Variable Light Chain Research Articles

Identification of a single base-pair mutation of TAA (Stop codon) → GAA (Glu) that causes light chain extension in a CHO cell derived IgG1

We describe here the identification of a stop codon TAA (Stop) → GAA (Glu) = Stop221E mutation on the light chain of a recombinant IgG1 antibody expressed in a Chinese hamster ovary (CHO) cell line. The extended light chain variants, which were caused by translation beyond the mutated stop codon to the next alternative in-frame stop codon, were observed by mass spectra analysis. The abnormal peptide peaks present in tryptic and chymotryptic LC–MS peptide mapping were confirmed by N-terminal sequencing as C-terminal light chain extension peptides. Furthermore, LC-MS/MS of Glu-C peptide mapping confirmed the stop221E mutation, which is consistent with a single base-pair mutation in TAA (stop codon) to GAA (Glu). The light chain variants were approximately 13.6% of wild type light chain as estimated by RP-HPLC analysis. DNA sequencing techniques determined a single base pair stop codon mutation, instead of a stop codon read-through, as the cause of this light chain extension. To our knowledge, the stop codon mutation has not been reported for IgGs expressed in CHO cells. These results demonstrate orthogonal techniques should be implemented to characterize recombinant proteins and select appropriate cell lines for production of therapeutic proteins because modifications could occur at unexpected locations.

Expression of recombinant anti‐colorectal cancer large single‐chain monoclonal antibody in insect cells

AbstractColorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733‐2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti‐colorectal cancer large single chain (lsc) mAb based on mAb CO17‐1A in the baculovirus‐insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17‐1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C‐terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus‐infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti‐colorectal lsc mAb recognizing human colorectal cancer cells.

S.13.04 Immunotherapy in amyotrophic lateral sclerosis

There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)–mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1G93A mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1G93A mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations.

Early Postnatal B Cell Ontogeny and Antibody Repertoire Maturation in the Opossum, Monodelphis domestica

Marsupials are a lineage of mammals noted for giving birth to highly altricial young, which complete much of their “fetal” development externally attached to a teat. Postnatal B cell ontogeny and diversity was investigated in a model marsupial species, the gray short-tailed opossum, Monodelphis domestica. The results support the initiation of B cell development late in gestation and progressing into the first two weeks of postnatal life. Transcription of CD79a and CD79b was detected in embryonic tissue prior to birth, while immunoglobulin heavy chain locus transcription was not detected until the first postnatal 24 hours. Transcription of the Ig light chains was not detected until postnatal day 7 at the earliest. The predicted timing of the earliest appearance of mature B cells and completion of gene rearrangements is consistent with previous analyses on the timing of endogenous antibody responses in newborn marsupials. The diversity of early B cell IgH chains is limited, as has been seen in fetal humans and mice, but lacks bias in the gene segments used to encode the variable domains. Newborn light chain diversity is, from the start, comparable to that of the adult, consistent with an earlier hypothesis that light chains contribute extensively to antibody diversity in this species.

454 pyrosequencing and bioinformatics analysis of the lineage of the broad and potent gp41-directed antibody 10e8

Results 37,669 IGHV3-15 heavy chain sequences and 91,951 IGLV3-19 light chain sequences were obtained and the distribution of these sequences analyzed by two key parameters – divergence from germline and sequence identity to 10e8. Grid-based clustering allowed for the selection of 61 heavy chain sequences and 48 light chain sequences. These were reconstituted with the appropriate 10e8 light or heavy chain partner, expressed and tested for MPER recognition. 12 heavy chain variants and 27 light chain variants were found to bind MPER, and several of the reconstituted antibodies neutralized a panel of HIV-1 isolates better than the original 10e8. Phylogenetic analyses identified clonally-related sequences, and at least three subgroups of 10e8-like heavy chains were observed. 10e8 light chains meanwhile also showed a few subgroups, though with less sequence variation.

Computer-aided antibody design.

Recent clinical trials using antibodies with low toxicity and high efficiency have raised expectations for the development of next-generation protein therapeutics. However, the process of obtaining therapeutic antibodies remains time consuming and empirical. This review summarizes recent progresses in the field of computer-aided antibody development mainly focusing on antibody modeling, which is divided essentially into two parts: (i) modeling the antigen-binding site, also called the complementarity determining regions (CDRs), and (ii) predicting the relative orientations of the variable heavy (VH) and light (VL) chains. Among the six CDR loops, the greatest challenge is predicting the conformation of CDR-H3, which is the most important in antigen recognition. Further computational methods could be used in drug development based on crystal structures or homology models, including antibody–antigen dockings and energy calculations with approximate potential functions. These methods should guide experimental studies to improve the affinities and physicochemical properties of antibodies. Finally, several successful examples of in silico structure-based antibody designs are reviewed. We also briefly review structure-based antigen or immunogen design, with application to rational vaccine development.

Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen

Phage display technology has been utilized for identification of specific binding molecules to an antigenic target thereby enabling the rapid generation and selection of high affinity, fully human antibodies directed towards disease target appropriate for antibody therapy. In the present study, single chain Fv antibody fragment (scFv) to hepatitis A virus (HAV) was selected from phage displayed antibody library constructed from peripheral blood lymphocytes (PBLs) of a vaccinated donor. The variable heavy (VH) and light chains (VL) were amplified using cDNA as template, assembled into scFv using splicing by overlap extension PCR (SOE PCR) and cloned into phagemid vector as a fusion for display of scFv on bacteriophage. The phage displaying antibody fragments were subjected to three rounds of panning with HAV antigen on solid phase. High affinity antibodies reactive to hepatitis A virus were identified by phage ELISA and cloned into a bacterial expression vector pET20b. The scFv was purified by immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The binding activity and specificity of the scFv was established by its non-reactivity towards other human viral antigens as determined by ELISA and immunoblot analysis. The scFv was further used in the development of an in-house IC-ELISA format in combination with a commercially available mouse monoclonal antibody for the quantification of hepatitis A virus antigen in human vaccine preparations. The adjusted r2 values obtained by subjecting the values obtained by quantification of the NIBSC standards using the commercial and the in-house ELISA kits by regression analysis were 0.99 and 0.95. 39 vaccine samples were subjected to quantification using both the kits. Regressional statistical analysis through the origin of the samples indicated International Unit (IU) values of 0.0416x and 0.0419x, respectively for the commercial and in-house kit respectively.

Construction of Human Nonimmune Library and Selection of scFvs Against IL-33

Interleukin (IL) 33 plays very important roles in inflammatory and allergic diseases. To select human single-chain Fv fragments (scFvs) against IL-33, a nonimmune phage library system was constructed. The full-length cDNA library was synthesized for amplification of the variable heavy chain (V(H)) and variable light chain (V(L)). By overlapping extension PCR for splicing V(H) and V(L), the full-length scFv library DNA were amplified and then transformed into Escherichia coli TG1. The scFv library was constructed successfully which contained 2.5×10(8) independent clones with full-length scFv inserts. The results of fingerprint maps of the scFvs by BstN I and DNA sequencing from the library at random proved that the library was diverse. The human IL-33 was amplified, expressed, and purified. The purified IL-33 with bioactivity was biotinylated and used as antigen for selection of scFv library by phage display. After three rounds of affinity selection, about 30 % of clones have specific binding activity with IL-33. Five of those with good binding activity were transformed into E. coli strain HB2151 for soluble expression. The selected scFvs were further identified by western blot and sequencing. Those selected scFvs could be used for further research of their effect on inflammatory and allergic diseases such as asthma by blockade of IL-33.

Rituximab Therapy Leads to Reduced Imprints of Receptor Revision in Immunoglobulin κ and λ Light Chains

Transient B cell depletion by rituximab (RTX) has become a specific treatment of rheumatoid arthritis (RA). Although phenotypic repopulation kinetics of B cell subsets are well documented, precise molecular analyses of the reconstituting immunoglobulin (Ig) genes encoding the B cell receptor in RA are sparse. A total of 708 individual CD19+CD27+ (memory) and CD19+CD27- (naive) B cells from 2 patients with RA were analyzed at baseline and 7 months after RTX at B cell repopulation. Ig light chain variable kappa (Vκ) and lambda (Vλ) light chain gene rearrangements were amplified, sequenced, and analyzed with a focus on receptor revision. The naive as well as the memory repertoire repopulated polyclonally with diverse use of variable light chain gene families and minigenes. During the reconstitution phase, B cells used significantly fewer Jκ distal Vκ genes (p = 0.0006), with a higher frequency of somatic hypermutation of rearrangements employing Jκ5 compared to baseline in memory B cells. The use of Vλ rearrangements in regenerating B cells was also biased toward use of Vλ genes of the proximal cassette. In general, reemerging CD27+ Ig light chain genes were substantially more highly mutated than before RTX therapy (p < 0.0001, baseline vs during reconstitution). Our data indicate that RTX therapy leads to generation of distinct Vκ/Jκ and Vλ/Jλ gene repertoires consistent with replenishment of antigen-experienced B cells by germinal centers. At baseline, the imprints of receptor revision appeared to be more striking, which indicates that receptor revision is active in patients with RA and can be reduced by RTX.

A novel disulfide-stabilized single-chain variable antibody fragment against rabies virus G protein with enhanced in vivo neutralizing potency

Rabies is a fatal infectious disease requiring efficient protection provided by post-exposure prophylaxis (PEP) with rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv) is a small engineered antigen binding protein derived from antibody variable heavy (VH) and light (VL) chains. This novel antibody format may potentially replace the current application of RIG to detect and neutralize rabies virus (RV). However, the broad use of scFvs is confined by their generally low stability. In this study, a scFv (FV57) was constructed based on the monoclonal antibody, MAB57, against RV. To enhance its stability and neutralizing potency, a disulfide-stabilized scFv, ds-FV57, was also derived by introduction of cysteines at VH44 and VL100. Furthermore, the cysteine at VL85 of ds-FV57 was mutated to serine to construct ds-FV57VL85Ser in order to avoid potential mis-formed disulfide bonds which would alter the affinity of the scFv. The stability and activity of all three proteins expressed in Escherichia coli were evaluated. All of the constructed scFvs could provide efficient protection against RV infection both in vivo and in vitro. However, the stability of ds-FV57VL85Ser was notably improved, and its in vitro neutralizing potency against RV infection was enhanced. Our findings from these stabilization modifications support the feasibility of developing scFvs for PEP treatment of rabies.

Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry

BackgroundThere has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL) with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes.MethodsCurrently anti-fragment antigen binding (Fab) conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig)-binding protein that binds to the variable light chains (kappa chain) of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv) and Fab fragments.ResultsWe used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2), two murine antibody derived CARs (anti-CSPG4, and anti-CD19), and two humanized mouse antibody based CARs (anti-ERBB2, and anti-PSCA). Transduced cells were stained first with biotin labeled protein L followed by phycoerythrin (PE)-conjugated streptavidin (SA) and analyzed by flow cytometry. For comparison, cells were stained in parallel with biotin conjugated goat-anti-mouse Fab or CAR specific fusion proteins. Using protein L, all CAR transduced lymphocytes exhibited specific staining pattern ranging from 40 to 80% of positive cells (compared to untransduced cells) and staining was comparable to the pattern observed with anti-Fab antibodies.ConclusionOur data demonstrate the feasibility of employing Protein L as a general reagent for the detection of CAR expression on transduced lymphocytes by flow cytometry.

Isolation and characterization of human polyreactive pneumococcal polysaccharide antibodies

Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique KD values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar KD values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype.

Exploring the Effect of Mutations on the Conformational Landscape of Amyloidogenic Antibody Fragments

Antibodies are composed of heavy chains and light chains. In Light Chain Amyloidosis (AL), antibody-secreting cells export only the light chains. These light chains are prone to misfold, forming amyloid fibers, which get deposited in various organs, leading to organ disfunction and patient death. The transition from the native state to the amyloid fiber requires partial or total denaturation. The structures of monomeric precursors to the oligomeric nuclei, or of those assembled at the ends of the fibers, are unknown. There is an inverse correlation between the stability of the native state and the speed of fiber formation, suggesting that lower stability allows the population of “excited states” of the native ensemble, some of which could be amyloid fiber precursors. Starting from the crystal structure of a variable light chain domain belonging to class 6a (one of the most common in clinical cases of AL), we generated point mutants that eliminate charges (R24G and D52A) or a proline (P7S). These mutants destabilize the native state, and speed up fiber formation (for R24G and P7S). We carried out MD simulations at three temperatures (298, 398 and 498K), to explore the effect of these mutations on the conformational landscape. We found many metastable unfolding intermediates, which have eluded experimental detection because their fluorescence is indistinguishable from that of the native state. A common early unfolding intermediate exposes strand D, which has a high potential for fiber formation according to ZipperDB. Those variants with a higher speed of fiber formation expose this area with greater frequency.We are thankful for computer resources at: Centro Nacional de Supercomputo, IPICyT; Kan Balam, UNAM; Sputnik, IBT-UNAM; Orion, FC and CIQ-UAEM.Funding: CONACYT 102182 and 133294.

Analyzing influence on the conformation of single-chain antibody with the differential length of linkers

To analyze the differential length of linkers of peptides on the conformation of single-chain antibody, and seek the length of linkers with which the conformation is the most similar to the wild type antibody. The similarity between the wild type antibody model and the single-chain antibody model was calculated by the comparison method of spherical shell layer; after comparing with conclusions of other algorithms, the length of the linker (connecting peptide) with which does little influence on the conformation of heavy chain variable (VH) and light chain variable (VL) of single-chain antibody was obtained. The similarity value was got when the number of layers range from 2 to 2000 and the length of the linker range from 0 to 9 by the method of layered spherical coordinate-based algorithm for similarity, and the average values ​​were 0.84974, 0.84101, 0.84970, 0.84532, 0.84285, 0.84235, 0.84077, 0.83857, 0.84237 and 0.83905 when n is equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9. The VH and VL of the single-chain antibody gain little influence on the conformation when n = 0 (n is even) and n = 3 (n is odd). When the length of G4S linker (n value) is 3, the influence on the conformation of single-chain antibody is least. Key word: Single-chain antibody, spherical coordinates, similarity, conformation and linker.

Abstract B189: Impact of site of conjugation on efficacy and pharmacokinetics of anti-NaPi3b antibody drug conjugate variants.

Abstract Introduction: Optimization of antibody-drug conjugates (ADCs) with therapeutic potential involves several critical parameters, including selection of the antibody, linker, cytotoxic drug, and drug-to-antibody ratio (DAR). To minimize the heterogeneity from conjugation, the antibody can be engineered at different conjugation sites to achieve a defined DAR at specific sites. Anti-NaPi3b-vc-MMAE (anti-NaPi3b-vc-E) is an ADC targeting sodium dependent phosphate transporter expressed in lung and ovarian cancers. Construction and production of the THIOMAB variant of antibody was reported previously. Briefly, a cysteine residue was engineered at Ala118 position of heavy chain (HC) or Val205 position of light chain (LC) to produce its THIOMAB HC and LC variants, respectively. The MMAE conjugates were produced with a drug to antibody ratio (DAR) of two. The purpose of this study was to evaluate the site of conjugation on ADC efficacy and antibody pharmacokinetics. Methods: The anti-tumor activity of anti-Napi3b ADC variants was evaluated in a mouse xenograft model of human ovarian cancer OVCAR3-X2.1 after single dose administration in a range of doses. The unconjugated antibodies including anti-NaPi3b (control) and anti-NaPi3b (HC and LC) and their respective ADC variants were administered intravenously to mice and rats; and the pharmacokinetic (PK) parameters measured by a total antibody assay were estimated by a two-compartmental model. Results: Based on comparisons of tumor growth inhibition, both LC and HC anti-Napi3b-vc-E showed clear dose-dependent inhibitory activity when given at 3 to 12 mg/kg versus the vehicle group. Non-NaPi3b-binding ADCs did not significantly affect tumor growth, indicating targeted anti-tumor activity. The activities of LC and HC variants were similar. Dose proportional pharmacokinetics of total antibody following administration of anti-NaPi3b-vc-E (HC and LC) were observed in mice and rats over the dose range tested. Total antibody clearance of the three unconjugated antibodies (control, thio HC, and thio LC) was comparable. Conjugation with MMAE only modestly increased the total antibody clearance in both LC and HC conjugates when compared to unconjugated antibodies. Overall, PK behavior of the HC and LC variants were similar. Conclusion: The results demonstrated that ADC variants with different sites of conjugation exhibited similar anti-tumor activities, which provided additional choice of conjugation sites for individual antibody. Cysteine mutation in either HC or LC of the unconjugated antibodies did not affect the PK when compared to the control. In addition, conjugation did not significantly change the overall clearance of the antibody, and the site of MMAE conjugations did not have a significant impact on the PK. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B189.

Antibodies 2.0

Antibodies 2.0

Isolation of a human anti-HIV gp41 membrane proximal region neutralizing antibody by antigen-specific single B cell sorting.

Broadly neutralizing antibodies are not commonly produced in HIV-1 infected individuals nor by experimental HIV-1 vaccines. When these antibodies do occur, it is important to be able to isolate and characterize them to provide clues for vaccine design. CAP206 is a South African subtype C HIV-1-infected individual previously shown to have broadly neutralizing plasma antibodies targeting the envelope gp41 distal membrane proximal external region (MPER). We have now used a fluoresceinated peptide tetramer antigen with specific cell sorting to isolate a human neutralizing monoclonal antibody (mAb) against the HIV-1 envelope gp41 MPER. The isolated recombinant mAb, CAP206-CH12, utilized a portion of the distal MPER (HXB2 amino acid residues, 673–680) and neutralized a subset of HIV-1 pseudoviruses sensitive to CAP206 plasma antibodies. Interestingly, this mAb was polyreactive and used the same germ-line variable heavy (VH1-69) and variable kappa light chain (VK3-20) gene families as the prototype broadly neutralizing anti-MPER mAb, 4E10 (residues 672–680). These data indicate that there are multiple immunogenic targets in the C-terminus of the MPER of HIV-1 gp41 envelope and suggests that gp41 neutralizing epitopes may interact with a restricted set of naive B cells during HIV-1 infection.

An anti‐hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.

Establishing a Link Between Amino Acid Sequences and Self-Associating and Viscoelastic Behavior of Two Closely Related Monoclonal Antibodies

To investigate the underlying cause for the observed differences in self-associating and viscoelastic behavior between two monoclonal antibodies, MAb1, and MAb2. Several mutants were designed by swapping charged residues in MAb1 with those present in MAb2 at their respective positions and vice versa. Rheological analysis was done at low and high shear rates. Dynamic light scattering quantified intermolecular interactions in dilute solutions; sedimentation equilibrium analysis determined the corrected weight average molecular weight (M (wc)) to assess the self-associating behavior in high concentration. The molecular charge was estimated from electrophoretic mobility measurements. Replacing the charged residues in the CDR of MAb1 resulted in a lower M (wc) and solution viscosity. The corresponding changes in either just the variable light (VL) or variable heavy (VH) chain showed only a partial decrease in viscosity, whereas changes in both VL and VH chains resulted in a dramatic reduction in viscosity. The converse case where the VL and VH chains of MAb2 were made to look like MAb1 did not self-associate or show increased viscosity. Exposed charged residues in the CDR of MAb1 are critical in determining the self-associating and highly viscous behavior observed at high concentrations.

Generation of recombinant guinea pig antibody fragments to the human GABAC receptor

To generate monoclonal antibodies to the human ρ1 GABAC receptor, a ligand-gated chloride ion channel that is activated by the neurotransmitter γ-aminobutyric acid (GABA), we recovered the immunoglobulin variable heavy chain (VH) and light chain (VL) regions of a guinea pig immunized with a 14-mer peptide segment of the N-terminal extracellular domain of the ρ1 subunit. Oligonucleotide primers were designed and used to amplify the VH and VL regions of guinea pig RNA by the reverse transcriptase polymerase chain reaction. The amplified and cloned VH and VL regions were transferred together into a phagemid vector, yielding a library of 5×106 members, which displayed chimeric fragments of antigen binding (Fabs) with guinea pig variable and human constant regions fused to protein III of M13 bacteriophage. Through affinity selection of this phage-display library with the biotinylated 14-mer peptide segment of GABAC, we isolated four different antibody fragments that bound specifically to the immunogenic peptide. Phage particles displaying two of these antibodies, but not negative controls, bound selectively to the surface of neuroblastoma cells expressing the ρ1 GABAC receptor. Such antibody fragments will be useful in future studies involving targeting of specific neural tissues that express the GABAC receptor.