Site-directed in vitro immunization leads to a complete human monoclonal IgG4 lambda that binds specifically to the CDR2 region of CTLA-4 (CD152) without interfering the engagement of natural ligands

Abstract

Background: The ability to acquire fully human monoclonal antibodies (mAbs) with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG) molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags) such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4) because proper hosts are lacking.

Results: Here we developed an optimized protocol for site-directed in vitro immunizing peripheral blood mononuclear cells (PBMC) by using a selected epitope of human CD152, an essential receptor involved in down-regulation of T cell activation. The resultant stable trioma cell lines constantly produce anti-CD152 mAb (gamma4lambdahuCD152), which contains variable (V) regions of the heavy chain and the light chain derived from the VH3 and V lambda human germline genes, respectively, and yet displays an unusual IgG4 isotype. Interestingly, gamma4lambdahuCD152 has a basic pI not commonly found in myeloid monoclonal IgG4 lambdas as revealed by the isoelectric focusing (IEF) analysis. Furthermore, gamma4lambdahuCD152 binds specifically, with nanomolar affinity, to an extracellular constituency encompassing the putative second complementarity determining region (CDR2) of CD152, whereby it can react to activated CD3+ cells.

Conclusion: In a context of specific cell depletion and conditioned medium,in vitro induction of human Abs against a conserved self Ag was successfully acquired and a relatively basic mAb, gamma4lambdahuCD152, with high affinity to CDR2 of CD152 was thus obtained. Application of such a human IgG4 lambda mAb with designated CDR2 specificity may impact upon and prefer for CD152 labeling both in situ and ex situ, as it does not affect the binding of endogenous B7 ligands and can localize into the confined immunological synapse which may otherwise prevent the access of whole IgG1 molecules.

Figure 1

Anti-CD152 derived from site-directed in vitro immunization exhibits specific binding towards human CD152 expressed both as a recombinant protein as well as a cell surface receptor. Panel A summarizes specific responses of PBMC's from five subjects receiving primary Ag stimulation alone (Nil) or from PBMC's treated with the indicated regimen. Bars indicate the median value of all subjects analyzed. Filled triangles represent specific frequencies of individual PBMC's. Original data can be retrieved from Additional File 1. Panel B illustrates a representative ELISA reactivity profile of culture supernatant. Diluted supernatants were tested in duplicate with 100 μL added to each well. Deviation between duplicate was less than 10% for any reported value. Panel C represents immunofluorescent staining of mitogen-activated CD3⁺ T cells (right thick peaks). PBMCs were isolated from normal blood donors and stimulated with indicated mitogens for 72 h to induce CD152 expression. Expression of mAb-recognized epitope was verified in gated CD3 population by flow cytometry. Data represent one experiment from three different normal subjects. Appropriate isotype controls were used for all Abs. The basal fluorescence obtained with the use of isotype control (IgG4λ myeloma protein) is indicated by the left thin peaks.

Figure 2

Immunoblot screening of peptide arrays denotes the mAb specificity against the Met 55-cored CDR2-like region of human CD152. 1 μg/mL of purified mAb was used to determine the binding epitope. Sequences of EYASPGKATEVRVTV, KVELMYPPPYYLGIG and QYIKANSKFIGITEL indicate CDR1-encompassing region, CDR3-encompassing region and T cell epitope (italic) used for site-directed immunization, respectively.

Figure 3

Immunological and biochemical natures of the mAb. Panel A shows isotyping and subtyping results by immobilizing anti-human Igs as indicated. The binding profile of the mAb was subsequently revealed by biotinylated CD152-muIg and avidin-peroxidase conjugates. Results indicate that the present mAb belongs to a type of IgG4λ. Panel B indicates the isoelectric point of the monoclonal IgG4λ anti-CD152 (lane 4 and 8), resolved by isoelectric focusing. Monoclonal human myeloma IgG1λ(lane 2 and 6) and IgG4λ (lane 3 and 7) were run in parallel for comparison. The electrophoretic patterns were visualized by either Coomassie brilliant blue staining (lane 1–5) or immunoblot (lane 6–8) with anti-human IgG conjugated with peroxidase and FAST™ DAB. The calculated isoelectric points for human IgG1λ, IgG4λ and anti-CD152 mAb to be approximately in the range of 7.92–8.79, 5.76–6.52 and 7.87–8.41, respectively, based on the calibration against the linear regression of standard protein markers. Panel C outlines the affinity determination by IAsys. Surface plasmon resonance obtained at 25°C for increasing concentrations of anti-CD152 mAb on purified, unlabeled CD152-muIg. The straight line in the inset was obtained from the k_obs plot versus ligated Ab concentration and yielded a k_diss (the intercept) of 16.81 and a k_ass value (the gradient) of 4.20 × 10⁹. Therefore produced a Kd (k_diss/k_ass) of 4 × 10^-9 M.

Figure 4

Partial deduced protein sequences of the mAb. Panel A represents the alignments of VH to known human VH sequences of the highest homology scoring. Panel B represents the alignments of VL to known human VL sequences of the highest homology scoring. FR, framework region; CDR, complementarity-determining region. Asterisks indicate amino acid identity to germline. Sequences are available from GenBank under accession numbers AY847516 (VH); AY847517 (VL); AB019439 (VH3–30 and VH3–33); BAC01778; S78058 and CAA38313. Homology searches were accomplished over the www using the program BLAST (Basic Local Alignment Search Tool) from NCBI (National Center for Biotechnology Information; Washington, DC.). A 12-aa CDRH3 sequenced Ala-His-Gly-Asp-Tyr-Gly-Arg-Asp-Gly-Met-Asp-Val was noted.

Figure 5

Binding of γ4λhuCD152 and CD80/CD86 agonists to human CD152 are not mutually exclusive. Panel A indicates the ligand competition assays that test the ability of monoclonal γ4λhuCD152 (solid line) and BNI3 (dashed line) anti-CD152 to compete for the CD80/CD86 and CD152 interactions. Biotinylated CD80-muIg or CD86-muIg plus indicated increasing concentrations of the mAbs (10^-3-10 μg/mL) were incubated in microtiter wells coated with purified CD152-muIg. Bound CD80/CD86 was detected with avidin-peroxidase conjugate and a peroxidase substrate. The data shown are representative of three experiments. Panel B illustrates the determination of in situ CD152 expression before and after PHA/PMA stimulation. Paraformaldehyde-fixed cells were first labeled with PE-anti-CD3 and BNI3 or γ4λhuCD152, and then incubated with FITC-conjugated anti-mouse IgG2a or anti-human IgGs, respectively. The percentage of each stained population after stimulation is denoted.