Comparison between Hybridoma and Fab/phage Anti-RhD: Their V Gene Usage and Pairings

Our 11 anti-RhD's in conjunction with 37 previously published RhD antibodies, produced by hybridoma technology were analysed for germline gene usage and restriction in VH and VL pairings. The 17 VH germline genes used by the hybridoma anti-RhD IgG were derived from 4 VH families (VH1, VH2, VH3 and VH4). Eighteen kappa chains were restricted to only 5 germline genes from only 2 Vκ families (Vκ1 and κ3). However, the 13 lambda chains were not as restricted, using 10 Vλ germline genes from 4 families (Vλ1, Vλ2, Vλ3 and Vλ8). Fifty six unique Fab/phage anti-RhD were also analysed. In all cases the Fab/phage VH germline genes were derived from the VH3 family (41/41). The 29 kappa chains were restricted to 4 germline genes primarily from Vκ1 (97%) germline genes from 5 families (Vλ1, Vλ2, Vλ3, Vλ4 and Vλ7). The VH germline genes of the Fab/phage were restricted to 4 of the 17 used by the hybridoma anti-RhD IgG (DP46, DP49, DP50 and DP77). Ninety percent of the Fab/phage were restricted to 1 of the 5 Vκ germline genes used by the IgG (DPK9). However, the repertoire of the Vλ germline genes used in these two systems is different, with analysis showing greater diversity in Vλ gene usage with 8 unique germline genes used by 76% Fab/phage compared to 4 unique genes used by 46% hybriboma anti-RhD.


Introduction
The RhD antigen is a highly immunogenic human red cell antigen. Alloimmunisation against the RhD antigen produces high affinity antibodies that cause haemolytic transfusion reactions (HTR) and haemolytic disease of the newborn (HDN) [1]. Currently, plasma-derived polyclonal anti-RhD is used for prophylaxis of RhD HDN although, human monoclonal RhD antibodies are being evaluated for possible replacement. The biochemistry and physiological function of the RhD antigen is not yet fully understood. Cloning and sequence analysis predicted a 417 amino acid protein of 45.5 kDa [2], with 12 hydrophobic transmembrane domains and cytoplasmic N and C termini resulting in 6 extracellular loops [3,4]. Epitope mapping of the RhD antigen, investigated by site directed mutagenesis indicated the presence of six external, distinct, non-overlapping epitopes [5].
Diversity of the antibody repertoire is a consequence of the recombination of the germline gene segments (V, D and J) during B cell development [6]. The rearranged V(D)J gene segments of the heavy and light chains are derived from functional germline genes consisting of 51 V H , 25 D, 6 J H and 31 V λ , 4 J λ or 40 V κ and 5 J κ genes [7][8][9][10][11]. Diversity is further increased when boundaries between the V(D)J gene segments undergo additions or deletions [12] and somatic hypermutation at the mature B cell stage.
In recent years a large number of hybridoma and Fab/phage anti-RhD have been sequenced in an attempt to identify usage of V H and V L germline genes, somatic hypermutations and affinity maturation. With construction of a database consisting of 48 hybridoma and 56 Fab/phage anti-RhD, restriction of the V H and V L gene repertoire and their pairings can be confidently compared and determined. The compiled IgG anti-RhD are represented by V H : n = 33, Vκ: n = 18 and V λ : n = 13 (λ chain of MD03 and the light chain of Oak-3 [14] was not determined) [13][14][15][16][17][18][19][20]. Fab/phage anti-RhD are represented by V H : n = 41, Vκ: n = 29 and V λ : n = 24 [21,22].

Comparison of V gene usage and pairing between hybridoma IgG and Fab/phage anti-RhD
It has been recognised that biased expression of V H and V L families or particular V genes occurs in nonimmunised individuals. Bias for the VH3 and Vλ2 family have been described in IgG and IgM peripheral blood B cells from normal healthy donors [23,24], although there is no evidence for preferential V H and V L pairings. In the case of autoantibodies there is evidence that there may be preferential use of certain V H genes. The anti-I/i cold agglutinins use DP-63 (VH4·21) but demonstrate different light chain usage with anti-I preferring VK3 family light chains [25][26][27][28]. Antibodies against gp120 of HIV-1 [29] showed preferential usage of the VH1, VH3 and VH5 families. Similarly, a limited set of VH3 family members have been found in the immune response to H. influenzae type b [30].
The favoured V H germline gene segments used by the 48 hybridoma anti-RhDs (33 IgG and 15 IgM) were mainly from the VH4 (44%, 21/48), VH3 (40%, 19/48) and VH1 (15%, 7/48) families. The V H germline gene segment preferred by the 15 IgM was DP-63 (14/15) and in all cases where the confirmed light chain was lambda, the V gene DPL16 (9/9) was preferred. This data suggests that the pairing of DP-63 with DPL16 is the most common primary immune response to the RhD antigen.
The 41 V H germline genes used by the 56 unique Fab/phage anti-RhD were derived in all cases from the VH3 family and the kappa chains were restricted to only 4 germline genes from the VK1 (28/29) and VK2 (1/29) families. The vast majority of these Fab/phage preferred the DPK9 germline gene for kappa chain and the "VH3-33 superspecies" gene for the heavy chain. The lambda chains however used 10 V L germline genes from the Vλ1 (13/24), Vλ2 (2/24), Vλ3 (5/24), Vλ4 (1/24) and Vλ7 (3/24) families. Table 2 compares the usage and frequency of the germline genes used in both the hybridoma and phage library systems. Only 4 out of the 17 V H germline genes used by hybridoma IgG were observed for Fab/phage. These 4 V H germline genes accounted for 100% of the Fab/phage but only 43% of the IgG. Ninety percent of the Fab/phage were restricted to 1 of the 5 V κ germline genes used by the hybridoma anti-RhD IgG. However, the repertoire of the V λ germline genes used between these two systems is different and with greater diversity. Two germline genes, DPL5 and DPL16 were common to both systems but only 24% of the Fab/phage and 54% of the anti-RhD IgG used these 2 genes. An additional 8 unique Table 2 Usage and frequency of germline V genes for hybridoma IgG and Fab/phage anti-RhD
V λ germline genes used by 76% Fab/phage were absent from hybridoma IgG. Thus the Fab/phage germline gene usage was restricted for the V H and V κ but different for the Vλ when compared with the hybridoma IgG anti-RhD's.

Comparison of canonical combinations between hybridoma IgG and Fab/phage anti-RhD
Canonical combinations of the V H and V L provides a better understanding of the conformation of the paratope which is responsible for binding of antigens.
Here we describe and compare the canonical combina-tion between hybridoma IgG and Fab/phage anti-RhD. Twenty four V H and V L gene pairings were identified for the hybridoma IgG compared to only 18 for the Fab/phage anti-RhD, resulting in 10 and 7 canonical combinations, respectively. The most commonly used combination by the kappa IgG anti-RhD was V H 1-3 : V κ 2-1 (67%, 12/18). This combination was found almost exclusively for the kappa Fab/phage (97%,29/30). A further 3 canonical structures were unique to kappa IgG but not Fab/phage. The V H 1-3 : V κ 2-1 structure has been reported most heavily used (21%) by randomly paired V gene segments from an non-immunised source [31]. Usage of this canonical combination was recently reported for antibodies raised against a second antigen (E) of the Rh system [32]. Two canonical combinations, V H 1-3 : V λ 11-7 and V H 1-1 : V λ 11-7 were used by the majority of the lambda IgG hybridomas although a different and unique structure (V H 1-3 : V λ 13-7A) was preferred by the Fab/phage (see Table 3). Hybridoma IgM anti-RhD were exclusive to the V H 1-1 : V λ 11-7 structure which was not favoured by Fab/phage and used by only one IgG. The V H 1-3 : V λ 14-7A (10%), V H 1-3 : V λ 11-7 (7%) and V H 1-3 : V λ 13-7A (5.5%) are the three most heavily used canonical structures by B cells from a non-immunised source [31].
It is apparent from this analysis that the V H and V L germline genes for Fab/phage are restricted only for V H and Vκ but different for the Vλ when compared with hybridoma IgG. This was further apparent when their pairing and canonical combinations were considered. In general, when creating phage libraries for a particular specificity, the germline gene usage and pairing for kappa libraries may be more restricted compared to lambda libraries.