3 RESULTS

3.1  LIBRARY CONSTRUCTION

3.1.1 Isolation of RNA

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107 cells/ml B lymphocytes - antibody producing cells - were isolated from 20 ml peripheral blood from an 8-1/2 year-old girl (SH) suffering from acute myocarditis with clinical signs of cardiac failure for library construction. She was diagnosed as a patient suffering from systemic lupus erythematosus and rheumatic fever. The blood was drawn 24 days after the patient had had IVIG therapy. RNA from peripheral blood lymphocytes after administration of IVIG would provide DNA dominated by currently secreted specificities, as cells actively secreting antibodies will contribute substantially more immunoglobulin RNA than from resting B cells that sit stationary in lymphoid tissues (Persson, 1993). Total RNA was extracted from the B lymphocytes for cDNA synthesis (Fig. 3.1).

Fig. 3.1 Gel electrophoregram of total RNA isolated from the peripheral blood lymphocytes of a patient with SLE and rheumatic fever. The quality of undegraded total RNA is shown by the double-sized 28S rRNA band to that of the 18S rRNA band. M, ΦX 174 molecular marker.

Since RNA molecules are unstable and denature readily, and since DNA molecules are stable to handle in cloning, the RNA was reverse-transcribed into cDNA, for subsequent use in PCR and routine cloning purposes.

3.1.2 Amplification and cloning of Fd, λ, and κ chain genes

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First strand cDNA syntheses in separate reactions (γ, κ and λ chains) were performed using 3' specific primers that were subsequently used in the PCR. To generate sufficient amount of DANN for cloning, the cDNAa was first amplified in PCR. The PCR primers are designed such that they incorporate restriction enzyme sites allowing for directed cloning with the proper reading frame. Analysis of the heavy chain PCR products on 2 % agarose gel revealed bands of expected sizes between 660-680 bp. The varying intensities may represent the level of expression of different antibody gene families presented by the patient at the time the material was collected (Fig. 3.2).

Fig. 3.2 Gel analysis of PCR amplified human genomic immunoglobulin heavy chain variable region segment. Amplification conditions are described in the materials and methods. M, ΦX 174 molecular marker; VH1, …..VH8, family specific Ig genes; ConGa, positive control primer for constant regions; Neg, negative control without template

The heavy chain PCR products were pooled, electrophoresed on 2% agarose, and the expected size of the heavy chain fragments excised and purified from the gel, Fig. 3.3 (A) and (B). The fragments were restricted by the heavy chain restriction enzymes, subjected to agarose gel electrophoresis, and finally purified from the agarose gel, Fig. 3.3 (C) and (D). Similarly, all light chain PCR fragments (kappa and lambda) were also pooled, restricted, and purified from agarose.

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The heavy and light chain PCR fragments were ligated into the pComb3H phagemid for the construction of the SH library which contained 3.3 x 107 members. The pComb3H phagemid used in this combinatorial library construction allow for display of the repertoire of cloned antibody fragments on the surface of helper phage (VSCM13), that in its genome carries the corresponding antibody DNA.

Fig. 3.3 2 % agarose gel of, (A) pooled heavy chain PCR products, (B) gel showing excised heavy chain PCR products, (C) Xho I/Spe I restricted heavy chain PCR products, and (D) gel showing excised restricted heavy chain products. M is the molecular marker ΦX 174.

In a random screening, 7/10 (70 %) clones contained both heavy and light chain DNA inserts as analysed by restriction enzyme analysis. Gene sequencing analysis of randomly picked clones (unpanned or non-antigen selected) from the unselected library revealed that the heavy chain antibody genes were in the right reading frame, but many of the light chain sequences had one or two stop codons in the reading frame. Therefore, light chain shuffling was done by retaining the heavy chain of the original repertoire, and new light chain genes were amplified and ligated into the plasmid. The new library contained 6.9 x 107 members and 8/10 clones had both heavy and light chain fragments as revealed by restriction enzyme analysis. To determine whether the unpanned SH library contained many different clones, the diversity was monitored by digesting 12 individual clones with BstN1 (New England, Biolabs, Berverly, MA., USA) restriction enzyme and the fragments analyzed on 2 % agarose (Fig. 3.4).

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Each of the clones analyzed by BstN1 restriction had a distinct restriction pattern from the other showing that the library was made up of different clones with diverse heavy and light chain gene usage. Cloning of target genes results in the presence of 'clones of interest' and inevitably wildtype plasmids which outcompete the desired clones. In order to successfully isolate clones that express antibody genes it was necessary to optimize the unselected library before its usage.

Fig. 3.4 BstN1 digestion of 12 randomly picked unselected clones from the library of patient SH. BstN1 frequently cleaves in the V regions but only 2 sites in the pComb3H phagemid vector. M1, 1 kb molecular marker; M2, ΦX 174 molecular marker.

3.2 LIBRARY OPTIMIZATION

Agarose gel electrophoresis of the total DNA from the unselected (original) library revealed 2 bands at or near the expected size of the library DNA (Figure 3. 5, lane 1). More often, the presence of 'bald' clones (clones containing no inserts but which have selective growth advantage) in a library results in over-amplification of the these clones to the disadvantage of clones containing inserts during further culture of the library (Fischer et. al., 1999). To find out which of these 2 bands contain clones with heavy and light chain inserts, the upper and lower bands were separately excised from the gel, purified, restricted and analyzed on agarose.

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Fig. 3.5 shows that the upper band contains both heavy and light chain inserts whilst the lower band was 'bald'. Hence, the upper band was used to transform bacteria, and the resultant library contained 5.6 x 106 members. This became the final constructed library from the patient with systemic lupus erythematosus and rheumatic fever (SH library), from which panning on an antigen (IVIG) resulted in the isolation of antigen specific Fabs, as well as the isolation of unselected clones.

Fig. 3.5 Restriction enzyme analysis of the original/unselected library to eliminated bald clones from the original library (lane 1). Lanes 2 and 3 respectively, are the unrestricted upper and lower bands purified from the original library. Lanes 4 and 6 show restricted heavy and light chain DNA fragments from the unrestricted upper fragment band (lane 2). Lanes 5 and 7 show that the unrestricted lower band (lane 3) contain no immunoglobulin chain DNA fragments. Xho I/Spe I were the heavy chain restriction enzymes whilst the light chain restriction enzymes were Sac I/Xba I. The molecular markers M1 and M2 have already been described in Figure 3. 4

3.3 BIOPANNING ON IVIG

Panning of the SH library for monoclonal Fab phages bound by IVIG was performed using 300μl of (30mg/ml) IVIG preparation (Sandoglobin, Sandoz, Basel, Switzerland)

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which were coated in 5ml Nunc immunotubes. A selection strategy was used in which the Fab phages were captured by the immobilized IVIG, and unbound phages washed off. Bound phages were then eluted with HCl/glycine buffer pH 2.2, neutralized with Tris HCL pH 9.0, and used to re-infect bacteria for propagation for the next rounds of panning, until a highly enriched population of phages carrying IVIG specific Fabs were obtained. The SH library was subjected to four rounds of panning on IVIG. To monitor the progress of the panning procedure, the percent of phage recovered at every round, as well as the enrichment of phages at a particular round over the previous were calculated from the number of clones that were plated on phage input and output carbenicillin plates, Table 3.1 (Siegel et. al., 1997).

Table 3.1 Panning of phage displayed Fabs from SH combinatorial antibody library on IVIG (After Siegel et. al., 1997).

Panning
round

Phage
Input (c.f.u)
a

Phage
Output (c.f.u)
b


% Bound
c


Enrichment
d

1

2.8 x 10 12

5.7 x 10 5

2.0 x 10 -5

-

2

1.5 x 10 12

3.9 x 10 5

2.6 x 10 -5

x1.3

3

1.2 x 10 12

2.6 x 10 6

2.2 x 10 -4

x8.5

4

1.3 x 10 12

9.8 x 10 6

7.5 x 10 -4

x3.4

aNumber of colony forming units (c.f.u) of phage incubated with antigen

Titration of phage input and output revealed that there was an enrichment of phages bound by IVIG at every round of panning (from panning 1 to panning 4). There was a 38-fold increase in percent phage bound in panning 4 over that obtained during the first round of panning. These data seemingly revealed that bacterial colonies that were infected with phages from a previous round of panning resulted in the enrichment of specific phage population expressing Fabs bound by IVIG. This gave the indication that the possibility to isolate monoclonal Fabs bound by IVIG exists.

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To isolate single clones producing monoclonal antigen-specific antibodies (Fabs bound by IVIG), total DNA from the fourth round of panning need to be transformed into bacteria. However, before the transformation, it was important to restrict the total DNA obtained from the fourth round on panning on IVIG to ascertain that the population of antibodies selected during the panning process contain both heavy and light chain inserts. In addition, it is also worthwhile to perform a polyclonal Fab phage ELISA on the antigen (IVIG) used in the selection process (panning) to gain a first hand knowledge about the specificity of the selected phages in the biopanning process.

3.4 POLYCLONAL FAB PHAGES BOUND BY IVIG

Though 'bald' clones in the unselected library were 'eliminated' or reduced as much as possible during the optimization of the library (section 3.2, Fig. 3.5), the re-emergence of such clones as well as clones with single inserts (light or heavy chain DNA, only) which have growth advantage over clones with double inserts (heavy and light chain DNA inserts) during various rounds of panning could not be underrated. Therefore, to have an idea whether clones selected at the end of the panning process were clones containing both heavy and light chain DNA inserts before transforming bacteria for the isolation of Fabs, the total DNA at the end of the last round of panning (panning four) was subjected to both heavy and light chain restriction enzyme analysis, and compared with the total DNA from the unselected library, Fig. 3.6

As visualized on the agarose gels, both Figures 3.6 (A) and (B) show that the IVIG-selected clones contain both antibody heavy and light chains DNA inserts of expected sizes of approximately 680 bp. After the digestion of the total DNA from panning 4 but before transforming bacteria with it for isolation of monoclonal Fab phages, it was also necessary to verify whether the selected clones during the panning process contained phages that bind IVIG. This was important to establish that the selected library is not full of 'plastic-binding' phages which inevitably are present in a library (Adey et. al., 1995). This was done by polyclonal Fab phage ELISA in IVIG-coated wells for the unselected library, as well as the selected libraries for all four rounds of panning on IVIG (Fig. 3.7).

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Figure 3.6 Enzymatic double restriction. (A) Total unselected library DNA, (B) total DNA IVIG panning IV. Restriction enzymes: light chain - Sac I/Xba I; heavy chain - Xho I/Spe I. M1, M2 molecular markers described in Figure 3.4

Figure 3.7 ELISA of polyclonal phages of the unselected (unpanned) repertoire of the systemic lupus erythematosus and rheumatic fever library, and selected against IVIG in panning round 1 (IVIG I), panning round 2 (IVIG II), panning round 3 (IVIG III), and panning round 4 (IVIG IV). Coated proteins consisted of IVIG as well as PBS/1% casein blocking buffer to detect nonspecific binding. Bound Fab-phages were detected with HRP-labeled anti-M13 antibodies. Results are expressed as absorbance at 405 nm with a multiscan automatic plate reader (MR5000, Dynatech Labs, Chantily, VA., USA)

Typical IVIG binding phage enrichment were observed from panning 1 to 4. As can be seen in figure 3.7, the initial 'high' background on PBS (plastic binding clones) gradually decreased from panning 1 to panning 4 indicating that the stringency of washing led to the retention of Fab phages specifically bound by IVIG. Only a few of sticky, non-specific binders remained after round four of panning. These phages were present at amuch higher proportion in the unselected repertoire. This also demonstrated one of the objectives of the Fab phage panning method, that is to say, to increase the 'signal-to-noise ratio' of specific versus non-specific binding ratio during the selection process (Siegel et. al., 1997). This result indicated that there was a probable chance of successful isolation of monoclonal Fabs bound by IVIG which would lead to their further characterization.

3.5 ISOLATION OF MONOCLONAL FAB PHAGES BOUND BY IVIG

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Total DNA from panning 4 was used to transform bacteria and individual colonies were picked and grown for 5 - 6 h, and super-infected with VCSM13 helper phage for Fab-phage rescue. This allowed for the display of cloned antibody fragments on the surface of phage particles that in its genome carries the corresponding antibody DNA. Culture supernatants from individual colonies from the fourth round of panning were analyzed for their reactivity with IVIG in ELISA. Plasmid DNA from positive clones were digested by restriction enzymes and analyzed by agarose electrophoresis. Clones that contain both heavy and light chain inserts (data not shown) were sequenced and only clones that contain functional heavy and light chain antibody variable region sequences were used further in the study. Representative clones showing specific reactivity to IVIG are shown in figure 3.8. This results also show that the panning procedure finally resulted in a highly enriched population of phage carrying specific antibody fragments with 'little' or 'minimal' unspecific binding.

One of the aims of this study was to compare the IVIG-binding Fabs isolated from the patient (SH) with rheumatic fever and systemic lupus erythematosus to those isolated from 3 patients with idiopathic thrombocytopenic purpura (Fischer et. al., 1999; Jendryeko et. al., 1998) cloned in our laboratory. One of the patients developed SLE. The IVIG-binding Fabs from patient SH was tested for their reactivity to human Fc fragments. Rheumatoid factors are antibodies that bind to the Fc portion of IgG immunoglobulins. They are found in patients with arthritis as well as patients with inflammatory and infectious diseases. Rheumatoid factors are also found in healthy people following immunization suggesting that they may play a role in normal immune functions such as in the clearance of circulating immune complexes (Potter and Capra, 1995). Rheumatoid factors in idiopathic thrombocytopenia are thought to stabilize (enhance) low affinity anti-platelet antibodies in binding to platelets. Yang et. al. (1999) postulated that some IgG components in IVIG interact with these IgG rheumatoid factors and thus interfere with their enhancing effects on anti-platelet antibodies.

None of the IVIG-binding Fabs from the patient with systemic lupus erythematosus and rheumatic fever library bound Fc fragments (figure 3.8) in contrast to IVIG-binding Fabs isolated from patients with idiopathic thrombocytopenic purpura (Fischer et. al., 1999; Jendryeko et. al., 1998).

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Figure 3.8 ELISA of representative individual (monoclonal) Fab-phage antibodies obtained after the fourth round of panning on IVIG. Wells were coated with IVIG, Fc fragments and PBS/1% Casein blocking buffer. Negative controls were pComb3H transfected E. coli phage supernatant (pComb3H), and SB medium containing VCSM13 helper phage (SB+M13). Bound Fab-phages were detected with HRP-labeled anti-M13 antibodies. Results were expressed as under Figure 3. 7. Clones selected repetitively included SH8 and SH44 (2X each); SH21 (9X), and SH89 (7X).

As a further step in characterizing and comparing the IVIG-binding Fabs isolated from patient SH to those from the patients with idiopathic thrombocytopenic purpura, the Fabs from SH was tested for their binding to platelets. Since platelets require a different surface (for coating) from IVIG for ELISA, the testing of the Fabs for their reactivity to platelets was done in a separate ELISA.

3.6 REACTIVITY OF IVIG-BINDING FABS ON PLATELETS

Idiopathic thrombocytopenic purpura is mediated by autoantibodies that react to antigens expressed on platelets leading to their clearance by phagoctosis. Thrombocytopenia and platelet dysfunction are frequently found in patients with SLE, in particular, in association with anti-platelet antibodies. These antibodies react with the major platelet glycoproteins, gpIIb-IIIa, interfering with platelet aggregation and adhesion (Xu et. al., 1995). To compare the extent of the repertoire used by the Fabs bound by IVIG from the library generated from the patient with systemic lupus erythematosus/rheumatic fever (SH) library, to that from the patients with autoimmune thrombocytopenia, the panel of selected Fabs from the SH library were tested also for binding to platelets in ELISA. IVIG-binding Fabs with reactivity to platelets had earlier been isolated from patients with autoimmune thrombocytopenia (one of whose illness progressed to SLE) in our group.

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Figure 3.9 Summary of two repetitive ELISAs with representative Fab phage supernatants of sequenced clones after the 4th round of panning with IVIG on human platelets. 50 μl/well of 3 x 107 cells/ml of purified platelets were coated by drying to flexible plates (Microtest III, Falco, Oxnard, California). Incubation with Fab phages and further development were as described for the IVIG ELISA. The blood group was A, Rh positive. Shown is the mean A450/650 after subtraction of the background staining on PBS/Casein coated wells. Positive controls were LO31 (V3-30 locus) and NK22 (V4-b locus), platelet-binding IVIG-derived Fabs from patients with autoimmune thrombocytopenia (Fischer et. al., 1999). Negative controls were pComb3H transfected phage supernatant (pComb3H) and SB medium containing VCSM13 helper phage (SB + M13). Bound Fab phages were detected with HRP-labeled anti-M13 antibodies.

None of the SH library IVIG-binding Fabs bound platelets. IVIG has a weak interaction with clone LO31, which was isolated from a patient with autoimune thrombocytopenia whose illness progressed to SLE (Jendreyko et. al., 1998; Fischer et. al., 1999). To analyze the IVIG-binding Fabs further, the DNA were sequenced. The sequencing showed that indeed full length antibody DNA has been cloned. This permitted their further characterization.

3.7 HEAVY CHAIN AMINO ACID HOMOLOGY

Sequencing of clones that were positive in the IVIG monoclonal Fab phage ELISA revealed 23 clones that had both functional heavy and light chain genes. Amino acid homology tree was constructed to show that the sequences were different from each other. Four individual clones were repetitively selected, these varied from two to nine, figures 3.10 and 3.11.

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Figure 3.10 Amino acid homology tree of representative VH Fab phages bound by IVIG from the SH library constructed by the McDNASIS software. Clones SH8 and SH44 (2X each), SH21 (9X), and SH89 (7X) were repetitively selected.

Comparison of the complete overlapping heavy chain amino acid sequences between all seven individual VH 3 family Fab phage loci bound by IVIG showed that Fabs 44 and 58 were the most closely related (88.1%) whilst the lowest homology was with Fab 89 (69.2%).

Figure 3.11 Amino acid homology tree of representative VL Fab phages bound by IVIG from the SH library constructed by the McDNASIS software. Clones SH8 and SH44 (2X each), SH21 (9X), and SH89 (7X) were repetitively selected.

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The light chain amino acid homology revealed that the most closely related Fabs were clones 55 and 8 which shared a homology of 87.7% whilst the lowest homologous clone was clone 58 (25.3%).

3.8  VH AND VL GENE SEGMENT USAGE OF IVIG SPECIFIC FABS

The variable region gene sequences from the Fab phages were aligned to human germline variable region sequences compiled in the VBASE directory to assign them to their closest germline counterparts with the aid of the DNAPLOT alignment software. A homology table was built to categorize the variable region genes into families, loci, and their deviation from their closest germline gene, table 3.2. The D and JH genes of each Fab are also provided. With regard to VH gene usage, only two VH3 germline genes were selected, namely V3-23 and V3-30, with 3-23 being the most frequently used 16/23 (70%). These two VH loci genes were the most frequently observed in IVIG-bindig Fabs from patients with autoimmune thrombocytopenia (Fischer et. al., 1999; Jendreyko et. al., 1998). Comparison of the 3-23 heavy chain sequences with known VH germline sequences revealed less divergence from the closest VH germline genes with homologies ranging between 96.7-99.6 %. The remaining 7/23 (30%) derived their VH loci from the gene segment V3-30, sharing 99.6% homology with their closest germline gene. Comparison of the SH IVIG Fab phages light chain variable region sequences with their closest germline gene segments revealed that the Vλ1, Vλ2, Vλ3, and Vκ2 families were represented. Among these, the VL 3 family predominated with about 19/23 (83%) clones being represented. Unlike the VH segments where only two VH loci were used, the light chain gene segments were derived from 4 different VL loci.

3.9 SH NON-ANTIGEN SELECTED (UNPANNED) CLONES

5 non-antigen or unpanned clones were sequenced to determine the diversity of the systemic lupus erythematosus and rheumatic fever library (Table 3.2). 4 clones belonged to the VH 3 family but only 1 (clone SH-u8a) was derived from the 3-23 locus compared with the IVIG binders where 16/23 were from this locus. The unselected clone SH-u8a, used a similar V-D-J rearrangement to that of the IVIG-selected clone SH-55. Nevertheless, they were two different clones as each paired with light chains from different VL families. The VH region segment from one non-antigen selected clone (SH-u2b), was not assigned to any particular locus after the database search. This clone however, paired with an identical light chain that was observed with nine (V 3-23) IVIG Fab clones. Another unpanned clone (SH-u3a) that belongs to VH1 family paired with a λ light chain that utilized a λ gene segment identical to that of the IVIG binders that used the VH 3-30 loci. The Vλ gene usage from the unpanned clones were Vλ1, Vλ2, Vλ3, and Vλ6.

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Table 3.2 Putative germline sequences of SH IVIG-selected and non-antigen selected antibody heavy and light chain variable regions from the patient with systemic lupus erythematosus and rheumatic fevera

Clone

V H family

V H

V H locus

Hom-ology %†

D

J H

V L family

V L

V L locus

Hom-ology %†

J L

IVIG-selected Fab phages

SH8‡

3

DP-47/V3-23…+ (Z12347)*

3-23

97.4

D6-19 (X97051)

JH4b (X86355)

VL3

DPL16/VL3.1…+ (Z22202)

3l

99.2

JL2/JL3a (M15641)

SH21‡

3

DP-47/V3-23…+ (Z12347)

3-23

99.6

D4 (J00232)

JH3b (X86355)

VL3

IGLV3S2+ (X71966)

3h

99.2

JL3b (D87017)

SH44‡

3

DP-47/V3-23…+ (Z12347)

3-23

97.4

D2-8/DLR1 (X97051)

JH4b (X86355)

VL2

2b2.400B5+ (Z73665)

2b2

91.7

JL2/JL3a (M15641)

SH51

3

DP-47/V3-23…+ (Z12347)

3-23

96.7

D4-11/DA1 (X97051)

JH4b (X86355)

VL1

DPL7/VL1.2

(IGLV1S2)+ (Z22193)

1e

99.6

JL3b (D87017)

SH55

3

DP-47/V3-23…+ (Z12347)

3-23

97.0

D6-25 (X97051)

JH4b (X86355)

VL3

DPL16/VL3.1…+ (Z22202)

3l

98.5

JL3b (D87017)

SH58

3

DP-47/V3-23…+ (Z12347)

3-23

99.3

D3-3/DXP4 (X97051)

JH1 (J00256)

VK2

DPK18/A17+ (X93635)

A17

97.8

JK2 (J00242)

SH89‡

3

DP-49/1.9III…+ (Z12349)

3-30/

3-30.5

99.3

D3-22/D21-9 (X97051)

JH6b (X86355)

VL3

3r.9C5/DPL23…+ (Z73647)

3r

99.6

JL2/JL3a (M15641)

Randomly picked clones from the unpanned SH library

SHu2a

3

DP-48/13-2+ (Z12348)

3-13

94.8

D6-19 (X97051)

JH5B (X86355)

VL2

2c.118D9/V1-2+ (X97462)

2c

95.9

JL2/JL3a (M15641)

SHu8a

3

DP-47/V3-23…+ (Z12347)

3-23

97.4

D6-25 (X97051)

JH4b (X86355)

VL6

6a.366F5/V1-22…+ (Z73673)

6a

98.1

JL2/JL3a (M15641)

SHu2b

3

p6

(M77305)

-

94.4

D4 (J00232)

JH4b (X86355)

VL3

IGLV3S2+ (X71966)

3h

96.4

JL3b (D87017)

SHu3b

1

DP10/hv1051…+ (Z12312)

1-69

97.0

D5-12/DK1 (X97051)

JH6b (X86355)

VL3

3r.9C5/DPL23…+ (Z73647)

3r

89.7

JL2/JL3a (M15641)

SHu7b

3

DP-38/9-1…+ (Z12338)

3-15

99.0

D3-10/DXP,1 (X97051)

JH4b (X86355)

VL1

1b.366F5/DPL5…+ (Z73661)

1b

96.4

JL2/JL3a (M15641)

*Numbers in parenthesis indicate European Molecular Biology Laboratory/GenBank accession numbers. Sequences are named according to the VBASE databank. Dots indicate abbreviated names and/or existence of synonyms; + indicates a mapped chromosomal location.
†Homology of the variable region genes
‡Clones selected repetitively included SH8 and SH44 (2X each); SH21 (9X), and SH89 (7X)

3.10 MUTATION RATES

A table of mutation rates (Table 3.3) was built to show where in the variable region (CDRs and FRs), the deviation (i.e. < 100% homology) from their germline counterparts observed in the previous section, occured for each clone. Somatic hypermutations leading to a single base substitution may either lead to replacement (R) of an amino acid or remain silent (S) if the resulting codon encodes for an identical amino acid. R/S ratios above 2.9 are considered high and indicative of antigen selection. However, the role of CDR3 which lies at the center of the antigen-binding site is difficult to access and were not included in this type of analysis. In addition, the first eight codons of the FR1 region were excluded from the analysis of somatic mutations because this region is complementary to the primers used for amplification (see figures 3.12 and 3.13) . Sequences from representative clones for the 23 Fabs were aligned on themselves to reveal individual variations among the clones (figures 3.12 and 3.13)

Figure 3.12 Alignment of VH amino acids of representative clones SH8, 21, 44, 51, 58, and 89 from IVIG-selected Fabs according to their highest homology. Complementarity determining regions (CDRs) according to the Kabat definition are indicated (Kabat et. al., 1991). The beginning of the sequences (ca. 8 amino acids) was determined by the PCR primers. * = critical amino acid positions known for binding to staphylococcal protein A (SpA) Graille et. al., 2000). The sequences are designated according to the library from which they were obtained, i.e., SH is the initials of the patient; g means IgG heavy chain; i means (IVIG), the antigen used in isolating the antibody. These sequence data are available from the European Molecular Biology Laboratory nucleotide sequence database under accession numbers AJ298606 - AJ298612.

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The VH CDR3 regions displayed an overall high degree of variation in length and sequence, ranging between nine (e.g. SHgi44) and 17 (SHgi21) amino acids.

Figure 3.13 Alignment of VL amino acids of representative clones SH8, 21, 44, 51, 58, and 89 from IVIG-selected Fabs according to their highest homology. Complementarity determining regions (CDRs) according to the Kabat definition are indicated (Kabat et. al., 1991). The beginning of the sequences (ca. 8 amino acids) was determined by the PCR primers. The sequences are designated according to the library from which they were obtained, i.e., SH is the initials of the patient; l means a light chain; i means (IVIG), the antigen used in isolating the antibody. These sequence data are available from the European Molecular Biology Laboratory nucleotide sequence database under accession numbers AJ298613 - AJ298619.

The pattern of somatic mutations in the CDR1 and CDR2 of the VH genes was indicative of replacement events in four Fabs namely SH8, 51, 55, and 58 with a high R/S ratio of >2.9 (Table 3.3 Mutation Rates). The remaining three Fabs had 0 R/S ratios. Two light chains, SH25 and SH55 had a higher R/S ratio >2.9 in the entire VL CDR1 and CDR2. On the whole, only two Fabs displayed a R/S ratio >2.9 over the entire VH region (SH51 and SH55) whilst one Fab (SH44) had a R/S ratio above 2.9 in the entire VL region.

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Analysis of the VH and VL genes in this study and earlier reports (Jendreyko et. al., 1998; Fischer et. al., 1999; Hoffman et. al., 2000) revealed that the interaction of IVIG with the Fabs might be dependent on the heavy chain since the light chains were from diverse families. In some cases, the Fabs bound by IVIG had no functional light chains. The interaction of IVIG with Fabs solely from the VH3 family in this study was characteristic of the interaction of B cell superantigens with immunoglobulins from the VH3 family (Silvermann, 1997). Thus, we next tested the IVIG-binding Fabs for their reactivity with B cell superantigens.

Table 3.3 Mutation rates in Variable region genes*

VH CDRs 1 and 2

VH FRs 1 - 3

Entire VH region

VL CDRs 1, 2, (3)

VL FRs 1 - 3

Entire VL region

Clone

No./total no. (%)

R/S (ratio)

No./total no. (%)

R/S (ratio)

No./total no. (%)

No./total no. (%)

R/S (ratio)

No./total no. (%)

R/S (ratio)

No./total no. (%)

IVIG-selected Fab phages

SH8†

4/66 (6.1)

3/1 (3)

3/204 (1.5)

1/2 (0.5)

7/270 (2.6)

0/78 (0)

0 (0)

2/177 (1.1)

1/1 (1)

2/225 (0.8)

SH21†

0/66 (0)

0 (0)

1/207 (0.5)

1/0 (∞)

1/273 (0.4)

1/78 (1.3)

0/1 (0)

1/177 (0.6)

1/0 (∞)

2/225 (0.8)

SH44†

0/66 (0)

0 (0)

7/207 (3.4)

4/3 (1.3)

1/273 (2.6)

12/75 (16.0)

9/3 (3)

9/177 (5.1)

6/3 (2)

21/252 (8.3)

SH51

5/66 (7.6)

5/0 (∞)

4/204 (2.0)

3/1 (3.0)

9/270 (3.3)

1/93 (1.1)

0/1 (0)

0/180 (0)

0 (0)

1/273 (0.4)

SH55

1/66 (1.5)

1/0 (∞)

7/204 (3.4)

4/3 (1.3)

8/270 (3.0)

3/84 (3.6)

3/0 (∞)

1/177 (0.6)

1/0 (∞)

4/261 (1.5)

SH58

1/66 (1.5)

1/0 (∞)

1/204 (0.5)

1/0 (∞)

2/270 (0.7)

5/90 (5.6)

2/3 (0.7)

1/186 (0.5)

1/0 (∞)

6/276 (2.2)

SH89†

1/66 (1.5)

0/1 (0)

1/207 (0.5)

0/1 (0)

2/273 (0.7)

1/78 (1.3)

0/1 (0)

0/177 (0)

0 (0)

1/255 (0.4)

Randomly picked clones from the unpanned SH library

Shu2a

7/63 (11.1)

7/0 (∞)

7/204 (3.4)

4/3 (1.3)

14/264 (5.3)

8/90 (8.9)

7/1 (7.0)

3/180 (1.7)

1/2 (0.5)

11/270 (4.1)

Shu8a

2/66 (3.0)

2/0 (∞)

5/204 (2.5)

2/3 (0.7)

7/270 (2.6)

2/84 (2.4)

2/0 (∞)

1/186 (0.5)

1/0 (∞)

3/270 (1.1)

Shu2b

6/66 (9.1)

4/2 (2.0)

9/204 (4.4)

6/3 (2.0)

15/270 (5.6)

5/72 (6.9)

3/2 (1.5)

4/177 (2.3)

3/1 (3.0)

9/249 (3.6)

Shu3b

4/66 (6.1)

4/0 (∞)

4/201 (2.0)

3/1 (3.0)

8/267 (3.0)

10/66 (15.2)

8/2 (4.0)

15/177 (8.5)

8/7 (1.1)

25/243 (10.3)

Shu7b

4/72 (5.6)

4/0 (∞)

2/204 (1.0)

0/2 (0)

6/276 (2.2)

4/75 (5.3)

3/1 (3.0)

5/174 (2.9)

4/1 (4.0)

9/249 (3.6)

*CDRs = complementarity determining regions; FRs = framework regions; no./total no. = number of mutations/total number of bases in the region; (%) = percent;
R/S = number of replacement mutations/number of silent mutations; ratio = R/S ratio; ∞ = infinity (mathematically incorrect).
†Clones selected repetitively included SH8 and SH44 (2X each), SH21 (9X), and SH89 (7X). Refer Table 3.2 for other definitions.

3.11 BINDING OF IVIG-SELECTED FABS TO KNOWN B CELL SUPERANTIGENS

A control study involving an investigation of IVIG Fabs from IgG and IgM phage display libraries from a healthy individual, and the results observed in this study as well as in previous studies involving interaction of IVIG with Fabs from 3 patients with autoimmune thrombocytopenia indicated that the preferential binding of IVIG to Fabs from the V3-23 and V3-30 loci might not be related to the healthy state of the donor or treatment with IVIG. Rather it was indicative of the unique binding of B cell superantigens to immunoglobulins from the VH3 family. This prompted us to examine the binding of the Fabs to Staphylococcal protein A (SpA) and HIV-1 gp120, two of the most characterized B cell superantingens (Silverman, 1997).

↓43

Figure 3.14 Summary of ELISAs with Fab phage supernatants comparing the binding to IVIG and the B cell superantigens staphylococcal protein A (SpA) and gp120. The VH germline origins of the Fabs are indicated. Each specific A450 was calculated by subtracting the value obtained for pComb3H phage supernatants on the respective antigen from the measured A450. Values on IVIG and SpA for clones marked with * were obtained in a second ELISA with appropriate controls, and all values on gp120 were obtained in an independent assay. However, all relative values were confirmed repeatedly in independently assays. SHu7b is a V3-15 unselected control Fab. GG30a is a V4-61 derived platelet-reactive IVIG binder isolated from a patient with idiopathic thrombocytopenic purpura. (Jendryeko et. al., 1998). M1-10 is a V3-30 unselected control Fab from a patient with Kawasaki disease (Leucht et. al., 2001). This clone has an amino substitution of glutamine (E) for lysine (K) at position VH57.

As demonstrated in Fig 3.14, some of the IVIG-selected Fab phages (SH8, SH44, and SH51) displayed a very strong binding to SpA, whilst interactions with gp120 were weak. Except SH44, binding to SpA did not correlate to binding with IVIG, for example, clones SH8 and SH51 showed stronger reaction to SpA than to IVIG. On the other hand, clones SH21(62), SH55, SH58, and SH89, showed higher binding to IVIG than SpA. Fab GG30a which is a VH4 family clone was negative on SpA, this is not an unexpected result. SH7ub an unselected clone, was nearly negative on IVIG but clearly bound SpA.


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