Light Chain Shuffling Research Articles

Engineering of pH-dependent antigen binding properties for toxin-targeting IgG1 antibodies using light-chain shuffling

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.

In vitro discovery of a human monoclonal antibody that neutralizes lethality of cobra snake venom

ABSTRACT The monocled cobra (Naja kaouthia) is among the most feared snakes in Southeast Asia due to its toxicity, which is predominantly derived from long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines, major limitations in safety, supply consistency, and efficacy create a need for improved treatments. Here, we describe the discovery and subsequent optimization of a recombinant human monoclonal immunoglobulin G antibody against α-cobratoxin using phage display technology. Affinity maturation by light chain-shuffling resulted in a significant increase in in vitro neutralization potency and in vivo efficacy. The optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.

Isolation and light chain shuffling of a Plasmodium falciparum AMA1-specific human monoclonal antibody with growth inhibitory activity

BackgroundPlasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirix™ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates.Material and methodsUsing antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1–3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays.ResultsThe selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2.ConclusionWe have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.

A therapeutic human antibody against the domain 4 of the Bacillus anthracis protective antigen shows protective efficacy in a mouse model

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

Antibody engineering to improve manufacturability

Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.

Abstract LB-306: Impacting Humoral Hypercalcemia of Malignancy (HHM) and associated PTH1R-mediated morbidities: Characterization of an anti-PTH1R antagonist monoclonal antibody to reverse hypercalcemia

Abstract Humoral Hypercalcemia of Malignancy (HHM) occurs in 20-30 percent of advanced cancers, both solid tumors and hematologic malignancies, and is the most common life-threatening complication of cancer. HHM is due to tumors secreting abnormally high quantities of parathyroid hormone-related protein (PTHrP), a ligand for PTH1R (a family B GPCR) resulting in hypercalcemia. A potent and long acting PTH1R antagonist could reverse the hypercalcemia in HHM. Using XOMA’s fully-human antibody phage display libraries, highly potent anti-PTH1R antagonist monoclonal antibodies were discovered, and screened by FACS for binding to CHO-K1 cells over-expressing human PTH1R. Human and murine receptor antagonism was demonstrated in cAMP accumulation and calcium flux assays with the human Saos-2 and the rat UMR106 osteosarcoma cell lines, which express endogenous PTH1R. The epitope demonstrating the greatest receptor antagonism was localized to the N-terminal extracellular domain of human PTH1R. Antibodies were affinity matured via light chain shuffling, resulting in leads with sub-nanomolar binding affinities as measured by SPR and verified by FACS. Functionally, PTH1R is expressed on osteoblasts and osteocytes. Stimulation with PTHrP leads to increased expression of M-CSF, RANKL and other factors that drive the differentiation and activation of bone resorbing osteoclast cells. It was shown that the lead anti-PTH1R mAb inhibited both PTH- and PTHrP-induced osteoclast differentiation by greater than 10-fold. In vivo proof-of-concept was achieved in rodent models where hypercalcemia was established in rats by SC infusion of PTHrP via an osmotic pump. IV administration of 2 and 10 mg/kg antibody reduced serum calcium levels by a minimum of 2 mg/dL within 48 hours in a dose-dependent manner. Additionally, in a murine C26 tumor model of hypercalcemia associated with elevated PTHrP, the anti-PTH1R mAb showed a dose-dependent correction of hypercalcemia at doses as low as 2 mg/kg with a sustained duration of action. In this model, the antibody given at 10 mg/kg IV completely reversed the hypercalcemia within 24 hours, lowering serum calcium from greater than 15mg/dL to approximately 6mg/dL. Additional PK/PD parameters were assessed in mouse and rat models. Patients with HHM typically have a poor prognosis and HHM is a frequent reason that cancer patients are readmitted to hospitals or enter hospice care. Left untreated, hypercalcemia in these patients can cause coma or death. The highly potent anti-PTH1R antagonist mAb described here has the potential to become a first in class therapy for HHM, ameliorating hypercalcemia-associated morbidities, and extending the utility of other anti-cancer agents. This promising therapeutic antibody is currently in late stage preclinical development. Citation Format: Amer M. Mirza, Agnes Choppin, Daniel Bedinger, Rachel Hunt, Robyn Cotter, Elizabeth Pongo, Kiran Ahluwalia, Catarina Tran, Llewelyn Lao, Kristin Lind, Sujeewa Wijesuriya, Lynn Webster, Fangjiu Zhang, Kirk Johnson, Toshihiko Takeuchi, Raphael Levy. Impacting Humoral Hypercalcemia of Malignancy (HHM) and associated PTH1R-mediated morbidities: Characterization of an anti-PTH1R antagonist monoclonal antibody to reverse hypercalcemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-306. doi:10.1158/1538-7445.AM2017-LB-306

IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde–Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope

The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde–acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library, and a derived Fab with the same HCDR3 rearrangement displayed identical MAA-binding properties. These data support the concept that OSE (MAA-epitopes), which are ubiquitous products of inflammation, may play a role in clonal selection and expansion of CLL B cells.

Light-chain shuffling from an antigen-biased phage pool allows 185-fold improvement of an anti-halofuginone single-chain variable fragment

Halofuginone is an antiprotozoal drug used in the treatment of coccidiosis in poultry, a contagious enteric disease caused by parasites of the Eimeria spp. To ensure that food is free from any halofuginone residues and safe for human consumption, a rapid method to detect these residues below the maximum residue limits (MRLs) in a variety of matrices is necessary. To address this need, we constructed an immune single-chain variable fragment (scFv) library from the RNA of a halofuginone-immunized chicken and selected halofuginone-specific scFv by phage display. The best clone isolated from the library had a limit of detection of 30 ng/ml as determined by enzyme-linked immunosorbent assay (ELISA). However, the minimum MRL for halofuginone in certain foodstuffs can be as low as 1 ng/ml, well below the sensitivity of the selected antibody. The selected antibody was then affinity maturated by light-chain shuffling to further improve the antibody’s assay performance. The halofuginone-specific heavy-chain pool of the biopanned library was assembled with the light-chain repertoire amplified from the original prepanned library. This resulted in a heavy-chain-biased library from which an scFv with the potential to detect halofuginone residues as low as 80 pg/ml was isolated, a 185-fold improvement over the original scFv. This new chain-shuffled scFv was incorporated into a validated ELISA (according to Commission Regulation 2002/657/EC) for the sensitive detection of halofuginone in spiked processed egg samples.

Single chain Fv fragment specific for human GM‐CSF: Selection and expression using a bacterial expression library

Single chain antibodies (scFvs) are replacing whole antibody molecules since they are easy to produce on large scale and amenable to genetic modifications. Here we report the development of an anti-human granulocyte macrophage colony-stimulating factor (hGM-CSF) scFv as an immunoassay bio-reagent, utilizing an easily scalable bacterial expression system. For this, the V(H) and V(L) gene repertoires were amplified from the immunoglobulin complementary DNA, derived from total RNA of mice splenocytes, pre-sensitized with the antigen. The scFv library was expressed under the strong T7 promoter in BL21 (DE3) Escherichia coli cells. Preliminary screening led to the selection of four potential candidates, which were later subjected to light chain shuffling. Cross-reactivity analysis involving the original and shuffled candidates resulted in the selection of one scFv (scFv196) with no cross-reactivity against E. coli antigens. The binding affinity of the scFv196 for hGM-CSF, measured by surface plasmon resonance, was found to be within the physiological range (K(D) =1.5 μM). The refolded scFv was also shown to recognize and bind the glycosylated antigen, a closer mimic of the physiological GM-CSF, potentiating its use in immunoassays. Expression studies using shake flasks suggested periplasmic export of the scFv196 protein.

Development of the dual-vector system-III (DVS-III), which facilitates affinity maturation of a Fab antibody via light chain shuffling

Light (L) chain shuffling is routinely used to analyze optimal L chains that pair with a specific heavy (Fd or H) chain, which ultimately leads to in vitro affinity maturation of a particular antibody. One of the major drawbacks to this procedure is that L chain libraries have to be created for each distinct H chain, which involves complicated cloning procedures. Herein, we designed of the dual-vector system-III (DVS-III), which is composed of a set of pLf1T-3 phagemid and pHg3A-3 plasmid, for L chain shuffling of any given human Fab antibody via phage display technology. To demonstrate the feasibility of our system, a human naïve L chain sublibrary, HuNL-D3, constructed in pLf1T-3 phagemid, was combined with the Fd of a human anti-IL-15 Fab, 4H10, subcloned in pHg3A-3 plasmid as a model system. After solution-phase sorting and biopanning the library we obtained eight Fab variants (4H10-LP1–7 and 4H10-LS). Among them, 4H10-LP4 exhibited the highest affinity which is about 36-fold higher than that of the parent molecule 4H10 (KD=6nM versus 200nM). Our results demonstrate that the DVS-III, along with the HuNL-D3 L chain sublibrary, can be served as a convenient approach for affinity maturation of any given human Fab antibody through L chain optimization.

Affinity maturation of human botulinum neurotoxin antibodies by light chain shuffling via yeast mating

Botulism is caused by the botulinum neurotoxins (BoNTs), the most poisonous substance known. Because of the high potency of BoNT, development of diagnostic and therapeutic antibodies for botulism requires antibodies of very high affinity. Here we report the use of yeast mating to affinity mature BoNT antibodies by light chain shuffling. A library of immunoglobulin light chains was generated in a yeast vector where the light chain is secreted. The heavy chain variable region and the first domain of the constant region (V(H)-C(H)1) from a monoclonal antibody was cloned into a different yeast vector for surface display as a fusion to the Aga2 protein. Through yeast mating of the two haploid yeasts, a library of light chain-shuffled Fab was created. Using this approach, the affinities of one BoNT/A and two BoNT/B scFv antibody fragments were increased from 9- to more than 77-fold. Subcloning the V-genes from the affinity-matured Fab yielded fully human IgG1 with equilibrium binding constants for BoNT/A and BoNT/B of 2.51 x 10(-11) M or lower for all three monoclonal antibodies. This technique provides a rapid route to antibody affinity maturation.

All Fuels & Energy testing Super Enzyme for making ethanol

Since Bacillus anthracis is a high-risk pathogen and a potential tool for bioterrorism, numerous therapeutic methods including passive immunization have been actively developed. Using a human monoclonal antibody phage display library, we screened new therapeutic antibodies for anthrax infection against protective antigen (PA) of B. anthracis. Among 5 selected clones of antibodies based on enzyme-linked immunosorbent assay (ELISA) results, 7B1 showed neutralizing activity to anthrax lethal toxin (LT) by inhibiting binding of the domain 4 of PA (PD4) to its cellular receptors. Through light chain shuffling process, we improved the productivity of 7B1 up to 25 folds. The light chain shuffled 7B1 antibody showed protective activity against LT both in vitro and in vivo. Furthermore, the antibody also conferred protection of mice from 3 × LD50 challenges of fully virulent anthrax spores. Our result expands the possibility of developing a new therapeutic antibody for anthrax cure.

Exceptionally Potent Cross-Reactive Neutralization of Nipah and Hendra Viruses by a Human Monoclonal Antibody

We have previously identified neutralizing human monoclonal antibodies against Nipah virus (NiV) and Hendra virus (HeV) by panning a large nonimmune antibody library against a soluble form of the HeV attachment-envelope glycoprotein G (sGHeV). One of these antibodies, m102, which exhibited the highest level of cross-reactive neutralization of both NiV and HeV G, was affinity maturated by light-chain shuffling combined with random mutagenesis of its heavy-chain variable domain and panning against sGHeV. One of the selected antibody Fab clones, m102.4, had affinity of binding to sGHeV that was equal to or higher than that of the other Fabs; it was converted to IgG1 and tested against infectious NiV and HeV. It exhibited exceptionally potent and cross-reactive inhibitory activity with 50% inhibitory concentrations below 0.04 and 0.6 μg/mL, respectively. The virus-neutralizing activity correlated with the binding affinity of the antibody to sGHeV and sGNiV. m102.4 bound a soluble form of NiV G (sGNiV) better than it bound sGHeV, and it neutralized NiV better than HeV, despite being originally selected against sGHeV. These results suggest that m102.4 has potential as a therapeutic agent for the treatment of diseases caused by henipaviruses. It could be also used for prophylaxis and diagnosis, and as a research reagent

Guided selection of an anti-gamma-seminoprotein human Fab for antibody directed enzyme prodrug therapy of prostate cancer.

The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug therapy in vivo. In this study we have achieved humanization of the anti-gamma-seminoprotein E(4)B(7) murine mAb by guided selection. Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients. The human Lc gene repertoire was first paired with the murine Fd gene of E(4)B(7) mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified gamma-seminoprotein antigen. The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four more rounds of panning, completely human Fab antibodies specific for gamma-seminoprotein were selected and further identified. First, using the E(4)B(7) Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a template, a human Fab antibody against gamma-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA, and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E(4)B(7) mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry analysis further confirmed that this newly constructed human anti-gamma-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue. Through guided-selection, we successfully produced a human anti-gamma-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody.

Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of thePseudonitzschia pungensToxin Domoic Acid

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V(H) and V(L) genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

Isolating human antibody against human hepatocellular carcinoma by guided-selection

With the pComb3X-displaying Fab antibody libraries, to achieve the humanization of murine HAb18 against HCC by guided selection. METHODS: With the optimized primers, the human Fd and CL repertoire genes were amplified by RT-PCR from PBMC of HCC patients. The Fd repertoire genes were paired with murine HAb18 CL gene to construct pComb3X-displaying hybrid Fab library. The recombinant HAb18GE was used as antigens to select the target antibodies and got the Fd fragments. Then the human CL genes were paired with the selected human Fds to construct human Fab library. After the panning, the complete human Fab antibodies were got and analyzed. RESULTS: With the murine HAb18 CL gene as template, the heavy chain Fd shuffling was achieved by panning the hybrid Fab library. Then with the selected Fds as template, the human Fabs were obtained through the light chain shuffling. Two of the resulting human Fabs (HuFab2 and HuFab11), with same Fd and different light chains, bound to HAb18G/CD147 specifically. The competitive ELISA, Western blotting, FCM, fluorescent cell staining and so on demonstrated that the human Fabs resembled its parental murine Fab in that they both perhaps recognized the same epitope. KD indicated (HuFab2= 210nm and HuFab11=280nm) the selected Fabs had available affinity. CONCLUSION: Through guided-selection, we got the available human Fab antibodies for the subsequent research. These results suggest that guided selection is a promising strategy in murine mAb humanization.

Light-chain shuffling results in successful phage display selection of functional prokaryotic-expressed antibody fragments to N-glycolyl GM3 ganglioside

Phage display technology makes it possible to introduce and rapidly screen diversity in antibody binding sites. Chain shuffling has been successfully used to humanize murine antibody fragments and also to obtain affinity matured variants. Here we report a different application of this method: the use of chain shuffling to overcome improper prokaryotic expression behavior of a hybridoma-derived single-chain antibody fragment. Construction and expression of such recombinant antibody fragments remain as empirical entities, hampered by the inability to express some antibody genes coming from eukaryotic cells in bacterial expression systems. Such problems are different for each combination of variable regions and can be serious enough to preclude the use of some hybridomas as sources of V regions to obtain recombinant antibody fragments. The particular binding properties and potential usefulness of some monoclonal antibodies make it highly desirable to bypass these technical limitations in order to develop smaller size therapeutic agents in the form of antibody fragments. The 14F7 mouse monoclonal antibody is one such attractive candidate due to its high specificity for the N-glycolyl GM3 ganglioside overexpressed in tumor cells and its ability to distinguish this antigen from closely related gangliosides like N-acetyl GM3. Our goal was to construct a phage-displayed single-chain Fv antibody fragment derived from 14F7. After cloning the original variable regions from the 14F7 hybridoma in a phagemid vector, we were unable to detect either binding activity or even expression of antibody fragments in bacteria, despite repetitive efforts. We constructed light-chain shuffling libraries, from which functional antibody fragments were readily selected. These combined the original 14F7 heavy chain variable region with a wide variety of unrelated murine and human light-chain variable regions. New antibody fragments retained the valuable properties of the monoclonal antibody in terms of fine specificity, affinity and tumor recognition. They were readily produced by bacteria, either in phage-displayed form or as soluble molecules, and provided a panel of potentially useful variants for cancer diagnosis and immunotherapy. Chain shuffling and phage display were found to be useful strategies for selecting antibody fragments on the basis of both prokaryotic expression and antigen binding criteria.

VH3 gene usage in neutralizing human antibodies specific for the Entamoeba histolytica Gal/GalNAc lectin heavy subunit.

A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomatic Entamoeba histolytica cyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- and N-acetyl-D-galactosamine (Gal/GalNAc)-specific lectin of E. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the V kappa 1 family, in which the closest V kappa germ line gene was 02/012 or L5, with the J kappa 1, J kappa 2, J kappa 4, or J kappa 5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation of E. histolytica trophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.

Improved production by domain inversion of single-chain Fv antibody fragment against high molecular weight proteoglycan for the radioimmunotargeting of melanoma.

Melanoma is among the few cancers with rising incidence. Currently there is no effective treatment for metastatic disease, but improved detection of melanoma has the potential to benefit the management of patients with early disease. Radioimmunodection by imaging with single-chain Fv (scFv) antibody fragments is one such emerging diagnostic method. However, the amount of scFv that can be produced at a scale suitable for use in patients is limiting. We have previously shown that the bacterial expression of a scFv derived from a monoclonal antibody (MAb) specific for melanoma-associated proteoglycan can be increased by light chain shuffling. In this report we show that a further increase in expression yield can be obtained by reversing the usual V(H)-V(L) orientation of scFvs to V(L)-V(H). Such seemingly minor changes have previously been reported to have unexpected effects on the in vitro and in vivo binding properties of recombinant antibodies. Our results show that reversal of the V domain orientation of the scFv improves expression by 150% without an adverse effect on melanoma binding in vitro and tumor targeting in vivo. Therefore, our results show that alteration of V domain orientation can improve the production yield of clinically useful antibody fragments. When used in combination with other antibody engineering approaches for increased antibody production changing the domain orientation is a simple strategy to achieve significant improvements in the production of scFvs for tumor radioimmunodetection for patient studies.

Light Chain Determines the Binding Property of Human Anti-dsDNA IgG Autoantibodies

We have previously prepared human anti-double-stranded (ds) DNA IgG Fab clones using phage-display technology. Nucleotide sequence analysis of genes of immunoglobulin (Ig) heavy and light chain variable regions in these Fab clones suggested that the DNA-binding activity of the clones depended on light chain usage. To confirm the role of the light chain in antibody binding to DNA, we constructed in the present study's new recombined Fab clones by heavy and light chain shuffling between the original anti-dsDNA Fab clones. Clones constructed by pairing Fdγ fragments with the light chain from a high DNA-binding clone showed high DNA-binding activities, whereas other constructed clones using light chains from low DNA-binding clones showed low DNA-binding activities. Our results indicate that light chains in anti-dsDNA antibodies can determine the DNA-binding activity of the antibodies. Ig chain shuffling of phage-display antibodies may be useful for investigating the molecular mechanisms for antigen–antibody binding of human autoantibodies.