The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.

Antigen-binding fragments (Fab) of mouse monoclonal antibodies to human interleukin-2 were obtained in preparative quantities by a modified procedure. These Fab-fragments were shown to be homogeneous according to the isoelectric focusing method. Various monocrystals of these free Fab-fragments and their complexes with the antigenic peptide corresponding to the 59-72 sequence of interleukin-2 were obtained. These were shown to be suitable for X-ray and were preliminarily studied by X-ray.

Some characteristics of the gel-entrapped hybridoma cells such as cell size, membrane immunoglobulin expression, and DNA content were studied by now cytometry and compared to those of the free-suspended culture. The entrapment of the cells in gel beads was found to result in the increase of the cell size which correlated to the DNA content and membrane immunoglobulin expression enhancement. The cell cycle analyses of the entrapped cell and the free-suspended cells, based on DNA histograms, were quite different. Despite the changes of the cell structure, the entrapped cells retained high viability, and produced monoclonal antibodies during a cultivation period.

Hybridomas producing monoclonal anti-idiotypic antibodies (anti-id MAbs) to N-acetylglucosaminyl-beta 1-4-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP) were developed. Three clones of hybridomas demonstrated the properties characteristic for the Ab2 beta type of anti-id antibody: they bound to Fab-fragments of high-affinity MAb to GMDP; dose-dependent inhibition of this binding by GMDP was observed; immunization of mice with these MAbs resulted in production of GMDP-specific antibodies. When these antibodies were used to stain blots from SDS-PAGE of macrophage lysate, the same receptor proteins were specifically stained as upon staining with 125I-labelled GMDP derivative.

Hybridoma E6/1.2 was produced by fusion of splenocytes from mice immunized with N-acetylglucosaminyl-(beta1-4)-N-acetylmuramyl-alanyl-D-isoglutamine (GMDP), conjugated to methylated BSA, with SP2/0 myeloma cells. GMDP-specific monoclonal antibody was IgGl subtype and had affinity constant 2.10(9) M-1. According to competitive ELISA, the presence of the intact disaccharide fragment and the alanyl residue was critical for the GMDP-antibody interaction.

Proton signals for nine synthetic peptide fragments of human interleukin-2 (region 59-78) were assigned for aqueous solutions both of pure peptides and their mixtures with LNKB-2 monoclonal antibody. The nonspecific magnetization transfer (NOE) between the antibody or its Fab-fragment and the peptides was studied upon large excess of free peptide over bound peptide. NOE spectra using modified pulse sequence, enabling to eliminate broad signals and achieve higher (peptide signal)/noise ratio were obtained. The saturation transfer experiments indicated that methyl groups of amino acid residues corresponding to Leu66,70,72, Val69 and Ala73 in interleukin-2 contact with the antibody binding site. Thus, the hydrophobic interactions are of major importance for the LNKB-2-IL-2 peptide complexes. The minimal IL-2 fragment which can still bind to LNKB-2 monoclonal antibody is -Leu70-Asn71-Leu72-.