Immunogenetic Analysis of Variable Regions Encoding AB1 andγ-Type AB2 Antibodies from the NeuGc-Containing Ganglioside Family

Abstract

The variable regions from P3, a murine monoclonal antibody (MAb) against NeuGc-containing gangliosides, and two anti-idiotype MAbs directed to P3 MAb were cloned and sequenced. Comparisons with previously reported sequences showed that P3 is a germline antibody encoded by genes from the V(H)Q52 and V(kappa)19 families. Analysis of nucleotides at the heavy chain CDR3 (H-CDR3) showed the presence of an extensive 3' N region that contains almost 50% of the nucleotides of this CDR. In addition, amino acid sequence analysis of the H-CDRs of this MAb revealed the presence of three arginines, two of which are present in the H-CDR3, that could be involved in the interaction of P3 MAb with its electronegative epitope on gangliosides. Anti-idiotype 1E10, which seems to define a "regulatory" idiotope on P3 MAb (it induces Id+ Ab3), represents a germline Ab2 that belongs to the V(H)J558 and V(kappa)10 gene families. By contrary, the anti-idiotype 3B11 is an extensively mutated antibody that belongs to the V(H)3660 and V(kappa)4/5 gene families, defining a "private" idiotope on P3 MAb. Even when different V genes contribute to the variable regions of 1E10 and 3B11 MAbs, they share an acidic motif E/D-D-Y/D-Y-D in H-CDR3, suggesting that both Ab2s recognize paratope positive residues on the Ab1. Therefore, complementary electrostatic interactions involving H-CDR3 from both Ab1 and Ab2, might provide a clue to understand the molecular basis for the generation of gamma-type anti-idiotype antibodies to V regions recognizing glycolylated ganglioside antigens.

Anti-ganglioside antibody-induced tumor cell death by loss of membrane integrity

“…It is possible that these arginines could also be relevant for cytotoxic activity. However, P3 mAb has a similar sequence in the H-CDR3 (RXXR), but this mAb was not able to induce direct cell death (47).…”

“…P3 mAb is restricted to the N-glycolyl sialic acid residue (48), whereas the epitope recognized by 14F7 mAb involved the disaccharide N-glycolyl sialic acid-galactose (46). Also, P3 mAb was encoded by a germ-line VH region (47), whereas 14F7 variable regions were heavily mutated (23), arguing an affinity maturation process. In fact, chimeric 14F7 antibody seemed to have a higher affinity for NGcGM3 than chimeric P3 antibody.…”

Induction of Anti-Anti-Idiotype Antibodies Against Sulfated Glycosaminoglycans Reduces Atherosclerosis in Apolipoprotein E–Deficient Mice

“…19 P3 mAb has an arginine motif at the heavy chain complementary determining region 3, which can be represented as R98-X-X-R100a (where numbers are according to Kabat system and X is any residue). 20 This segment is crucial for the binding of murine P3 mAb to gangliosides, 21,22 and it is also found in the principal PG-binding sequence (site B) in the apoB moiety of LDL. 23 Chimeric mouse/human variant of P3 mAb that retain its properties was generated, 24 and its mutants E99→R (chP3R99) and R98→S (chP3S98) present the same reactivity against 1E10 Ab2 mAb but a higher and lower reactivity, respectively, against their antigens compared with the wild chimeric P3 antibody.…”

Lonely killers

“…80,81 In contrast to 14F7, the chimeric version of P3 82 was unable to kill GM3(Neu5Gc)-expressing cells. 66 Originally a germ-line IgM, 83 its lower affinity was the explanation given to this observation. 66 This was later proved with a more reactive mutated variant of chimeric P3.…”