoprotein, TAG-72. In this study, to select a completely human antibody light chain against TAG-72, guided selection strategy using phage display was used. The heavy chain variable region (VH) of AKA was used to guide the selection of a human TAG-72-specific light chain variable region (VL) from a human VL repertoire constructed from human PBL. Most of the selected VLs were identified to be originated from the members of the human germline VK1 family, whereas the VL of AKA is more homologous to the VK4 family. Competition binding assay of the selected Fabs with mouse CC49 suggested that the epitopes of the Fabs overlap with that of CC49. In addition, they showed better antigen-binding affinity compared to parental AKA. The selected human VLs may be used to guide the selection of human VHs to get completely human anti-TAG72 antibody.     

A Monoclonal Antibody Specific for Reovirus Outer-Capsid Protein ς3 Inhibits ς1-Mediated Hemagglutination by Steric Hindrance

Reovirus virions are nonenveloped icosahedral particles consisting of two concentric protein shells, termed outer capsid and core. Outer-capsid protein sigma1 is the viral attachment protein and binds carbohydrate molecules on the surface of host cells. Monoclonal antibody (MAb) 4F2, which is specific for outer-capsid protein sigma3, blocks the binding of sigma1 protein to sialic acid and inhibits reovirus-induced hemagglutination (HA). To determine whether MAb 4F2 inhibits HA by altering sigma1-sigma3 interactions or by steric hindrance, we analyzed the effect of 4F2 immunoglobulin G (IgG) and Fab fragments (Fabs) on HA induced by reovirus strain type 3 Dearing (T3D). The concentration of 4F2 IgG sufficient to inhibit T3D-induced HA was 12.5 microg per ml, whereas that of Fabs was >200 microg per ml. Dynamic light scattering analysis showed that at the concentration of IgG sufficient to inhibit HA, virion-antibody complexes were monodispersed and not aggregated. The affinity of 4F2 Fabs for T3D virions was only threefold less than that of intact IgG, which suggests that differences in HA inhibition titer exhibited by 4F2 IgG and Fabs are not attributable to differences in the affinity of these molecules for T3D virions. We used cryoelectron microscopy and three-dimensional image analysis to visualize T3D virions alone and in complex with either IgG or Fabs of MAb 4F2. IgG and Fabs bind the same site at the distal portion of sigma3, and binding of IgG and Fabs induces identical conformational changes in outer-capsid proteins sigma3 and mu1. These results suggest that MAb 4F2 inhibits reovirus binding to sialic acid by steric hindrance and provide insight into the conformational flexibility of reovirus outer-capsid proteins.     

IgG Fab Fragments Forming Bivalent Complexes by a Conformational Mechanism That Is Reversible by Osmolytes

Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing cross-linking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches, including size exclusion chromatography, SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte l-proline might impact bivalent Fab complexation. We found that l-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of preformed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of preformed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.

In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

In vitro evidence indicating a role for the Fc region of IgG in the mechanism for the long-term maintenance and regulation of antibody levels in vivo

The synthesis of anti-human serum albumin (HSA) antibody was induced spontaneously in cell cultures prepared from the draining lymph nodes of rabbits immunized months earlier with polymerized HSA. Serum from HSA-immunized rabbits suppressed this response. Removal of specific antibody from immune serum eliminated suppression and the addition of specific IgG restored suppression, indicating that the feedback phenomenon may be explained by an effect of specific IgG antibody. Fab and F(ab′)₂ fragments masked antigen as effectively as IgG; however, they were markedly inferior to IgG in mediating suppression. Furthermore, Fab competed with IgG and interfered with IgG mediated suppression. The addition of small amounts of antigen to antibody-suppressed cultures induced an antibody response. The level of induction was proportional to the antigen-antibody ratio. However, 80 to 100 times the antibody concentration needed to mask all antigenic determinants was needed in order to eliminate induction of antibody synthesis. High concentrations of antigen-antibody complexes at equivalence also suppressed the spontaneous response. This suppression was similar to antibody mediated suppression at the spontaneous response in that the Fc region of IgG was required.     

Neutralizing Human Fab Fragments against Measles Virus Recovered by Phage Display

Five human recombinant Fab fragments (Fabs) specific for measles virus (MV) proteins were isolated from three antibody phage display libraries generated from RNAs derived from bone marrow or splenic lymphocytes from three MV-immune individuals. All Fabs reacted in an enzyme-linked immunosorbent assay with MV antigens. In radioimmunoprecipitation assays two of the Fabs, MV12 and MT14, precipitated an approximately equal 80-kDa protein band corresponding to the hemagglutinin (H) protein from MV-infected Vero cell cultures, while two other Fabs, MT64 and GL29, precipitated an approximately equal 60-kDa protein corresponding the nucleocapsid (N) protein. In competition studies with MV fusion, H- and N protein-specific monoclonal antibodies (MAbs), the H-specific Fabs predominantly blocked the binding of H-specific MAbs, while the N-specific Fabs blocked MAbs to N. In addition, N-specific Fabs bound to denatured MV N protein in Western blotting. The specificity of the fifth Fab, MV4, could not be determined. By plaque reduction assays, three of the five Fabs, MV4, MV12, and MT14, exhibited neutralizing activity (80% cutoff) against MV (LEC-KI strain) at concentrations ranging between approximately 2 and 7 microg x ml(-1). Neutralization capacity against MV strains Edmonston and Schwarz was also detected, albeit at somewhat higher Fab concentrations. In conclusion, three neutralizing Fabs were isolated, two of them reactive against the H glycoprotein of MV and another reactive against an undefined epitope. This is the first study in which MV-neutralizing human recombinant Fab antibodies have been isolated from phage display libraries.     

Expression of humanized Fab fragments that recognize the IgE-binding domain of human Fc(epsilon)RIalpha in COS and CHO cells

Interfering with the binding of IgE to high-affinity IgE receptor alpha chain (Fc(epsilon)RIalpha) is a straightforward strategy for the specific prevention of the IgE-mediated allergic reaction specifically. A Fab fragment (Fab) of a humanized antibody against the membrane proximal IgE-binding domain of human Fc(epsilon)RIalpha inhibits the release of histamine from human basophils. We established an efficient expression system in which to produce directly the humanized anti-human Fc(epsilon)RIalpha Fabs without papain-digestion of the whole antibody. Four Fabs with different C-termini of CH1 were expressed directly in COS-7 cells transfected with expression vectors with or without the Fc gene downstream of a stop codon inserted within the hinge gene. The secretion of Fabs when transfected without the Fc gene was remarkably enhanced compared to that when transfected with the Fc gene. The ability of Fabs to inhibit IgE-Fc(epsilon)RIalpha binding when transfected without the Fc gene was equivalent to that of purified Fab prepared by papain-digestion of the whole antibody. No significant differences among the four Fabs were observed in secretion or activity. Clones of CHO-transfectant cells that secreted the Fabs constitutively were acclimatized to a serum-free medium. Analysis of the binding interface between the Fab and human Fc(epsilon)RIalpha will provide useful information for the design of therapeutic reagents for allergy and asthma.     

Exchanging human Fcγ1 with murine Fcγ2a highly potentiates anti-tumor activity of anti-EpCAM antibody adecatumumab in a syngeneic mouse lung metastasis model

An important mode of action shared by human IgG1 antibody therapies is antibody-dependent cellular cytotoxicity (ADCC). ADCC relies on the interaction of the antibody’s Fc portion with Fc-gama receptors (FcγR) on immune effector cells. The anti-tumor activity of human IgG1 antibodies is frequently assessed in mouse models. Binding of human IgG1 to murine FcγRs is however of reduced affinity. We here show that ADCC of adecatumumab (MT201), a fully human IgG1 antibody specific for epithelial cell adhesion molecule (EpCAM/CD326), is drastically lower if human peripheral blood mononuclear cells are replaced by murine splenocytes as effector cells. When the variable domains of adecatumumab were genetically fused to a murine IgG2a backbone (yielding mu-adecatumumab), ADCC with murine effector cells was much improved, but at the same time significantly reduced with human effector cells. The serum half-lives of adecatumumab and mu-adecatumumab were determined in mice and dosing schedules established that gave similar serum trough levels during a 4-week antibody treatment. The anti-tumor activities of adecatumumab and mu-adecatumumab were then compared side-by-side in a lung metastasis mouse model established with a syngeneic B16 melanoma line expressing human EpCAM at physiologically relevant levels. Treatment of mice with mu-adecatumumab led to an almost complete prevention of lung metastases, while the human version of the antibody was much less active. This shows that adecatumumab has high anti-tumor activity when tested in a form that is better compatible with the species’ immune system. Moreover, our data suggest to routinely compare in mouse models human IgG1 and murine IgG2a versions of antibodies to properly assess the contribution of ADCC to overall anti-tumor activity.     

人源单克隆抗人免疫缺陷病毒1型抗体Fab段基因的获得

应用噬苏体抗体库技术有效地筛选出了多株抗ＨＩＶ－１人源单克隆抗体。以逆转录聚合酶链反应（ＲＴ－ＰＣＲ）从ＨＩＶ－１感染者外周血淋巴细胞中扩增抗体轻重链可变区基因,插入载体ｐＣＯＭＢ３,建立噬菌体抗体库。分别以ＨＩＶ－１ｇｐ１２０和ｇｐ１６０为固相抗原,经过多轮筛选,从中获得了多株抗ＨＩＶ－１ｇｐ４１、ｇｐ１２０和ｇｐ１６０的单克隆抗体Ｆａｂ段基因。抗ＨＩＶ特异性噬菌体抗体随抗体库的筛选高度富集,抗     

Cholestatic Liver Disease after Rituximab and Adalimumab and the Possible Role of Cross-Reacting Antibodies to Fab 2 Fragments

Background

Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group.

Methods

Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin.

Results

Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients’ sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected.

Conclusion

This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug clearance in patients on Tmab therapy.     

Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses

Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT(50)) titer, ranging from 0.26 to 1.33 microg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 microg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT(50) titer of approximately 3.2 to 4.2 microg/ml. This humanized antibody represents an attractive candidate for further development of immunoprophylaxis against DENV and perhaps other flavivirus-associated diseases.     

Development of ELISAs for quantification of HMFG1-specific human anti-mouse IgG and IgM antibodies

The aim of this study was to develop and validate ELISAs for quantification of HAMA-IgM and HAMA-IgG in serum of patients with ovarian cancer who enrolled in a large international randomized phase III trial of intraperitoneal Yttrium-90-labeled HMFG1 murine monoclonal antibody therapy. The capture antibody of these 2 assays was the murine antibody HMFG1, while mouse anti-human IgM-HRP or mouse anti-human IgG(Fc)-HRP served as tracer antibodies. A pool of HAMA-positive serum samples was used to prepare a series of assay standards and another pool served as reference preparation. The analytical sensitivity of the HAMA-IgM assay was 2.5 arbitrary units per mL (AU/mL) and 4.7 AU/mL for the HAMA-IgG ELISA. Diluted serum samples showed good parallelism with the HAMA-IgM and HAMA-IgG standard dose-response curves. Within-assay coefficient of variation was 7.5% for HAMA-IgM and 6.5% for HAMA-IgG. Between-assay variation was 14.2% for HAMA-IgM and 15.3% for HAMA-IgG. The developed HAMA-IgM and HAMA-IgG ELISAs show satisfactory reliability criteria (sensitivity, parallelism and precision) and are suitable for monitoring of HAMA-IgM and HAMA-IgG responses in ovarian cancer patients. These ELISAs will be used to monitor the development of HAMAs in patients who received radioimmunotherapy with murine HMFG1.     

Modification of the first constant domain of heavy chain enabled effective folding of functional anti-forskolin antigen-binding fragment for sensitive quantitative analysis

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (C_H1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between V_H-C_H1 and V_L-C_L was improved by the C_H1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91–62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.     

Design and characterization of novel dual Fc antibody with enhanced avidity for Fc receptors

Monoclonal antibodies (mAbs) have become an important class of therapeutics, particularly in the realm of anticancer immunotherapy. While the two antigen-binding fragments (Fabs) of an mAb allow for high-avidity binding to molecular targets, the crystallizable fragment (Fc) engages immune effector elements. mAbs of the IgG class are used for the treatment of autoimmune diseases and can elicit antitumor immune functions not only by several mechanisms including direct antigen engagement via their Fab arms but also by Fab binding to tumors combined with Fc engagement of complement component C1q and Fcγ receptors. Additionally, IgG binding to the neonatal Fc receptor (FcRn) allows for endosomal recycling and prolonged serum half-life. To augment the effector functions or half-life of an IgG1 mAb, we constructed a novel “2Fc” mAb containing two Fc domains in addition to the normal two Fab domains. Structural and functional characterization of this 2Fc mAb demonstrated that it exists in a tetrahedral-like geometry and retains binding capacity via the Fab domains. Furthermore, duplication of the Fc region significantly enhanced avidity for Fc receptors FcγRI, FcγRIIIa, and FcRn, which manifested as a decrease in complex dissociation rate that was more pronounced at higher densities of receptor. At intermediate receptor density, the dissociation rate for Fc receptors was decreased 6- to 130-fold, resulting in apparent affinity increases of 7- to 42-fold. Stoichiometric analysis confirmed that each 2Fc mAb may simultaneously bind two molecules of FcγRI or four molecules of FcRn, which is double the stoichiometry of a wild-type mAb. In summary, duplication of the IgG Fc region allows for increased avidity to Fc receptors that could translate into clinically relevant enhancement of effector functions or pharmacokinetics.