Antibody CB4-1 Research Articles

Thermodynamic Analysis of the Binding of 2F5 (Fab and Immunoglobulin G Forms) to Its gp41 Epitope Reveals a Strong Influence of the Immunoglobulin Fc Region on Affinity

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.

Isothermal Titration Calorimetry Reveals Differential Binding Thermodynamics of Variable Region-identical Antibodies Differing in Constant Region for a Univalent Ligand

The classical view of immunoglobulin molecules posits two functional domains defined by the variable (V) and constant (C) regions, which are responsible for antigen binding and antibody effector functions, respectively. These two domains are thought to function independently. However, several lines of evidence strongly suggest that C region domains can affect the specificity and affinity of an antibody for its antigen (Ag), independent of avidity-type effects. In this study, we used isothermal titration calorimetry to investigate the thermodynamic properties of the interactions of four V region-identical monoclonal antibodies with a univalent peptide antigen. Comparison of the binding of IgG1, IgG2a, IgG2b, and IgG3 with a 12-mer peptide mimetic of Cryptococcus neoformans polysaccharide revealed a stoichiometry of 1.9-2.0 with significant differences in thermodynamic binding parameters. Binding of this peptide to the antibodies was dominated by favorable entropy. The interaction of these antibodies with biotinylated peptides manifested greater enthalpy than for native peptides indicating that biotin labeling affected the types of Ag-Ab complexes formed. Our results provide unambiguous thermodynamic evidence for the notion that the C region can affect the interaction of the V region with an Ag.

Proteochemometric modelling of antibody-antigen interactions using SPOT synthesised peptide arrays

Proteochemometrics is a technology for the study of molecular recognition based on chemometric techniques. Here we applied it to analyse the amino acids and amino acid physico-chemical properties that are involved in antibodies' recognition of peptide antigens. To this end, we used a study system comprised by a diverse single chain antibody library derived from the murine mAb anti-p24 (HIV-1) antibody CB4-1, evaluated on peptide arrays manufactured by SPOT synthesis. The binding pattern obtained was correlated to physico-chemical descriptors (z-scales) of antibodies and peptides amino acids using partial least-squares projections to latent structures. Cross terms derived from antibody and antigen descriptors were included, which substantially improved the proteochemometric model. The final model was statistically highly satisfactory with a correlation coefficient R(2) = 0.73 and predictive ability Q(2) = 0.68. The physico-chemical properties of each interacting amino acid residue of both the peptides and the antibodies being essential for the antigen-antibody recognition could be retrieved from the model. The study shows for the first time the feasibility of using proteochemometrics to analyse the molecular recognition of antigens by antibodies.

QSAR of multiple mutated antibodies

The aim of this study was to develop predictive quantitative structure-activity relationship (QSAR) modeling for antibody-peptide interactions. A small single chain antibody library was designed and manufactured around the murine anti-p24 (HIV-1) monoclonal antibody CB4-1 by use of statistical molecular design (SMD) principles and site directed mutagenesis, and its affinity for a p24 derived antigen was determined by fluorescence polarization. A satisfactory QSAR model (Q(2) = 0.74, R(2) = 0.88) was derived by correlating the affinity data to physicochemical property scales of the amino acids varied in the library. The model explains most of the antibody-antigen interactions of the studied set, and provides insights into the molecular mechanism involved in antigen binding.

SPOT synthesis: Reliability of array-based measurement of peptide binding affinity

Peptide arrays prepared by the SPOT synthesis technology have emerged as a proteomic tool to study molecular recognition and identify biologically active peptides. However, it was previously not clear how accurately signal intensities obtained by probing peptide arrays for protein binding really reflect the dissociation constants of the protein–peptide complexes. Using the monoclonal antibody CB4-1 as a model system, we systematically compared dissociation constants of antibody–peptide complexes with signal intensities obtained using the SPOT technology. By analyzing a set of peptides possessing different affinities to the antibody, we determined the strengths of the SPOT screening method. The accuracy of the measured results was improved by taking regional trends in the membrane surface into account. A model based on the mass action law compares well with the experimental results. Interestingly, the applied concentrations of the binding partners do not directly correspond to the effective concentrations in the assay. We show that the SPOT technology is an accurate method for assigning the spots’ measured signal intensities to three different binding affinity classes. The dissociation constants of the intermediate region were found to be between p K dis = 5 and p K dis = 7. Altering the experimental parameters causes a directed change of this region.

Linker peptide and affinity tag for detection and purification of single-chain Fv fragments

The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1. This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution. The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography. The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL). Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced. All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm. Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs.

Interaction of epitope-related and -unrelated peptides with anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab fragment.

The binding of four epitope-related peptides and three library-derived, epitope-unrelated peptides of different lengths (10-14 amino acids) and sequence by anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab fragment was studied by isothermal titration calorimetry. The binding constants K(A) at 25 degrees C vary between 5.1 x 10(7) M (-1) for the strongest and 1.4 x 10(5) M (-1) for the weakest binder. For each of the peptides complex formation is enthalpically driven and connected with unfavorable entropic contributions; however, the ratio of enthalpy and entropy contributions to deltaG(0) differs markedly for the individual peptides. A plot of -deltaH(0) vs -TdeltaS(0) shows a linear correlation of the data for a wide variety of experimental conditions as expected for a process with deltaC(p) much larger than deltaS(0). The dissimilarity of deltaC(p) and deltaS(0) also explains why deltaH(0) and TdeltaS(0) show similar temperature dependences resulting in relatively small changes of deltaG(0) with temperature. The heat capacity changes deltaC(p) upon antibody-peptide complex formation determined for three selected peptides vary only in a small range, indicating basic thermodynamic similarity despite different key residues interacting in the complexes. Furthermore, the comparison of van't Hoff and calorimetric enthalpies point to a non-two-state binding mechanism. Protonation effects were excluded by measurements in buffers of different ionization enthalpies. Differences in the solution conformation of the peptides as demonstrated by circular dichroic measurements do not explain different binding affinities of the peptides; specifically a high helix content in solution is not essential for high binding affinity despite the helical epitope conformation in the crystal structure of p24.

Identification of distinct antibody epitopes and mimotopes from a peptide array of 5520 randomly generated sequences

We used a relatively small library of 5520 randomly generated single 15-mer peptides prepared by SPOT synthesis as an array of 28.5×19.0 cm to identify epitopes for three distinct monoclonal antibodies, namely anti-p24 (human immunodeficiency virus (HIV)-1) monoclonal anibody (mab) CB4-1, anti-interleukin-10 (IL-10) mab CB/RS/13, and anti-transforming growth factor α (TGFα) mab Tab2. Initially identified peptide ligands mostly had very low affinities for the antibodies with dissociation constants around 10 −4 M. Subsequent identification of residues critical for the antibody interactions involved complete L-amino acid substitutional analyses. Several substitutions resulted in analogs with dissociation constants in the low micromolar and high nanomolar range. Specifically binding peptides with key residue patterns matching the wild-type epitopes were identified for all three antibodies. In addition, for antibody CB4-1 mimotopes that showed no homology to the known epitope were selected. Our results suggest that a very limited library diversity, although far from covering the entire sequence repertoire, can suffice to rapidly and economically select peptidic antibody epitopes and mimotopes.

Evolutionary transition pathways for changing peptide ligand specificity and structure.

We identified evolutionary pathways for the inter- conversion of three sequentially and structurally unrelated peptides, GATPEDLNQKL, GLYEWGGARI and FDKEWNLIEQN, binding to the same site of the hypervariable region of the anti-p24 (HIV-1) monoclonal antibody CB4-1. Conversion of these peptides into each other could be achieved in nine or 10 single amino acid substitution steps without loss of antibody binding. Such pathways were identified by analyzing all 7 620 480 pathways connecting 2560 different peptides, and testing them for CB4-1 binding. The binding modes of intermediate peptides of selected optimal pathways were characterized using complete sets of substitution analogs, revealing that a number of sequential substitutions accumulated without changing the pattern of key interacting residues. At a distinct step, however, one single amino acid exchange induces a sudden change in the binding mode, indicating a flip in specificity and conformation. Our data represent a model of how different specificities, structures and functions might evolve in protein-protein recognition.

Conformation, pH-induced conformational changes, and thermal unfolding of anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab and Fc fragments

Conformation, acid-induced conformational changes and stability of the murine monoclonal antibody CB4-1 directed against the human immunodeficiency virus type 1 capsid protein p24, and its Fab and Fc fragments, were analysed by circular dichroism (CD), fluorescence, and differential scanning calorimetry (DSC) measurements. CD spectra show the characteristics expected for β-proteins. Lowering the pH to 3.5 reduces the stability, but does not change the conformation. Between pH 3.5 and 2.0 conformational changes and the formation of new structures are indicated. Deconvolution of the bimodal DSC curves of CB4-1 reveals five ‘two-state’ transitions at pH 7.5. At pH 5 and below, only four transitions are found. Half transition temperatures T m and molar enthalpy changes Δ H m gradually decrease at pH 4 and 3.4. At pH 2.1, two low-temperature ( T m=36.9 and 44.1°C) and two high-temperature ( T m=74.6 and 76.8°C) transitions are identified. The Fab and Fc fragments behave similarly. Deconvolution of their monophasic DSC curves yields two ‘two-state’ transitions for each fragment. T m and Δ H m values gradually decrease at pH 4.0 and 3.4; and at pH 2.1 and 2.8 for Fab and Fc, respectively, one of the transitions is found at high temperature ( T m=67.2 and 75.9°C for Fab and Fc, respectively).

Stepwise transformation of a cholera toxin and a p24 (HIV-1) epitope into D-peptide analogs.

We have transformed two peptide epitopes into D-peptide analogs: VPGSQHIDS derived from cholera toxin recognized by the antibody TE33, and GATPQDLNTML from the HIV-1 capsid protein p24 recognized by the antibody CB4-1. The transformation process was performed by stepwise substitution of each single epitope position by all 19 D-amino acids and glycine followed by antibody binding studies and selection of one D-analog for further transformation. Thus, each transformation step introduced one novel D-position into the peptide. For both epitopes complete D-analogs were obtained. The cholera toxin-derived variant dwGsqhydp binds to the antibody TE33 with higher affinity than its original epitope, whereas in the case of the p24-derived analog saGdwwGkssl lower affinity was detected. Both D-peptides are completely stable in serum for several days. Antibody interaction models for both D-molecules were generated by computer-assisted modelling based on the crystal structures of the starting complexes. Compared with the L-peptides, the binding conformation of dwGsqhydp is very similar, whereas saGdwwGkssl displays a completely different interaction mode.

Molecular Basis for the Binding Promiscuity of an Anti-p24 (HIV-1) Monoclonal Antibody

Multiple binding capabilities utilized by specific protein-to-protein interactions in molecular recognition events are being documented increasingly but remain poorly understood at the molecular level. We identified five unrelated peptides that compete with each other for binding to the paratope region of the monoclonal anti-p24 (HIV-1) antibody CB4-1 by using a synthetic positional scanning combinatorial library XXXX[B 1,B 2, B 3,X 1,X 2,X 3]XXXX (14 mers; 68,590 peptide mixtures in total) prepared by spot synthesis. Complete sets of substitution analogs of the five peptides revealed key interacting residues, information that led to the construction of binding supertopes derived from each peptide. These supertope sequences were identified in hundreds of heterologous proteins, and those proteins that could be obtained were shown to bind CB4-1. Implications of these findings for immune escape mechanisms and autoimmunity are discussed.

P-A5-08 Effects of maleylation of HIV-1 capsid protein rp24 on antibody CB4-1 binding and conformation: Welfle H, 1 Ehrhard B, 2 Misselwitz R, 1 Welfle K, 3 Hausdorf G, 1 Glaser RW, 2 Schneider-Mergener J. 2 1Inst. Biochem., 2Inst. Med. Immunol., 3Inst. Biol., Humboldt-Univ. Berlin (D)

P-A5-08 Effects of maleylation of HIV-1 capsid protein rp24 on antibody CB4-1 binding and conformation: Welfle H, 1 Ehrhard B, 2 Misselwitz R, 1 Welfle K, 3 Hausdorf G, 1 Glaser RW, 2 Schneider-Mergener J. 2 1Inst. Biochem., 2Inst. Med. Immunol., 3Inst. Biol., Humboldt-Univ. Berlin (D)

Chemical modification of recombinant HIV-1 capsid protein p24 leads to the release of a hidden epitope prior to changes of the overall folding of the protein.

It was found that the affinity of a monoclonal antibody directed against a recombinantly expressed HIV-1 capsid protein p24 (rp24) strongly increased after chemical modification of the Iysine residues of rp24 with different amounts of maleic anhydride. The extent and the sites of modification were analyzed by MALDI-TOF mass spectrometry. Unmodified rp24 and the differently modified rp24 samples were tested for binding the murine monoclonal antibody CB4-1 which recognizes the epitope GATPQDLNTML comprising residues 46-56 of rp24. An increase in the number of modified lysine residues led to enhanced binding affinity of CB4-1. Most pronounced effects were observed after substitution of the first amino groups: an average number of three modified residues per protein molecule increases the binding affinity by a factor of 23, but the substitution of the remaining nine residues increases the binding affinity only by a factor of 11. Fully modified rp24 variant proteins were bound by CB4-1 with Kd values comparable to that of the peptide epitope. Conformation and stability of the unmodified rp24, highly (rp24F, 9 residues; rp24G, 11 residues) modified, and fully modified protein (rp24I, 11 lysine residues and N-terminus) were analyzed by circular dichroism (CD) and fluorescence spectroscopy under different solvent conditions. Little difference in conformation and unfolding behavior was observed between the unmodified and highly modified rp24, which differ drastically in the antibody binding behavior. The fully modified sample, however, displayed a significant decrease in alpha-helical content. Thus, the epitope seems to be hidden (cryptotope) in the unmodified rp24 in a low-affinity binding conformation and becomes displayed at low levels of chemical modification which obviously induce subtle structural changes prior to changes of the overall folding observable by spectroscopic means.