Prof. Dr. Jürgen Mlynek
President of the Humboldt-University of Berlin

• 1  INTRODUCTION

  • 1.1  GENERATION OF ANTIBODIES

  • 1.2 V_H(D)J_H RECOMBINATION AND THE HUMAN IMMUNOGLOBULIN LOCI

    • 1.2.1 The human antibody IgH locus

    • 1.2.2 The human variable light chain locus

  • 1.3 PHAGE DISPLAY TECHNOLOGY

    • 1.3.1 Antibody production by combinatorial libraries

    • 1.3.2 Production of antibody fragments by Phage Display

    • 1.3.3 Phagemid pComb3H

    • 1.3.4 Recombinant antibody gene expression in E. coli

  • 1.4 SYSTEMIC LUPUS ERYTHEMATOSUS

  • 1.5 IDIOPATHIC THROMBOCYTOPENIC PURPURA

  • 1.6 INTRAVENOUS IMMUNOGLOBULINS (IVIG)

  • 1.7 THE IDIOTYPIC-ANTIIDIOTYPIC NETWORK IN AUTOIMMUNITY

  • 1.8 AIMS OF THIS STUDY

• 2 MATERIALS AND METHODS

  • 2.1  PATIENT

  • 2.2 ISOLATION OF LYMPHOCYTES FROM PERIPHERAL BLOOD LYMPHOCYTES

  • 2.3 RNA ISOLATION FROM LYMPHOCYTES

  • 2.4 SYNTHESIS OF Fd, λ AND κ cDNA BY REVERSE TRANSCRIPTION

  • 2.5 RT-PCR AMPLIFICATION OF Fd, λ, AND κ CHAINS

    • 2.5.1 Purification and Quantification of PCR products

    • 2.5.2 Restriction of PCR products

  • 2.6 PREPARATION OF E. coli STRAIN XL1-Blue FOR CLONING

  • 2.7 PREPARTION OF pComb3H VECTOR

    • 2.7.1 Transformation of E. coli strain XL1-blue by High Voltage-Electroporation (Electrotransformation).

    • 2.7.2 pComb3HSS: QIAGEN MIDI plasmid purification

  • 2.8 PREPARATION OF LIGHT CHAIN VECTOR FOR CLONING

  • 2.9 TEST LIGATION-LIGHT CHAIN INSERT

    • 2.9.1 Light Chain Library construction: ligation of Light Chain insert to Light Chain vector

    • 2.9.2 Light Chain Library: Single clones minipreps culture

  • 2.10 THE COMPLETE FAB LIBRARY CONSTRUCTION

  • 2.11 PREPARATION OF THE NAIVE SURFACE DISPLAYED FAB PHAGES

  • 2.12 BIOPANNING

    • 2.12.1 Coating of antigens

    • 2.12.2 Day 1

      • 2.12.2.1 Bacterial culture

      • 2.12.2.2 Panning methodology

    • 2.12.3 DAY 2 (and other subsequent rounds of panning)

  • 2.13 ELISA PROCEDURES

  • 2.14 DETERMINATION AND ANALYSIS OF NUCLEOTIDE AND AMINO ACID SEQUENCES

• 3 RESULTS

  • 3.1  LIBRARY CONSTRUCTION

    • 3.1.1 Isolation of RNA

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

  • 3.2 LIBRARY OPTIMIZATION

  • 3.3 BIOPANNING ON IVIG

  • 3.4 POLYCLONAL FAB PHAGES BOUND BY IVIG

  • 3.5 ISOLATION OF MONOCLONAL FAB PHAGES BOUND BY IVIG

  • 3.6 REACTIVITY OF IVIG-BINDING FABS ON PLATELETS

  • 3.7 HEAVY CHAIN AMINO ACID HOMOLOGY

  • 3.8  V_H AND V_L GENE SEGMENT USAGE OF IVIG SPECIFIC FABS

  • 3.9 SH NON-ANTIGEN SELECTED (UNPANNED) CLONES

  • 3.10 MUTATION RATES

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

• 4 DISCUSSION

  • 4.1  GENERAL REMARKS

  • 4.2 GENETIC ORIGIN OF IVIG-SELECTED HEAVY AND LIGHT CHAINS

  • 4.3 BIASED V_H GENE REPRESENTATION IN THE Ig REPERTOIRE

  • 4.4 BINDING OF IVIG COMPARED TO KNOWN B CELL SUPERANTIGENS

  • 4.5 SUMMARY AND CONCLUSION

  • 4.6 ZUSAMMENFASSUNG UND SCHLUSSFOLGERUNG

• LIST OF ABBREVIATIONS

• DECLARATION

• LIST OF PUBLICATIONS

• CURRICULUM VITAE

• ACKNOWLEDGEMENT

• CITED LITTERATURE

• Table 1.0 The 1982 revised criteria for SLE (Tan et. al., 1982).

• Table 1.1 Human variable and constant region PCR primers used for phage antibody library construction (Kang et. al., 1991b).

• Table 1.2: Sequencing primers.

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

• 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 fever^a

• Table 3.3 Mutation rates in Variable region genes*

• Fig. 1.0 Overview of the Phage display system with pComb3H. Amplified light and heavy chains permits the production of phages expressing antibodies whose genetic information are carried within the phage coat. Desired antibodies are selected by successive rounds of biopanning, and subsequently ELISA on the antigen of interest. Soluble Fabs are produced upon restriction of phage coat protein gene III from the phage genome.

• Fig. 1.1 pComb3H is a modified derivative from the original pComb3 phagemid. The lac Z promoter drives the synthesis of the light chain and Fd/gene III transcript. The two ribosome binding sites initiate the translation of the two separate polypeptide chains; a complete light chain (VL - CL), and the Fd fragment of a heavy chain (VH - CH1) fused to the carboxy-terminal domain of the gene III protein. Random combination of light chain and Fd fragment PCR products which have been independently amplified are cloned into the plasmid to generate a combinatorial antibody library. The leader peptides omp A and pel B target both polypeptides to the bacterial periplasm where the soluble light chain fragment and the membrane-bound Fd fragments associate via a disulfide bond. The Sac I and Xba I restriction enzyme sites are provided for cloning light chain fragments whilst Fd fragments are inserted at Xho I and Spe I restriction sites. Cleavage of pComb3H by the restriction enzyme Spe I (A|CTAGT) and Nhe I (G|CTAGT) which have identical cohesive ends that can be re-ligated results in the removal of the gene III product of M13 producing soluble Fabs. The wild type vector is provided in the form pComb3H-SS where the SS designation is for stuffer fragments. The stuffer fragments have not been well characterized in detail but they lack the restriction sites found in the vector. (After Rader and Barbas, 1997).

• 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.

• 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

• 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.

• 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.

• 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

• 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)

• 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).

• 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.

• Figure 3.10 Amino acid homology tree of representative V_H 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.

• Figure 3.11 Amino acid homology tree of representative V_L 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.

• Figure 3.12 Alignment of V_H 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.

• Figure 3.13 Alignment of V_L 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.

• 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 V_H 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 V_H57.