Production of a Human Monoclonal Antibody Reactive Against Verotoxin- 1

Shanen L. Carter

A thesis submitted in conformity with the requirements for the degree of Master of Science

University of Toronto 1998

Copyright by Shanen Carter, 1998

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Production of a Human Monoclonal Antibody Reactive Against Verotoxin-1, degree of Master of Science, 1998, Sitanen L. Carter, Graduate Department of Medical and Molecuiar Genetics, Micmbiology Program, University of Toronto

Abstract

Verotoxins are produced dunng E-coli 0157:H7 infections and are

associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS). The

elderly and very young children are most likely to develop complications such as

HUS, which can lead to severe rend darnage or death. HUS is too infrequent to

justify the expense of irnmunizing entire populations, however, the development

of an immunotherapy would be very useful dunng E. coli 0157:H7 outbreaks in

closed populations such as daycare centres and nursing homes and for isolated

cases of E. coli 0157:H7 infections. Passive imrnunization of people at risk of

developing HUS would be made possible by the developmeni of human

monoclonal antibodies directed against verotoxins.

In this sîudy. irnrnunoglobulin (Ig) genes From human antibodies specific

for verotoxin- 1 were subcloned during the construction of a combinatorid library

of Ig heavy and light chahs using a recombinant phage antibody system. This is

the fint step in the production of human monoclonal antibodies that may have

therapeutic value in the treatrnent of E. coli 0 157:H7 infections.

TABLE OF CONTENTS

LIST OF FIGURES LIST OF TABLES LIST OF ABBREWTIONS 1.1 LNTRODUCTION

1.2 Epiderniology 1.3 Transmission 1.4 Hemorrhagic colitis 1.5 Hemolytic uremic syndrome 1.6 Thrombotic thrornbocytopenic purpura 1.7 Verotoxins: structure and functions 1.8 Role of verotoxins in pathogenesis of HUS 1.9 Human immune remonse to verotoxins

L

Antibody structure and hinctions Production of monoclonal antibodies Engraftment of SCID mice Human-mouse heteromyelornas Epstein-Barr virus immortaiization of B lymphocytes Production of antibodies using recombinant DNA technology Synsorb Therapy Project rationale Project objectives

2.1 M A T E U C S AND METHODS 2.2 Strains 2.3 ELISA for the detection of antibodies specific for VT 1 2.4 Isolation of peripherd blood lymphocytes 2.5 Animal expenments

2.5.1 Engraftment of NOD-SCID mice 2.5.2 Toxoiding VTI 2.5 -3 immunizing NOD-SCID rnice 2.5.4 Andyzing mouse sera

2.6 Cell culture 2.6.1 Production of Epstein-Barr v i n s 2.6.2 EBV imrnortalization of B lymphocytes

2.7 Isolation of total RNA 2.8 Making a scFv library

2.8.2 First strand cDNA synthesis 2.8.3 Amplification of VH and VL genes

2.8.4 Purification of PCR products 2.8.5 Quantification of purified PCR products 2.8.6 Preparation of linker fragments 2.8.7 Assernbly of VH and VL genes 2.8.8 Addition of restriction sites 2.8.9 Selective purification 2.8.10 Restriction enzyme digest of scFv fragments 2.8.1 1 Ligation of scFv with pCANTABSE

2.9 Construction of an Fab Library 2.1 O Preparation of electrocompetent cells 2.1 1 Electroporation 2.12 Screening Library

2.12.1 Rescue of recombinant phage 2.12.2 Panning phage 2.12.3 Rescue of library following panning 2.12.4 Re-panning 2.12.5 Reinfection with enriched phage 2.12.6 Screening ennched clones

3.1 RESULTS 3.2 NOD-SCID engraftment 3.3 EBV immortalization of human B lymphocytes 3.4 Construction of scFv library #1

3.4.1 Screening of library # 1 3.5 Construction of scFv library #2

3 -5.1 Screening of library #2 3.6 Screening Nguyen library 3.7 Construction of an Fab library

4.1 DISCUSSION 4.2 Engrafting NOD-SCID Mice 4.3 EBV Irnrnortalization of Human PBLs 4.4 Cloning the Variable Heavy and Light Genes 4.5 Construction of a Single Chain Variable Fra,gment Library 4.6 Cornparison of scFv Library to Winter's Library 4.7 Screening of Nguyen Library 4.8 ConcIusion 4.9 Future Considerations

5.1 REFERENCES

LIST OF FIGURES

1 . 1 Structure of verotoxins 1 -2 Structure of antibodies and antibody fragments 1.3 Filamentous phage and Fab expression 1.4 Construction of scFv fragments 3.0 CD45 staining on human lymphocytes 3.1 Amplification of VH and VL genes 3.2 Linking VH and VL genes 3.3 pCANTABSE vector 3.4 Amplification of VH and VL genes for library #2 3.5 Linking VH and VL genes for library #2 3.6 Amplification of heavy and light chains for Fab library

LIST OF TABLES

2.1 Oligonucleotide primers used for PCR of hurnan Ig genes 2.2 Primer sequences used for production of an Fab library 3.1 TT antibody levels in Hu-PBL-NOD-SCID mice 3.2 VTI antibody levels in Hu-PBL-NOD-SCID mice 3.3 VT 1 antibody levels in tissue culture supematants 3.4 VT I antibody levels in 24 day old EBV-immortalized B ce11 cultures 3.5 TT antibody levels in 24 day old EBV-immortalized B ce11 cultures 3.6 VTI antibody levels in 21 and 28 day old EBV-immortalized B ce11 cultures 70 3.7 Phage eluted after panning with VTI 79 3.8 Phage eluted after panning library #2 against VTI 85 3.9 Panning of Nguyen library with VTl 87

LIST OF ABBREVIATIONS

bp C cDNA CDR EBNA EBV EHEC Fab Fc Gb3 H HC HUS IL IFN i.p. J D A L LMP MHC NK NOD NPN PBL PBS PCR scFv scm STn TGF TNF TT TTP VH VL. VT VTEC

base pair constant complementary DNA complementaity determining region Epstein-Barr nuclear antigen Epstein-Barr virus entemhemorrhagic E. coii antigen binding fragment crystallizing fragment g Iobotriaosy lceramide heavy hemorrhagic colitis hemolytic urernic syndrome interieukin in terferon intraperi toneally joining region kilodalton light latent membrane protein major histocompatibility cornplex natural killer non-obese diabetic p-nitrophen y 1 phosphonamidate antigen 1 peripheral blood lymphocyte phosphate buffered saline polymerase chain reaction single chah variable fragment severe combined immunodeficient synthetic carbohydrate antigen transforming growth factor tumor necrosis factor tetanus toxin thrombotic thrombocytopenic purpura variable heavy variable light verotoxin verotoxin producing E. coii

1 . 1 Introduction

E. coli can be grouped according to the Kauffman schema which identifies

different svains on the b a i s of their somatic and flagellar antigens. There are

approximately 17 1 lipopolysaccharide (O) antigens and 56 flagellar (H) antigens

(Levine et al. 1987). E. coli 0157:H7 is a newly recognized and virulent

pathogen which was shown to be the predominant cause of hemorrhagic colitis

(HC) as recently as 1982 (Wells et ai. 1983; Riley et ai. 1983) and of hemolytic

uremic syndrome (HUS) (Kannali. 1989; GriWn et al. 199 1). Other E. coli

serotypes have since been implicated as causes of HC and they are now referred

to as enterohemorrhagic E. coli (EHEC) (Levine. 1987). EHEC are differentiated

from other diarrheogenic E. coli by their ability to produce verotoxins (VT)

(Konowaichuk et al. 1977) and the formation of attaching and effacing lesions.

It is thought that a new therapy c m be developed that rnay neutralize the VTs

thereby amelionting complications associated with such infections.

Two prospective studies in Calgary (Pai et ai. 1988) and in Washington

state (Ostroff et al. 1989) have described the epiderniological attributes of EHEC

infections. In Calgary EHEC were the most cornmon cause of bacteriai diarrhea.

EHEC infection had the highest incidence in children under 5 years of age and in

geriatric patients. In addition it was most prevalent in the summer months. In

Washington state 0157:H7 was the fourth most frequent cause of bacterial

dimhea with 60% of 0157:H7 cases occuning in the summer months. The

highest attack rate also occurred in children under 5 years of age and in penons

over 60 years of age.

Not al1 age groups are at an equal risk for developing 0 157:H7 associated

diseases (Ostroff et ai. 1989). One group at an increased risk are the elderly in

nursing homes. It has been demonstrated that the highest attack rate is often in

the very old (Carter et al. 1987) as well, these patients are twice as likely to

become il1 as the staff caring for them (Riley. 1987; Pavia et al. 1990). Deaths

are more common in the elderly with 0157:H7 infection and do not occur in the

nursing home employee population (Carter et ai. 1987; Pavia et al. 1990). The

second group at an increased risk for developing 0 157:H7 associated diseases are

children in daycare centres and schools where the most susceptible are the

youngest children (Spika et al. 1986; Cimolai et al. 1990). It seems that this

organism has a predilection for patients at the extremes of age.

1.3 Transmission

There is a wide animal reservoir for E. coli O157:H7 organisms. Beef and

dairy cattle, pork, lamb, poultry, and diarrheic cats al1 act as reservoirs (Doyle et

al. 1987; Abaas et ai. 1989). VT-producing E. coli strains can produce HC in

cattle, however, E. coli 0157:H7 can also be can-ied asymptomatically in their

intestinal tract. This organism c m be transmitted via different vehicles including:

undercooked rneat, unpasteurized milk and apple cider, contaminated lake and

potable water. and improperly washed lettuce and bean sprouts (Preston et al.

1 997; Fukushima et al 1997). Person-to-peaon spread also occurs as secondary

E. coli 0 157:H7 infections have k e n seen in nursing homes, day care centres and

in 5% of households with sporadic infections (Spika et al. 1986; Ostroff et al.

1989).

1.4 Hemorrhagic Colitis [HC)

The earliest clinical symptoms of HC are nonbtoody diamhea and severe

abdominal cramps and pain. The hallmark of HC. bloody diarrhea occurs 1-2

days after infection. There may also be vomiting or a low grade fever (Ostroff

et al. 1989). The incubation period is 3-4 days and the average duration of illness

is 7 days (Riley et al. 1983: Ostroff et ai. 1989). Treatment for HC is supportive

care as it is mostly a self-limiting disease.

1.5 Hemolvtic Uremic Syndrome (HUS)

In the 1980s the first observation was made that VTEC was linked with

HUS (Karmali et al, 1983, 1985). Subsequent studies have confirmed an

association between HUS and E. coli 0 157:H7 infections (Cleary. 1988; Neill et

al. 1985) . HUS is the most important sequela of EHEC infections. It is defined

as a syndrome of microangiopathic hemolytic anetnia, thrombocytopenia and acute

rend failure. HUS most cornmonly affects children between 1-4 years of age and

it may result in persistent rend or neurologic disease. HUS commonly occurs 7

days after the onset of HC, at this time the organisms may no longer be present

in stool cultures. HUS occurs in approxirnately 540% of patients with sporadic

HC, however, during outbreaks of HC the risk of developing HUS increases

(Ostroff et ai. 1989; Griffin et al. 1988; Spika et al. 1986; Gransden et al. 1986).

Although HUS is occasiondly associated with persistent rend disease, it

frequently requires dialysis for treamient which is expensive and unpleasant for

the patient.

1.6 Thrombotic Thrombocytopenic Purpura (TTP)

TTP is clinically sirnilar to HUS, however, it is usudly diagnosed in adults

rather than children (Tm. 1995). TTP is rarely preceded by diarrhea (Griffin and

Tauxe. 199 1) but it is often associated with a fever. As weII, there is less renal

injury and more neurologic involvement compared with HUS (Karmali. 1992)

1.7 Verotoxins: Structure and Functions

Verotoxins are composed of two subunits which are noncovalently linked,

a single 3 1 kDa enzymatic A subunit and a pentamer of 7 kDa receptor binding

E3 subunits (Donohue-Rolfe et al. 1984). VTI B subunit and VT1 holotoxin have

been crystallized (Stein et al. 1992; Fraser et al. 1994). See figure 1.1. The A

subunit is activated by proteolytic cleavage resulting in 2 fragments, A, and A2.

The A, fragment inactivates the 60s ribosome leading to the inhibition of protein

synthesis while the A, fragment remains associated with the B subunit (Reisbig

et al. 1981). The B subunit of the toxin binds to the eukaryotic ce11 suxface

receptor glycolipid globotriaosylcerarnide, Gb3 (Jacewicz et al. 1986; Lingwood

et al. 1987).

There are two types of verotoxins: VTI which is almost identical to and

cross-neutralizable with shiga toxin and VT2 and its variants VT2e and VT2c,

which share some homology to shiga toxin, however they are not cross-

neutralizable with shiga toxin antiserum (Kannali et al. 1989). AI1 verotoxins

bind to Gb3. but VT2e binds preferentially to globotetraosylceramide, Gb4

(DeGrandis et al. 1989).

Figure 1 . 1 Structure of verotoxins - Figure A is a top view of the holotoxin, a-carbon structure only. The 5 B subunits form the periphery and the A subunit rests above the centre of the pentameric structure. Figure B is a side view of the holotoxin. The A subunit is shaded yellow. The amino-terminus (N) and carboxy-terminus (C) of the A subunit are labelled. This figure has k e n taken with permission from Bast et al. 1997.

1.8 Role of Verotoxins in the Pathogenesis of HUS

Two virulence factors leading to the pathogenesis of VTEC infections are

the ability of E. coli 0 1 57:H7 organisms to attach to intestinal epithelial cells and

their ability to produce verotoxins. It is the binding of the toxins to Gb3 that

leads to their cytotoxic activities.

Although no circulating VT has k e n observed in the blood it is thought

that HUS is mediated by systemic toxemia (Richardson et al. 1988). It is not

known how VT travels from the intestinal lumen to gain access to the

bloodstream. however, the most likely hypothesis is that VT could translocate non-

specifically across the epithelial cells via transcytotic vesicles (Acheson et al.

1996). VT could then travel through the blood and bind to Gb3 in the kidney.

This is thought to be the initial event leading to the pathological effects of HUS.

It has been demonstrated that there are high levels of Gb3 in cultured

human renal microvascular endothelid cells and that these cells are sensitive to

the cytotoxic effects of VT (Obrig et al. 1993) As well it has been shown that

Gb3 is present in human glomenili (Boyd et ai. 1989). This supports a receptor

mediated action in darnage to endothelium in human renal glomeruli and in

development of microangiopathic lesions.

In addition, cytokines may contribute to the microvascular darnage observed

in HUS patients. Increased levels of TNF-a, IL-6 and IL-8 in the plasma and

TNF-a and IL-@ in the urine of HUS patients have k e n detected (Karpman el

al. 1995; Lopez et al. 1995; van de Kar el al. 1995). Treatrnent of human

monocytes with VT in vitro resulted in ce11 activation and release of the above

mentioned cytokines (van Setten et al. 1996). In addition, it has been found that

TNF-a and IL- 1 enhance the cytotoxicity of VTl towards cultured endothelid

cells by upregulating microvascular Gb3 expression (van de Kar et al. 1991).

These results suggest a hinctional role of cytokines in the progression of

disease.

The binding of verotoxins to their receptors plays an essential role in

verotoxin cytotoxicity. One therapeutic goal is to prevent the toxins from binding

to their receptors in an attempt to prevent further complications such as. HUS.

1.9 Huma. Immune Response to Verotoxins

The immune response in humans following VTEC infections can be quite

unpredictable. Some patients develop anti-VT I antibodies immediately after

VTEC infections and others do not. In one study only 9.1 % of patients with

known VTI exposure developed anti-VTI IgG antibodies (Karmali et al. 1994).

There are reports of patients with HUS who had no antibodies against VTl 13

days after the onset of diarrhea (Karmali et al. 1994). In one case a patient

showed evidence of seroconversion a few months after the initiai infection. Some

people may develop a delayed aniibody response for reasons that are still

unknown.

On the other hand, some people have high levels of antibodies to VTI

which may help to protect them against reinfection at a later time (Karmali et al.

1994). This is common with farm families who are exposed to VTEC more

frequently through the consumption of unpasteurized milk. h one study, 4 1% of

individuals living on dairy farms had antibodies to VTI (Wilson et ai. 1996). It

is thought that their repeated exposure has led to the production of protective

antibodies. as some of the antibodies are able to neuvalize the toxin by preventing

VTI from binding to its receptor (Karmaii et al. 1994).

Rabbits previously immunized with VT 1 or 2 and challenged

intravenously with either toxin are able to clear the toxin by the liver or spleen,

preventing it from locaiizing in the gastrointestinal tract or the central nervous

system (Bieiaszeska et al. 1997). This provides evidence of VT1 and VT2 cross-

neutralization in vivo in the rabbit mode1 and the results suggest that cross-

neutralization is a function of antibodies directed against VT A subunits, as rabbits

immunized with either VT 1 or 2 A subunit were able to clear both toxins, but

those immunized with VT B subunit could only clear the homologous hototoxin.

Hypotheses have evolved which attempt to explain the reasons for poor

antibody responses to VTs. One theory is that there is not enough toxin in the

blood to elicit an immune response (Levine et al. 1992). This occurs during C.

tetani infections. Enough tetanus toxin is produced to elicit biological effects.

However. an antibody response is not tnggered.

An immune response against VT may be MHC restricted (Levine et al.

1992). It has k e n shown that in mice the VTl B subunit antibody response is

dependent on the MHC H-2 haplotype (Bast et al. 1997). Another theory is that

VT may bind to germinal centre lymphocytes. killing IgG committed B cells and

thereby inhibiting the maturation of antibody producing cells (Cohen et al. 1990).

The last theory is that no antibodies are produced against VT in order to avoid

any autoimmune reactions (Maloney and Lingwood. 1994). Amino acid sequences

in VT B subunits resemble both an extracelluar portion of the type 1 interferon

receptor, IFNAR 1, (Lingwood et al. 1992) and sequences of CD 19 (Maloney and

Lingwood. 1994).

The fact that neutralizing antibodies against VT have been found is

encouraging. Commercial preparations of intravenous human immunoglobulin

contain antibodies to VTI and have been used therapeutically to treat a srnail

number of chiIdren with HUS (Sheth et al. 1990). It is not known whether

antibodies against VT2 are produced, as none have been detected to date.

However, human blood does contain a serum lipoprotein that has VT2 neutralizing

rictivity (Caprioli et al. 1994).

1. IO Antibody Sû-ucture and Functions

Irnrnunoglobulins, are multi-functional molecules made up of 2 identical

55 or 70 kDa heavy chains and 2 identical 24 kDa light chains. One light chain

is attached to each heavy chah by a disulfide bond and the two heavy chains are

attached to each other by 2 disulfide bonds. See figure 1.2.

Humans have 5 different classes or isotypes of antibodies. The constant

regions of dl antibodies within an isotype share extensive regions of amino acid

sequence identity. This shared identity is responsible for the cornmon properties

and effector functions within an isotype. The isotype is determined by the class

of heavy chain for each antibody. Therefore, it follows that there are 5 different

types of heavy chains: y, p, cc, E, and 6. Conversely. there are only 2 isotypes of

antibody light chains, called K and h. The light chains do not mediate effector

functions of antibodies.

The amino acid sequences of the amino terminal domains of both heavy

and light chains are cdled variable (V) regions and the more conserved areas in

the remainder of each chain are cailed the constant (C) regions. There are 3

highly divergent siretches within the V regions of both heavy and light chains

called hypervariabie regions or cornplernentarity-determining regions (CDR).

These 3 regions are flanked by more conserved frarnework regions. The 3 CDRs

of both heavy and light chains form the antigen binding site of the antibody

molecule. The carboxy terminal domains of antibodies rnediate the effector

functions such as: activation of complement. opsonization, and antibody-

dependent ceIIular cytotoxicity.

Digestion of an antibody with papain produces two Fab (antigen binding

fragment) fragments and one Fc (crystallizing fragment) fragment. Digestion with

pepsin produces one F(ab'), fragment with two antibody binding sites and one Fc

fia-ment. Single chain Fv fragments consist of one variable light chain joined

via a peptide linker to one variable heavy chain. See figure 1.2.

Figure 1.2 Structure of antibodies and antibody fragments

Single Chain Fv

1.1 1 Production of Monoclonal Antibodies

Hybndoma technology has been used to produce large quantities of mouse

monoclonal antibodies with defined antigen specificities (Kohler et al. 1975).

These mouse monoclonal antibodies are now widely used for medical research

purposes and to a smaller extent for clinical purposes, such as immunotherapies.

The major problem with the use of mouse monoclonal antibodies for

immunotherapy is that they are immunogenic, causing hurnan anti-rnouse antibody

responses (Honimi et al. 1993). Modifications can be made which lessen the

i mmunogenicity of these antibodies. Mouse-human c himeric an tibodies

(Boulianne et al. 1984) or humanized rnouse monoclonal antibodies (Verhoeyen

et ai. 1988) can be made, however, neither are as good as human monoclonal

antibodies for reducing the recipient's immune response against these molecules.

There are a handful of approaches currently being used to produce hurnan

monoclonal antibodies. Human hybridoma cell lines have been made (Kohler and

Milstein. 1975), however, they are dificult to maintain because they c m

spontaneously lose chromosomes (Larrick et al. 1989). Severe cornbined

immunodeficient mice have ken engrafted with human peripheral blood

lymphocytes (PBLs) and then immunized with a certain antigen to elicit a human

immune response to the chosen antigen (Sandhu et al. 1994). This source of

lymphocytes can be used for the production of human monoclonal antibodies

(Carlsson et al. 1992). Human B lymphocytes specific for a ceratin antigen have

been immortalized with Epstein-Barr virus (EBV) to produce human monoclonal

antibodies (Mosier et al. 1988). More recently. recombinant DNA technology has

been used to produce human monoclonal antibodies and antibody fragments

against an antigen of choice (Huse et al. 1989; Persson et al. 1991: Marks et al.

1991).

1.12 Ennraftment of Severe Combined immunodefecient Mice

Severe combined immunodeficient (SCID) mice are devoid of functional

humoral or cell-mediated immune responses, because they lack B and T

lymphocytes (Bosrna et al. 1983). However, they do have macrophages and

natural killer cells. Their immune deficiency is due to a recessive gene mutation

which affects the recombination activity in the germline antibody and T ce11

receptor genes (Kirchgessner et al. 1995). The SCID defect allows for human

immune system transfer. An efficient method to engraft human penpheral blood

lymphocytes in SCXD mice has been developed (Sandhu et al. 1994). This animal

model of the human immune response is useful since, obviously, it is not ethical

to adrninister experimental biological materids to human volunteers in order to

elicit an immune response. The Hu-PBL-SCID mouse model cm allow human

antibodies of desired specificity to be safely generated in animals.

The presence of functioning natural killer (NK) cells is one barrier to high

levels of Hu-PBL engraftment in SCID mice (Shpia et ai. 1994; Dorshkind et al.

1985) Pretreatrnent of SCID mice with anti-asialo GM-1 antiserum and radiation

work in concert to decrease the number of active NK cells, thereby increasing the

efficiency of engraftment (Shpitz et al. 1994; Sandhu et al. 1994). However, non-

obese diabetic severe combined immunodeficient (NOD-SCID) mice were chosen

for the present study because they have very few NK cells (Shultz et al. 1995).

Therefore, a high eficiency of engraftment can be achieved using radiation

pretreatment done.

Secondary immune responses have been generated in Hu-PBL-SCID mice

against antigens such as tetanus toxin, hepatitis B core antigen and keyhole limpet

hemocyanin (Maricham and Donnenberg. 1992; Duchosal et al. 1992). More

recently, primary immune responses have dso k e n generated in Hu-PBL-SCID

mice against antigens such as keyhole limpet hemocyanin, circurnsporozoite

malaria parasite and a synthetic carbohydrate antigen (STn), which is a B ce11

epitope of the tumor-associated antigen, TAG72 (Sandhu et al. 1994). This means

that the Hu-PBL-SCID mouse model provides a way to bypass the ethical

restraints of immunizing human volunteen while still enabling the rich memory

cornpartment of human antibody responses to be tapped. thereby allowing for

novel human immune responses to be elicited. Following detection of the

production of human antibodies, specific monoclonal Fab fragments can then be

isolated from the mice by repertoire cloning (Duchosal et al. 1992). This

recombinant DNA technology will be discussod later.

1.1 3 Human-Mouse Heteromvelomas

Monoclonal antibodies against many different antigens have been produced

by fusing human B cells with mouse myeloma ce11 lines (Kohler et al. 1975). The

problem with this technique is that it leads to a preferential loss of human

chromosomes and instability of the hybrids (Roder et al. 1986).

1.14 Epstein-Barr Virus Immortalization of B Lymphoc-es

One method of producing human monoclonal antibodies is carried out by

immortalizing human B cells with Epstein-Barr virus (EBV). EBV is a double

stranded DNA virus of the herpesvims family. It infects human B cells by

binding specificdly to CD2 1 also known as the type 2 complement receptor, CR2

(Cooper et al. 1988). Following EBV infection a series of moIecular events occur

which act in concert to allow the B ce11 to proliferate in culture indefinitely.

A cellular activation cascade is initiated which includes: uniquely shaped

transmembrane ca2+ currents, Na+ /++ antiports not normally used in activated B

cells. tyrosine phosphorylation and transcriptional activation of p5fikk, hsp7* and

hsp9' (Cheung et d. 1993). In addition viral latency genes are expressed.

Transcnpts of at least 4 latency genes are present within 30 min of EBV infection:

Epstein-Barr nuclear antigens (EBNA-1 and EBNA-2) and latent membrane

proteins (LMP- 1 and LMP-2) (Cheung et al. 1993). Interference with any of the

above events will prevent transformation (Dosch et al. 1990).

The EBV BCRFl gene has k e n found to be important for viral latency.

It is dso known as the viral interleukin 10 gene (VIL-IO) as it shares most

functions with its homologue, human IL- IO (Moore et al. 1990). The VIL- IO gene

is expressed within 6 hours of transformation (Miyazaki et al. 1993). VIL-10

works as a potent B ce11 growth prornoter (Roussset et al. 1992) and it suppresses

IL-2 (Taga et al. 1992) and IFN-y production (Ding et al. 1992). IL-2 activates

T cells and IFN-y upregulates expression of MHC class 1 molecules in infected

cells.

EBNA-5 is also required for B ceIl transformation. It binds both

retinoblastoma and p53. 2 major tumor suppressor proteins (Szekely et al. 1993).

By irnpairing retinoblastoma and p53 which both hnction as ce11 cycle

checkpoints the B cells may be allowed to enter the mitotic cycle (Szekely et al.

1993).

Another change which promotes B ce11 immortalization is the loss of high

affinity TGF-P receptors on the surface of EBV infected B cells (Kumar et al.

1991). TGF-P acts as a negative regulator for proliferation of B cells, therefore,

the loss of these receptors lead to TGF-P resistance and uncontrolled growth of

the B ce11 (Kumar et al. 1991).

There are many drawbacks to this method of obtaining monoclonal an tibodies.

EBV immortalization of B cells produces unstable ce11 Iines which can lose their

ability to produce antibodies at any time (Roder et al. 1986). Resting B cells are

irnmortalized in preference to activated B cells (Aman et al. 1984), which is

problematic because immortalization of antibody producing cells is desired.

Finally, the efficiency of transformation is very low with less than 10% of EBV-

infected B cells reaching a state of stable transformation (Miyazaki et al. 1993).

1.15 Production of Antibodies Using Recombinant DNA Technologv

This past decade has brought with it much progress in the field of

recombinant DNA technology and antibody engineering. It is now possible to

clone the antibody genes of a B lymphocyte producing an antibody of interest and

express these genes as antibodies or antibody fragments in mammalian cells, yeast

or bacteria (Better et al. 1988; Skerra et al. 1988). This represents immortalization

of human heavy (H) and light (L) chain genes without the problems associated

with maintaining human-mouse heterornyelomas or EBV-immortalization of B

lymphocytes.

In 1989, Huse and colleagues made a combinatorid library of Fab

fragments of the mouse antibody repertoire (Huse et al. 1989). Their expression

libraries were constructed in bacteriophage h. 106 H chain genes from one library

were randomly recombined with 106 L chah genes from a second library

producing a Fab expression library of 2 . 5 ~ 107 clones. 9x 104 clones from this

library were screened for their ability to bind p-nitrophenyl phosphonamidate

antigen 1 (NPN) using nitrocellulose filters. 6 clones were found which bound

specifically to NPN (Huse et al. 1989).

In 199 1, Penson and colleagues produced an Fab combinatorial library of

human H and L chain genes using 5x10~ peripheral blood lymphocytes from a

human volunteer who had been imrnunized against tetanus toxin. Their Fab

library consisted of 1d clones which were screened using nitrocellulose filters.

1 in 5000 clones tested could bind to tetanus toxin (Persson et al. 1991).

Fab combinatorial librarïes cloned in h vecton are useful because they

allow for the expression of Fab fragments which behave as whole antibodies with

respect to antigen recognition. As well, the combinatorial properties of H and L

chahs serve as an important source of divenity. However. this method

necessitates the screening of large numbers of individual clones on nitrocellulose

filters which can be very laborious and time consuming.

In vivo, B lymphocytes display antibodies on their surface and contain the

antibody genes within the cell. Likewise, filamentous phage can provide the sarne

genetic display package and can be selected with antigen. The filamentous phage

system c m also rnimic B lymphocytes in the ability to express soluble or surface

displayed antibodies. This is accomplished by the insertion of an amber stop

codon between the variable (V) genes and gene III which codes for g3p coat

protein. See figure 1.3. When the phages are grown in suppressor strains of E.

coli, the stop codon is read through and full size fusion proteins are produced and

expressed on the tip of the phage. Nonsuppressor strains recognize the arnber stop

codon and terminate transcription. This event produces soluble antibody

fragments which are secreted into the bacterial periplasm (Hoogenboom et ai.

1991) and can then be secreted into the culture medium (Marks et ai 1991).

Figure 1.3 Filamentous phage and Fab expression

Antibody genes

- . gene III

Barbas and colleagues developed the filamentous phage expression system

to overcome the limitation of screening large numbers of clones by plaque lifts.

They cloned human H and L chain fragments independently and randornly

recombined them in a phagemid vector, pComb3 (Barbas et al. 199 1). Phagemids

are plasmids that can be packaged into phage particles, but require the use of

helper phage (Vieira and Messing. 1987). The pComb3 expression vector allows

Fab fragments to be displayed as fusion proteins. The Fab fragment is attached

to the arnino-terminus of the filamentous phage coat protein, g3p, which in turn

is attached to the phage tip. 3-5 copies of g3p are present on each phage (Glaser-

Wuttke et al. 1989). Screening of these clones is accomplished by panning the

clonally mixed phage over a solid-surface coated with purified antigen. Phage

which do not contain reactive scFv are washed away and the bound phage are

eluted with acid.

Another phagemid, pComb8. has also been used as an Fab expression

vector (Chang et al. 1991; Huse et al. 1991). In this case the Fab fragments are

fused with the phage coat protein g8p of which there are approximately 2700

copies per phage (Glaser-Wuttke et al. 1989). The pCornb8 system is less

advantageous than the pComb3 systern because the increase in copy number,

which increases the avidity of the phage for the antigen and decreases the required

affinity of the individual antigen Fab interaction, yields a much higher nonspecific

background. In this way multivalent display cm be useful when searching for a

rare or low affinity Fab molecule.

In 199 1, Marks and colleagues developed a system where they cloned

single chain variable fragments (scFv) using human PBLs from a non-immune

individual. scFvs consist of the VH gene bound to the VL gene by a 90 bp

neutral DNA linker. Their method was an improvement on the previous

techniques since only one combinatorid library was made rather than making

individual libraries for H and L chain genes and recombining them later. They

produced a library of IO' clones displayed on the surface of filamentous phages

and detected high affinity scFv binders to their antigen of choice, nirkey egg-white

lysozyme (Marks et al. 1991). They hypothesized that a single large phage

display library could be used to isolate human antibodies against any antigen using

their techniques.

Cloning of human VH and VL genes is performed by RT-PCR as follows.

First strand cDNA synthesis employs primers that bind to the highly conserved 5'

end of the constant (C) domains. This reverse transcription reaction requires only

one primer for each isotype. The first framework region within the 5' end of VH

and VL chains is another highly conserved area (Orlandi et al. 1989) for which

complementary backwards primen have been designed. Prirners that bind to the

highly conserved 5' end of the joining (J) regions have aiso been designed and are

used together with the backwards primers to ampli@ H and L chains. The VH

and VL genes are linked together via PCR using a 90bp DNA fragment which

codes for an inert, flexible linker peptide. The full length scFv fragments cm be

cloned into the phagernid expression vector and screened for specific antigen

bi nders (Marks et al. 199 1 ). Figure 1.4 diagrams these procedures.

In many cases hiIl length imrnunoglobulins may be more desirable than

Fab fragments or scFv fragments. These fragments can be converted to full length

monoclonal antibodies by removd of the VH and VL domains from the bacterial

expression vector and subcloning into individual marnmalian expression vectors

containing the corresponding irnmunoglobulin H and L chain constant regions

(Ames et al. 1995; Tsui et ai. 1996). The new vector c m then be CO-transfected

into a marnrnalian ce11 line such as, COS cells which secrete the monocIona1

antibodies so that they can be purified from the supernatant. For large scde

preparations bacterid (E. coli) or yeast (Saccharomyces cerevisiae) expression

systems c m be used.

Figure 1.4 Construction of a çcFv library

[a) 1 st strand cDNA synthesis mRNA

1 st strand VH-CHI cDNA i 1 st strand V,-CL cDNA .

Primary PCRs HuVHBACK +

1 st strand VH cDNA f- 4 HuJHFOR

1 st strand V ~ C D N A \L HuJLFOR

1

: VLCDNA a

(Cl PCR assembly HuVHBACK -

1

VL f--- - Linker DNA

Reamplification with primers containing restriction sites

Vii ScFv linker

1.16 Svnsorb Therapy

VTI and VT2 bind with high affinity to Synsorb-Pk, a silicon dioxide

sand-like material covdentiy coupled to a synthetic Pk trisaccharide (Armstrong

et al. 1991). It was hypothesized that Synsorb-Pk may be usehil in preventing

HUS and a phase I study was conducted to determine possible side effects

associated with oral consumption of Synsorb-Pk by healthy adults. No adverse

side effects were reported Synsorb-Pk recovered from stools retained its ability

to absorb VT and neutralized VT in vitro when rnixed with stools from HUS

patients (Armstrong et al. 1995)

Unfominately phase II trial results were not as encouraging. The patients

enrolled in this study suffered from diarrhea. However, many of them were not

infected with E. coli 0157:H7. Synsorb-Pk did not significantly reduce the

duration of illness for these patients. These results were presented in part at the

VTEC '97 Meeting (Armstrong et al. 1997). It is important to give patients

immunotherapies as soon as an 0 157:H7 infection is suspected, keeping in mind

that these cases are more predominant in the summer months. There are two

methods used for rapid detection of OL57:H7 infections: a toxin test and an 0157

antigen detection test.

1.17 PROJECT RATIONALE

HUS is too infrequent to justify the expense of irnmunizing entire

populations. However, the development of an immunotherapy would be very

useful for VTEC outbreaks in closed populations such as daycare centres and

nursing homes and for isolated cases of VTEC infections. Passive immunization

of people at risk of developing HUS would be made possible by the development

of human monoclonal antibodies directed against verotoxins.

1.18 PROJECT OBJECTIVES

The objectives of this study were to: identify human B cells producing

anti-VTI antibodies and to clone the variable heavy (VH) and variable light (VL)

genes for these VT-specific antibodies into an appropriate expression vector.

Initially B cells were to be immortalized and RT-PCR would be performed using

RNA from specific clones. In addition, Hu-PBL-NOD-SCID mice were

immunized with VT in order to enhance the number of VT specific human B

cells.

2.2 Strains

E. coli TG 1 : K 1 2A(lac-pro), supE, thi, hsdAYF' (truD3 6, pro AB, lacIq.

1acZA.M 15) (Maniatis et al. 1989) was used for propagating the recombinant phage

expression vector, pCANTABSE (Pharmacia 27-9401-0 1). B95-8 cells (Miller et

al. 1973; ATCC #CRL 16 12) were used to propagate Epstein-Barr virus, M 13KO7

helper phage (Vieira et al. 1987; Pharmacia 27-9401B) was used to coinfect TG1

ceils.

2.3 ELISA for the detection of antibodies specific for VTI or TT

Each well of an Immulon- 1 96 well microtitre plate (Dynatech Laboratories

Inc.) was coated with O. lpg of VTl or tetanus toxin (TT) in 100 pi

carbonate/bicarbonate buffer pH 9.6 (Sigma). The plate was covered.incubated at

4°C ovemight and washed 3 times with wash buffer ( IxPBS-0.05% Tween 20

(BDH Inc.)). Each well was blocked with 5% BSA (electrophoresis grade, Sigma)

in IxPBS-0.05% Tween 20. The plate was incubated ovemight at room

temperature, then washed 3 times with wash buffer. The sera were diluted 1/200

in IxPBS-0.05% Tween-3% BSA and lûûpi of the dilution was added to each

well. The plate was incubated for 1 hour at 37°C and washed 3 times with wash

buffer. The anti-human IgG antibody conjugated to HRP (MO-Rad) was diluted

1/5000 in 1xPBS-O.OS%Tween-3%BSA and 100pi was added to each well. The

plate was incubated for 1 hour at 37°C and washed 3 times with wash buffer.

100@ of soluble peroxidase substrate (OPD tablets, Sigma) was added to each

well, the plate was covered with foi1 and incubated at room temperature for 15-20

min. 30pI of 3M sulfuric acid (BDH Chemicals) was added to each well and then

the opticai density of each well was measured at 490 nm using a microplate reader

(Dy natech).

2.4 Isolation of ~eripherai blood lymphocvtes

lOml of peripheral blood was taken from a volunteer using a vacutainer

tube containing sodium heparin. The whole blood was diluted 1: 1 with lxPBS

without Ca2+ and Mg+ (media preparation, Mount Sinai Hospital). LM of diluted

blood was overlaid on 3mI of Ficoll-Paque (Pharmacia) and spun at 1800rpm for

30 min at room temperature. The plasma was discarded and the bufi coat layer

was transferred to a new tube. The tube was filled with lxPBS and spun at

1800rpm for 5 min at room temperature. The pelleted cells were resuspended in

lxPBS and washed twice.

3.5 Animal Experiments

2.5.1 Engraftment of NOD-SCID mice

3 6-8 week old NOD-SCID mice (Jackson Labs, Maine) were irradiated

with a dose of 3 Gy y-radiation and immediately given an i.p. injection of 2 . 5 ~ 10'

human peripheral blood lymphocytes suspended in 0.5ml of RPMI 1640 (media

2.5.2 Toxoiding VT1

Glutaraldehyde (Sigma) was added to a punfied VTI suspension to make

a 0.1 % glutaraidehyde solution. The suspension was mixed well and incubated

at 4°C overnight.

2.5.3 Immunizing NOD-SCID mice

2 mice were imrnunized with toxoided VTl and 1 mouse with IxPBS.

50pg of toxoided VTL in 100N PBS was mixed with 100N of a 1: 10 mixture of

complete Freund's adjuvant (Gibco): incomplete Freund's adjuvant (Gibco). 2Wpi

of this mixture was injected i.p into a NOD-SCD mouse. Altematively, 100@ of

PBS was mixed with 100pI of the sarne 1:10 mixture of Freund's cornplete and

incomplete adjuvants and 200p.i was injected i.p. into a control NOD-SCID mouse.

When immunizing mice with TT the same procedures were used.

2.5.4 Anahsis of mouse sera

Blood was taken from the tail vein of the 2 surviving mice 3 weeks after

immunization. The mouse sera were then tested for the presence of human anti-

VT 1 IgM antibodies using the ELISA that was described in section 2.3.

2.6 CeII culture

2.6.1 Production of Epstein-Barr virus

106 B95-8 cells were suspended in 5ml of RPMI 1640 + 1OBFCS. The

celIs were cultured for 1 week in a COT hurnidified incubator at 37°C. Medium

was replaced weekly. The cells were grown for 6-7 weeks, at which tirne they

were transferred to a 30°C CO, hurnidified incubator and grown for 1 week

without meduim replenishment. Cells were pelleted. the supernatant was clarified

through a 0 . 4 5 ~ filter (Sarstedt), and lm1 aliquots were stored at -70°C.

2.6.2 EBV imrnortalization of B iym~hocvtes

Sera from 15 volunteers were tested for the presence of anti-VTl

antibodies by ELISA. as described in section 2.3. Whole blood was taken from

a donor found to have anti-VTl IgG antibodirs. the PBLs were isolated as

described in section 2.4 and infected with EBV as follows: the PBLs were

washed 3 times. resuspended in 3mi of supernatant containing EBV that had been

warmed to 3PC and incubated for 2 hours at 37°C in the C O incubator. After

infection. the cells were plated in 96-well tissue culture plates (ICN Biomedicals

Inc.) at either 30,000 cells/well or 100,000 cells/welI aliquots. The cells were lefi

in culture for 3 weeks.

After 3 weeks, 50p.i of supernatant was removed from each well, rnixed

with 50p1 of blocking buffer (1XPBS-0.05%Tween20-3%BSA) and tested for the

presence of anti-VT 1 or anti-TT antibodies by ELISA as described in section 2.3.

l00pl of fresh RPMI 1640 with 20% FCS was added to each well and the plates

were incubated for another week, at which time the wells that initially contained

anti-VT 1 antibodies were tested again to detexmine whether the cells continued to

produce antibodies. Cells from wells that rernained positive for the presence of

specific antibodies by an ELISA were collected and pelleted for subsequent RNA

extraction.

2.7 Isolation of totd RNA

Isolation of total RNA was performed using TRIzol Reagent (GibcoBRL)

according to the manufacturer's total RNA isolation protocol. 1 ml of TRIzol was

used for resuspending and homogenizing 5x10~ PBLs. The cells were incubated

at room temperature for 5 min after which time 200pl of chloroform was added

for every 1 ml of TRIzol used. The tubes were shaken vigorousiy by hand for 15

seconds and then incubated for 3 min at room temperature. The sarnples were

then spun at 12 O x g for 15 min at 5°C. The upper aqueous phase was

transferred to a new RNase free 1Sml tube. The RNA was precipitated from the

aqueous phase by adding 500pI of propanol per lm1 TRIzol Reagent used. When

total RNA was isolated from less than 106 cells 10pg of RNase-free glycogen

(Boehringer Mannheim) was added to the aqueous phase prior to RNA

precipitation. The sarnples were rnixed by vortexing and incubated for 10 min at

room temperature followed by a spin at 12 000 x g for 10 min at 5°C. The

supernatant was removed and the RNA pellet was washed with lm1 of 70%

DEPC-ethanol. The sample was spun at 7 500 x g for 5 min at 5"C, the

supernatant was removed by aspiration and the pellet was air dried for 10 min.

The RNA pellet was resuspended in 20pI of DEPC-H,O and incubated for 1 O min

at 55°C before being stored at -70°C. RNA integrity was confirrned by 1%

agamse gel electrophoresis separation.

2.8 Constructing a scFv Library

2-8.2 First strand cDNA synthesis

Fint strand syntheses were accomplished using isotype specific constant

region primen for the heavy chains and a K constant region primer for the light

chains. In an RNase free tube, 20pmol of specific primer (Cy, Cp, or CK), 3pl of

total RNA from the 20pi preparation (approxirnately 0.5pg) and 8pI of DEPC-HIO

were rnixed and heated for 10 min at 70°C. The tube was then placed on ice for

I min and the contents spun briefly. 1pi of RNAguard (Pharmacia), 4ul of 5x

first strand buffer(GibcoBRL), 2pl of O. 1M DïT (GibcoBRL) and lpi of lOmM

dNTP mix (Pharmacia) were added, gently rnixed and incubated for 2 min at

42°C. lpl of Superscnpt II reverse transcriptase (GibcoBRL) was gently mixed

into the reaction tube and incubated for 50 min at 42°C. This was followed by

an incubation for 15 min at 70°C and storage at -70°C. The primes used for first

strand synthesis and the remaining PCR amplification reactions are listed in table

3.1 (Winter et al. 1991).

Table 2.1 Oligonucleotide primea used for PCR of human immunoglobulin genes

A. First strand cDNA synthesis Human heavy chah constant region primers

HuIgG 1 -4CH 1 FOR 5'-GTC CAC CIT GCT GTT GCT GGG CIT-3' HuIg MFOR 5'-TGG AAG AGG CAC GTT C l T TTC TTT-3'

Human K constant region primer HuGKFOR 5'-AGA CTC TCC CCT GTT GAA GCT CTC-3'

B. Primary PCR Human VH back primers HuVX 1 ai3 ACK 5'-CAG GTG CAG CTG GTG CAG TCT GG-3' HuVH2aB ACK 5'-CAG GTC AAC T'TA AGG GAG TCT =-3' HuVH3aBACK S'-GAG GTG CAG CTG GTG GAG TCT GG-3' HuVH4aBACK 5'-CAG GTG CAG C'TG CAG GAG TCT GG-3' HuVHSaBACK S'-GAG GTG CAG CTG TTG CAG TCT GC-3' HuVH6aBACK 5'-CAG GTA CAG C'TG CAG CAG TCA GG-3'

Human JH forward primers HUM 1 -2FOR 5'-TGA GGA GAC GGT GAC CAG GGT GCC-3' HuJH3FOR 5'-TGA AGA GAC GGT GAC CAT TGT CCC-3' HuJH4-5FOR 5'-TGA GGA GAC GGT GAC CAG GGT TCC-3' HdH6FOR 5'-TGA GGA GAC GGT GAC CGT GGT CCC-3'

Human VK back primers HUVK 1 aBACK 5'-GAC ATC CAG ATG ACC CAG TCT CC-3' HuViCLaBACK 5'-GAT GTT GTG ATG ACT CAG TCT CC-3' HuVK~~BACK 5'-GAA A T GTG TTG ACG CAG TCT CC-3' HuVK~~BACK 5'-GAC ATC GTG ATG ACC CAG TCT CC-3' HuVKS~BACK S ' - G u ACG ACA CTC ACG CAG TCT CC-3' H u V K ~ S A C K 5'-GAA A'IT GTG CTG ACT CAG TCT CC-3'

Human JK forward primers HUJK 1 FOR 5'-ACG T'TT GAT TTC CAC CTT GGT CCC-3' HuJIQFOR 5'-ACG TTT GAT CTC CAG CTT GGT CCC-3' HuJK~FOR 5'-ACG IT' GAT ATC CAC l'TT GGT CCC-3' HuJK~FOR 5'-ACG l'TT GAT CTC CAC CTT GGT CCC-3' HuJKSFOR 5'-ACG T'IT AAT CTC CAG K G TGT CCC-3'

TabIe 2.1 continued.

C. P CR assembly Reverse JH for scFv linker RHuTH 1-2 5'-GCA CCC TGG TCA CCG TCT CCT CAG GTG G-3' RHuPH3 5'-GGA CAA TGG TCA CCG TCT CTT' CAG GTG G-3' RHuJH4-5 5'-GAA CCC TGG TCA CCG TCT CCT CAG GTG G-3' RHuTH6 5'-GGA CCA CGG TCA CCG TCT CCT CAG GTG C-3' Reverse VK for scFv linker RHuVKI~E~ACKFV 5'-GGA GAC TGG GTC ATC TGG ATG TCC GAT CCG CC-3' RHuVIQ~BACKFV 5'-GGA GAC TGA GTC ATC ACA ACA TCC GAT CCG CC-3' RHuVK~~BACKFV 5'-GGA GAC TGC GTC AAC ACA A I T TCC GAT CCG CC-3' EWuVic4aBACKFv 5'-GGA GAC TGG GTC ATC ACG ATG TCC GAT CCG CC-3' RHuVicSaBACKFv 5'-GGA GAC TGC GTG AGT GTC G l T TCC GAT CCG CC-3' RHuVK~~BACKFV 5'-GGA GAC TGA GTC AGC ACA A ï T TCC GAT CCG CC-3'

D. Rearnplification with primers containing restriction sites

Human VH back primes HuVH 1 aBACKSfi

HuVH2aBACKSfi

HuVH3aBACKSfi

HuVH4aBACKSfi

HuVHSaBACKSfi

HuVH6aBACKSfi

Human JK forward HUJK 1 B ACKNot

HuJK2BACKNot

Huk3BACKNot

Hdic4BACKNot

HuJ SBACKNot

5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG CAG CTG GTG CAG TCT GG-3' 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTC AAC TTA AGG GAG TCT GG-3' 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC GAG GTG CAG CTG GTG GAG TCT GG-3' 5'-GTC CTC GCA ACT GCG GCG CAG CCG GCC ATG GCC CAG GTG CAG CTG CAG GAG TCG GG-3' 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTG CAG CTG T'TG CAG TCT GC-3' 5'-GTC CTC GCA ACT GCG GCC CAG CCG GCC ATG GCC CAG GTA CAG CTG CAG CAG TCA GG-3'

primen 5'-GAG TCA 'TTC TCG ACT TGC GGC CGC ACG TTT GAT TTC CAC CTT GGT CCC-3' 5'-GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT CTC CAG C'Tl' GGT CCC-3' S'-GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT ATC CAC TTT GGT CCC-3' S'-GAG TCA TTC TCG ACT TGC GGC CGC ACG TTT GAT @TC CAC CTT GGT CCC-3' 5'-GAG TCA ?TC TCG ACT TGC GGC CGC ACG TTI' AAT CTC CAG TCG TGT CCC-3'

2-8.3 Amdification of V H and VL genes

7 primary polymerase chain reactions were performed, 6 for the heavy

chains and 1 for the light chains. Each reaction consisted of a unique primer pair.

In a 200pi PCR tube (Stratagene) lpl of back primer (2ûpmoVpl), 1pl of forward

primer (2OpmoVpi). 2jd of first strand cDNA (2pmol), lOjd of 10 x PCR

NEBuffer2 (New England Biolabs) and 56p.l of HPLC grade HZO (Fisher

Scientific) were rnixed gently by pipetting. Each reaction mixture was overlaid

with 50p1 of minera1 oiI (Fisher Scientific) and heated to 94°C for 5 min in a

thermal cycler (S tratagene Robocycler). In a 1 Sm1 eppendorf nibe the master mix

was prepared, 30pl for each reaction mixture. This consisted of lpi of VENT

polymerase (2000U/ml, New England Biolabs), 4pI of lOmM dNTP mix

(Pharmacia Biotech) and 25p.l of HPLC HzO. The master mix was heated to 65°C

and 30pl was added to each PCR tube undemeath the minerai oil by pipetting up

and down to mix. The reaction mixtures were left to cycle 40 times (94°C for 1.5

min, 57°C for 1.5 min and 72°C for 1.5 min) with a final 10 min incubation at

72°C. PCR products were stored at 4°C if they were not used immediately.

2.8.4 Purification of PCR products

The PCR products were separated on a 296 low melting point agarose gel

(ultrapure, GibcoBRL) with ethidium bromide (50pg/100ml gel). The gels were

run at lOOV until the Coomasie blue dye (BIO-Rad) from the loading buffer had

migrated 15cm. The gels were photographed using a üV light source, the specific

bands containing the heavy and light chahs were cut out of the gel, and each was

purified using a Geneclean II kit (BI0 10 1 Inc.). Briefly, each agarose band was

transferred to a tube, weighed and 3 volumes of Na1 stock solution added. Tubes

were incubated at 55OC for > 5 min, until the agarose was completely melted. 10

of Glassmilk suspension was added to each tube and mixed by vortexing

briefly. The tubes were incubated on ice for 30 min with vortexing every 2 min.

The pellets were washed 3 times by centrifuging each tube at 14 000 rpm in a

rnicrocentrihige for 2 min, aspirating the supematant and then resuspending the

pellet in 500N of NEW wash buffer. The DNA was collected by resuspending

the pellet in 30p1 of HPLC H,O, incubating at 55OC for 5 min, centrihiging for 3

min and carefully removing the supematant containing the DNA. The elution was

repeated once and the second supematant cDNA fractions were pooled.

2.8.5 Quantitation of purified PCR products

1/20 of each purified cDNA product was separated using a 1.5% agarose

oel (ultrapure, BIO-Rad) with ethidium bromide (50pV100ml gel). The gel a

electrophoresis was perfonned using 100 V until the Coomasie blue dye had

migrated 10 cm. In adjacent wells known concentrations of a 600 base pair

fragment were separated simultaneously to estimate the concentration of PCR

product. using a W light source.

2.8 -6 Preparation of Iinker frapments

To make the scFv linker DNA, 24 separate polymerase chah reaction

mixtures were prepared using each of the 4 reverse .TH primers in combination

with each of the 6 reverse V,prirners. In a 200pl PCR tube lpI of back primer,

1 pl of forward primer, 2pi of DNA template (2ng. gift from Dr. Honimi), IO@ of

10 x PCR NEBuffer 2 and 56pI of HPLC H20 were mixed and overlaid with 50pl

of minerd oil. The PCR tubes were incubated at 94OC for 5 min and then 30pi

of master mix was added to each tube. with pipetting up and down, under the

minera1 oil. The tubes were then cycled 40 times (94OC for 1.5 min. 57°C for 1.5

min and 72°C for 1.5 min), followed by a 10 min incubation at 72°C. The

products of each reaction were separated on a 15% acrylarnide gel dong with a

marker, pBR322 MspI digest (New England Biolabs). Each 90 base pair band

was cut from the gel over a W light source and the DNA was extracted from the

acrylamide using a QIAEX II gel extraction kit (Qiagen). Briefly, DNA was

diffused out of the gel using 2 volumes of their diffusion buffer and incubating

each sample for 30 min at 50°C. The samples were centrifuged at 14 000 rpm for

I min and the supernatant containing the DNA was passed dirough g la s wool

using a sterile syringe. 6 volumes of buffer QXl was added to each tube which

was vortexed briefly. 20pl of Qiaex II silica particles were added to each tube

and incubated for 20 min, with vortexing every 2 min. The samples were

centrifuged for 1 min, washed twice with buffer PE and air dried for 15 min. The

DNA was eluted twice from each pellet using 40pI of lOrnM Tris-HCI, pH 8.0 and

incubating for 5 min at 50°C. After punfucation the arnount of linkers were

quantitated using a 1.2% agarose gel, so that a mixture containing equimolar

concentrations of each of the 24 linkers could be prepared.

2.8.7 Assemblv of VH and VL genes

Samples from each of the 6 pnmary PCR heavy chah reactions were

attached with an aliquot from the light chain reaction using the linker fragments.

20 pmol each of heavy chains, Iight chains and Iinkers were mixed in a 200@

PCR tube dong with 8p.f of 10 x PCR NEBuffer 2 and the volume was brought

up to 50pi with HPLC -0. This mixture was overlaid with 50pl of mineral oil

and incubated at 94°C for 5 min and then 65°C for 5 min. Master mix was heated

to 65°C for 1 min and 30pl was added to each PCR reaction mixture. The tubes

were cycled 10 times (94°C for 1.5 min, 65°C for 3 min and 72°C for 2 min). The

respective primer mixes were then added to each reaction. They contained 0.5p.i

of forward primer (HuJKForMix). 1pl of back primer (VHI-6aBack), 2pi of 10 x

PCR NEBuffer 2 and 16.5pi of HPLC H20. The tubes were cycled 30 times

(94°C for 1.5 min, 67°C for 3 min and 72°C for 2 min) and then incubated at 72°C

for 10 min.

3.8.8 Addition of restriction sites

After purifying and quantitating the linked fragments as described above

restriction sites were appended using a polymerase chain reaction. 2ûpmol of

linked fragments, IO@ of 10 x PCR NEBuffer 2, lpi of back primer

(HuVH IaBSfi), lpI of forward primer (HuJianixFNot) and sufficient HPLC HtO

to bring the volume up to 70pi, were rnixed in a 200pI PCR tube. The mixture

was overlaid with SON of mineral oil and incubated at 94°C for Smin. 30pI of

master mix was added to each tube after k ing heated to 65°C. The tubes were

cycled 40 times (94°C for 1.5 min, 57°C for 1.5 min and 72°C for 1.5 min) and

then incubated at 72°C for 10 min,

2.8.9 Selective purification

1/10 the volume of 10 x STE buffer ( 1 M NaCl, 200mM Tris-HCl pH 7.5,

1OOm.M EDTA) was added to the PCR products followed by 1 equal volume of

4M ammonium acetate (Sigma). 2.5 volumes of 100% ethanol (Commercial

Alcohols Inc.) equilibrated at room temperature was added and then the tube was

centrifuged in a microcentrifuge at 10 000 x g for 20 min. The supernatant was

aspirated, the pellet air dried and resuspended in 20pI of TE buffer.

2.8.10 Restriction enzyme digest of scFv fragments

To estimate the concentration of scFv fragments, 1/20 of each scFv sample

was electrophoresed using a 1.5% agarose gel with ethidium bromide alongside

a known arnount of a 750 base pair fragment (Recombinant Phage Antibody

System Module, Pharmacia). An equimolar mixture was made containing al1 of

the different scFv groups and it was used for the first digestion. 10pl of Sfi 1 (20

000U/ml. New England Biolabs), IO@ of 10 x N B 2 buffer (New England

Biolabs). Ipi of purified BSA concentrate (LOmglml, New England Biolabs),

lOOng of scFv and d H 0 to bnng the volume up to 100N were added together and

incubated at 50°C for 1 hour.

The second digest was then performed. IO@ of Not 1 (50 000U/ml, New

England Biolabs). 5pl of 10 x NEB2 buffer. ISjd of 10 x substitute buffer (0.5M

NaCl. 0.4M Tris-HCI pH 7.9). 0SpI of purified BSA concentrate and 19.5pi of

dH,O were added to the mixture from the first digestion. This was incubated at

37°C for 1 hour and then at 65°C for 20 min and stored at 4°C.

2.8.1 I Ligation of scFv with pCANTAB5E

The digested scFv fragments were purified by selective precipitation and

their concentration was quantitated as descnbed in 2.9.10. 15ng of scFv was

ligated with 25 ng of pCANTAB5E (SOng/ml, RPAS Pharmacia) in 50pi volume

containing 5p1 of 10 x ligation buffer (RPAS Pharmacia), 5pi of lOmM ATP

(Pharmacia), 1.5p.I of T4 DNA ligase (6 000 Weiss unitdml, Pharmacia) and

dH20. The reaction was incubated at 16°C for 1 hour, at 70°C for 10 min and

then placed on ice for 5 min.

2.9 Construction of an Fab Library

Using total RNA isolated from the PBLs of a volunteer fÏrst strand cDNA

synthesis reactions were perfonned. In an RNase free 1.5d tube, 4pi of total

RNA was mixed with 2pi of random primers (Gibco) and 6pi of HPLC grade

H,O, incubated for 10 min at 70°C and chilled on ice for 1 min. The procedure

was continued as described in section 2 - 8 2

The heavy and light chain genes were amplified using the primers Iisted

in table 2.2. p, y, h, and a chahs were PCR amplified as follows: 10pl of 10 x

PCR buffer (Gibco), 3pl of 5 W MgCl, (Gibco), 2pi (20pmol) of back primers,

2pi (20pmol) of forward primer, 2pl of first strand synthesis cDNA and 5 1pi of

HPLC grade H,O were rnixed in a 200~1 PCR tube. Each reaction mixture was

overlaid with 50~1 of minera1 oil and heated to 94°C for 5 min. 30pi of master

rnix was added to each tube: this consisted of 2pl of 1 ûmM dNTPs, 0 .5~1 of Taq

DNA polymerase (Gibco) and 27.5pl of HPLC grade -0. The tubes were cycled

40 times (94°C 1.5 min, 54°C 1.5 min, 72°C 1.5 min) with a final IO min

incubation at 72°C.

The PCR products were purified according to section 2.8.4 and cloned into

the pCR-Script cloning vector, according to the instruction manual (Stratagene).

Table 2.2 Primer sequences used for production of an Fab library

Forward primers VHA 5'-AGG TGC AGC TGC TCG AGT CTG G-3' VHB 5'-AGG TGC AGC TGC TCG AGT CGG G-3' W C 5'-AGG TGC AAC TGC TCG AGT CTG G-3' VHD 5'-AGG TGC AAC TGC TCG AGT CGG G-3' VHK 5'-GTG CCA GAG CTG AGC TCG TGA TGA CCC AGT CTC CA-3' W h 5'-CTG CAC AGG GTC CTG GGC CGA GCT CGT GGT GAC TCA-3'

Back primers p p e 5'-AGC ATC ACT AGT ACA AGA TIT GGG CTC-3' p p e 5'-AGG CTï ACT AGT GCA CAC CAC GTG 'TTC-3' Aspe 5'-GCA TTC TAG ACT ATT ATG AAC ATT C'TG TAG GGG C-3' icspe 5'-TCC TTC TAG ATI' ACT AAC ACT CTC CCC GTT GAA GCT (SIT

TGT GAC GGG GCG AAC TC-3'

2.10 Preparation of electrocompetent cells

Electrocompetent E. coli TG1 cells were prepared as previously described

(Current Protocols in Molecular Biology, Supplement 32, Fall 1995).

2.1 1 Electroporation

2pi of the ligation mixture was added to 50pl of electrocompetent TG1

cells in a 0.2cm cuvette (BIO-Rad). The electroporator (BIO-Rad) was set at

25uF. 2.5kV and 200R. I d of fresh 2 x YT+Z%glucose (media preparation,

Mount Sinai Hospital) was added to each cuvette and the cells resuspended. The

contents of each cuvette were pooled in a 50 ml polypropylene tube (Sarstedt) and

the culture was incubated for 1 hour at 37°C. A lûO@ aliquot of culture was used

for a series of IO-fold dilutions, which were plated on LB+amp plates (media

preparation, Mount Sinai Hospital) and incubated ovemight at 30°C to determine

the frequency of transformants.

2.12 Screening Library

2.1 2.1 Rescue of recombinant phage

After the 1 hour incubation above, ampicillin (Sigma) and M 13K07 helper

phage (Pharmacia) were added to the culture at concentrations of lûûuglrnl and

4 x 1 0 ~ pWml respectively. The culture was incubated again for 1 hour at 37°C.

The cells were sedimented and resuspended in lOml of 2 x YT medium with

100pg/ml of ampicillin and 50pglml of kanarnycin, in a sterile 50ml polypropylene

tube (Sarstedt). The culture was incubated overnight at 37OC. The cells were

sedimented and the recombinant phage were PEG precipitated from the

supematant. 2ml of PEGINaCl (20g polyethylene glycol 8000-BDH Chemicds,

14.6g NaCI-BDH Chernicds, brought up to l O O d with m20) was mixed with

lOml of the supernatant and placed on ice for 1 hour. The mixture was spun at

10 000 x g for 20 min at 4"C, the supematant was aspirated and discarded together

with any traces of PEG on the sides of the tube. The pellet was resuspended in

1 ml of 1 xPBS-3%BSA, filtered through a 0.45- filter (Gelrnan Sciences) and

incubated overnight at 4°C.

2.12.2 Panninn ~hage

5 wells of a 96-well Irnmulon microtitre piate (Dynatech Laboratories Inc.)

were coated with O. 1 pi of VT 1 (2pg/ul) in 100~1 of carbonatehicarbonate buffer

pH 9.6 (Sigma). The plate was incubated overnight at 4°C and the wells were

washed 6 times with washing buffer ( IxPBS-0.058Tween20 (BDH Chernica1s)-

O.l%TritonX (Sigma). The 5 wells plus 1 other uncoated well were filled with

blocking buffer (1xPBS-0.05%Tween20-0.1 %TritonX-3% BSA(Sigma)J% skim

milk powder (Carnation)) and incubated for 2 hours at room temperature. The

wells were washed 4 times with wash buffer and 95p.l of recombinant phage

suspension was added to each of the 6 wells. The plate was covered and

incubated for 2 hours at 37°C. Each weIl was washed 80 times with wash buffer.

The bound phages were eluted by the addition of 100pi of lOOmM triethylamine

(Fisher Scientific) to each well. The plate was incubated for 5 min at room

temperature and then 6pl of 3M Tris pH 7.5 (media preparation. Mount Sinai

Hospital) was added to each well to neuualize the contents. The eluted phages

from each well were removed and used to inoculate 2ml of log phase TG1 cells.

2.12.3 Rescue of library following panning

The E. coli TG1 cultures were grown for 1 hour at 37°C. lOOpg/d of

ampiciilin, glucose (ACP Chernicals Inc.) to a final concentration of 2% and

M 13K07 helper phage at 4x log pfu/ml, were added. The culture was incubated

for 1 hour at 37°C. The cells were sedirnented and resuspended in l h l 2 x YT-

AK medium (IOOpg/rd of ampicillin and 50pg/rnl of kanamycin (Sigma)). The

culture was incubated ovemight at 37°C. The cells were sedimented and then the

phage were precipitated as described above.

2.12.4 Repanninq

Panning was performed a total of 3 times. During the last panning

protocol, the wells were washed 30 times with 1 x PBS-O.O5%Tween20-

O.l%TritonX and then 50 times with 0.5M NaCl in 1 x PBS-O.O5%Tween20-

O. 1 %TritonX.

2.12.5 Reinfection with enriched ~hag;e

After the final panning the eluted phage were used to infect lOml of log

phase TG1 cells. The culture was grown for 1 hour at 37°C. A lOul aliquot was

used for a senes of IO-fold dilutions which were plated on LB-glucose-amp plates

(2% glucose. 100pg/d of ampicillin) and incubated ovemight at 30°C. Frozen

stock cultures were aiso made. 800pl of the cells were added to 200pi of 80%

glycerol (Sigma). The suspension was gentiy rnixed and stored at -70°C.

2.12.6 Screening enriched clones

300 well isolated colonies were picked from the LB-arnp-glucose plates

and used to inoculate individual culture tubes (Falcon 2063) containing 500pl

2xYT-AG (100pg/ml ampicillin, 2% gglcose). The cultures were capped tightly

and incubated ovemight at 30°C. 40pi from each overnight culture was used to

inoculate 400~1 of 2xYT-AG plus M 1 3KO7 (5x 10' pWml). These cultures were

grown for 3-4 houn at 37°C with shaking. The cells were pelleted, the

supernatants were aspirated and the pellets were each resuspended in 400pl of 2

x YT-AK (1ûûpg/ml of ampicillin, 50pg/ml of kanamycin). These cultures were

incubated ovemight at 37OC. The cells were centrifuged and the supernatants

containing the recombinant phage were saved.

50pl of each supernatant was rnixed with 50pl of blocking buffer (1 x PBS-

0.05%Tween20-3QBSA-5% skim milk powder) and incubated for 1 hour at room

temperature. ELISAs were performed on each sample. 96-well Irnrnulon

microtitre plates (Dynatech Laboratorïes Inc.) were coated ovemight at 4OC with

O. I pg of VT 1 in 100p.l of carbonate/bicarbonate buffer pH 9.6 (Sigma). Plates

were washed 3 times with wash buffer (1 x PBS-û.O5%Tween20) and blocked

ovemight at room temperature with 1 x PBS-0.05%Tween20-5%BSAA The plates

were washed 3 times with wash buffer and 100@ of each recombinant phage

suspension was added to 1 well. The plates were incubated for 2 hours at 37'C

and then washed 6 times with wash buffer. 100@ of HRP anti-Ml3 conjugate

diluted 1/5000 in blocking buffer was added to each well and the plates were

incubated for 1 hour at roorn temperature. The wells were washed 6 times with

wash buffer and then 100pi of soluble peroxidase substrate (Sigma) was added to

each well. The plates were incubated for 1 hour at room temperature and the

optical densities of well constituents were recorded . using a microplate reader

(Dynatech).

3.2 NOD-SCID enmaftment

NOD-SCID mice were engrafted with human PBLs in order to induce a

human secondary immune response. The donor was boosted with tetanus toxoid

and donated blood 2 weeks later, at which time the number of circulating B cells

specific for tetanus toxin would have increased. 3 mice were engrafted with

3.0~10' PBLs and immunized with tetanus toxoid in adjuvant the next day. 2

mice were immunized with 26pg of toxoided tetanus and 1 control mouse with

PBS. The mice were bled by their tail veins 2 and 4 weeks Iater and the serum

was tested by ELISA for the presence of human IgG specific for tetanus toxin.

One of the mice immunized with tetanus toxoid died before the 2 week penod

ended, Table 3.1 shows the ELISA results. There were no detectable human anti-

tetanus IgG present in the mouse serum collected 2 or 4 weeks after

immunization.

In an attempt to induce a human primary immune response, an individual

who had been exposed to VTI but was not VTI seropositive, person 2, donated

PBLs which were used to engraft 3 NOD-SCID mice. The next day, 2 mice were

immunized with 20 pg of toxoided VTl in adjuvant and 1 control mouse with

PBS. 2 weeks later the mice were bled by their tail veins and the serum was

tested for the presence of human anti-VT1 IgG and IgM by ELISA.

Irnrnunohistology slides demonstrate the presence of human lymphocytes in the

mice spleens, lungs and liven. See figure 3.0. The ELISA results are shown in

Table 3.2. The ELISA demonstrated that there was no dif5erence between the

antigen coated wells and the background OD readings for any of the samples. As

well, the OD readings of the mice immunized with VTI did not differ from the

OD readings from the mouse immunized with PBS.

Table 3.1 Tetanus toxin antibody levels in Hu-PBL-NOD-SCID rnice as determined by ELISA

serum sarnple ( 1 :40 dilution)

a background OD @ 490nm = 0.05 b background OD @ 490nm - 0.02 c mouse A immunized with tetanus toxin d mouse B immunized with tetanus toxin e serum frorn a TT immunopositive person

mouse Bd

positive controle

2 weeks postimrnunizationa

4 weeks postimmunizationb

0.05

> 1.75

0.03

> 1.80

Table 3.2 Verotoxin-l antibody levels in Hu-PBL-NOD-SCID mice as determined by ELISA

semm sarnple Antibody level ( 1 :40 dilution)

O. 10 (0.06)~

0.18 (0.1 1)

II positive controlc 11 > 1-41 (0.37)

a mice A and B immunized with VT1 b mouse C immunized with PBS c serurn from a VTI immunopositive person d background OD @ 490nm

Figure 3.0 Detection of CD45 in tissue sections from Hu-PBL-NOD-SCID mice - The levels of engraftment of Hu-PBL-NODSCID rnice were assessed immunohistologicaily. Figure A is a tissue section of a lung fiom a Hu-PBL- NOD-SCID mouse demonstrating IabeIIed human cefls. Human lymphocytes appear red as their CD45 surface markers were Iabelled with horse radish peroxidase. Figure B is a tissue section of a lung from a non-engrafted NOD- S C D mouse.

3.3 EBV immortalization of human PBLs

An individual who had ben recently exposed to verotoxin. person 2,

donated lOml of pcripheral blood every 3 4 days for 2 weeks. The PBLs were

isolated from the whole blood and irnmediately infected with EBV. The cells

were then seeded in 96-well tissue culture plates at densities of 30 000 cells/weII

and 100 000 cells/welI, in a volume of 2ûûp.i. The cells were cultured for 295-3

weeks, at which time the supernatant from each well was tested for the presence

of anti-VT1 antibodies by ELISA. 50p.l of supernatant from each well was rnixed

with 50pi of PBS+3%BSA and the entire 100pl was used in an ELISA. 2 wells

were found to contain anti-VT 1 IgM 17 days after EBV infkction. However, only

1 remained positive on day 24. See Tables 3.3 and 3.4. Total RNA was extracted

from the cells remaining in well 8C 24 days after EBV infection.

Table 3.3 VTI antibody Ievels in 17 day old EBV-immortalized B ce11 cultures as determined by ELISA

II Antibody Ievels OD @ 49ûnm 11 !+Pl

positive controla

a serum from a VTI immunopositive person b semm from a VT I immunonegative penon

Table 3.4 VT 1 antibody levels in 24 day old EBV-immortalized B ce11 cultures as determined by ELISA

S ample IgM levels OD @ 49hm

IgG levels OD @ 490nm

1

a background OD @ 490nm b serum from a VTI immunopositive person

positive controlb

well 4D (0.02)

d a

O. 16 (0.02)

> 1.66 (0.05)

0.03

In order to become proficient at EBV immortalization of Hu-PBLs, long-

term culniring of these cells and detection of antibodies in the supernatant, a

source of circulating B cells producing a specific antibody was needed. A

volunteer who wa. receiving a tetanus toxoid booster imrnunization donated l h l

of peripheral blood 7, 10 and 14 days after the boost. The PBLs were isolated

and infected with EBV immediately. The cells were seeded in 96-well tissue

culture plates with 100 000 cells/well and cultured for 21 days before the

supernatants were tested. 50pl of each supernatant was rnixed with 50pl of

PBS+3%BSA and the entire volume was assayed by ELISA. 24 wells were found

to contain anti-TT IgG. The optical densities at 490nm of each of the 24 positive

supernatant samples ranged from 0.60 to >1.17. The c e k in these welIs were

transferred to a 24-well tissue culture plate and grown for another 7 days, at which

time the supematants were retested using the same volumes and only 5 wells

remained positive. See Table 3.5. Total RNA was isotated from the cells in the

5 wells and pooled.

Table 3.5 TT antibody Ievels in 24 day old EBV-immortalized B ce11 cultures as detennined by ELISA

Antibody levels OD @ 490nm

/ ( well 2B 11 0.27

II well 3F 11 0.36

II well 5A 11 0.22

supernatant from a well with no TT specific cells

An individuai who had had a VTEC infection many years ago and

exhibited a high anti-VTI IgG titre was located, person 1. This individual

donated 200ml of peripherai blood from which the PBLs were isolated and EBV

immortalized on the same day. The semm was tested and was found to contain

a high level of circulating anti-VT1 IgG. The cells were seeded at a concentration

of 100 000 cells/well and cultured for 21 days before their supematants were

tested for the presence of ami-VT1 antibodies. By ELISA 7 wells were positive

at day 21 and only one well remained positive on day 78. See Table 3.6. Total

RNA was extracted from the cells in the one positive well on day 28.

Table 3.6 VT 1 antibody levels in 2 1 and 28 day old EBV-immortaiized B ce11 cultures as detennined by ELISA

Antibody level, day 2 1 OD @ 490nm

weII 2A

well 4C

well 5C

well 7D

0.12

O. 13

well 11B

weII 12D

positive controla

a semm from a VTI immunopositive person b semm from a VTI immunonegative person

- - - -. - - - - --

NIA

0.04

O. 16

O. 17

negative c o n t r o ~ ~

0.04

0.06

0.14

0.26

1.75

0.46

0.05

1.68

0.03 0.02

3.4 Construction of scFv library #1

EBV-immortalized B cells producing anti-VT1 IgM were detected within

a mixed B ce11 population. Total RNA was isolated from the population of B

cells and was used to constmct a library of single chain variable fragments. RNA

was reverse transcribed to produce cDNA which was used to PCR-arnplify the VH

and VL genes. There are 6 different families of heavy chains within any class of

antibody and hence 6 separate PCR amplifications were performed. Only one

amplification reaction was needed for the light chains as there is only one family

of this class of chain. 5 of the 6 heavy chain amplifications were successful.

Single bands of the correct size. approximately 350bp for the heavy chain and

325bp for the light chain, were obtained after amplification of the VH and VL

genes. See Figure 3.1.

The V H and VL genes were joined in five separate polymerase chain

reactions using a mixture of 24 unique, flexible 90bp DNA linker fragments. The

linker fragments bound the 3' J region of the VH gene to the 5' V region of the

VL gene. 4 of the 5 linking reactions were successful. The linked genes were

PCR-amplified which resulted in 750bp products representing single chain variable

fraawents. See Figure 3.2.

Figure 3.1 Amplification of VH and VL genes. PCR products were separated on an agarose gel. Amplificahon of VL-r and VH genes produced 325bp and 350bp fragments respectively.

Lane A VL K gene B VH6 genes C VH5 genes D VH4 genes E VH3 genes F VH2 genes' G VHl genes F Molecular Weight (MW) marker

* could not ampli@

Figure 3.1 Amplifcation of VH and VL genes

Figure 3.2 Linking VH and VL genes. PCR products were sepatated on an agarose gel. Linked fragments are labelled as 750bp bands.

Lane A V H ~ - K B VH5-K* C VH4-K D VH3-K E V H 1 -K

F MW marker

* could not link and amplify

Figure 3.2 Linking VH and VL genes

A B C D E F

Restriction sites were appended onto the linked fragments by PCR-

amplification. This produced 800bp products which were digested and cloned into

the pCANTABSE vector. 150 ng of scFv DNA ligated with 250 ng of

pCANTABSE vector yielded a scFv library consisting of 4x 103 clones. Figure 3.3

is a schematic diagram of the vector.

Figure 3.3 pCANTABSE vector

This figure has been repnnted and modified from the Phannacia Biotech Recombinant Phage Antibody System instruction manual.

3-4.1 Screenine; librarv #I

Phage particles were panned on a solid phase support using purified VT- 1.

The number of bound phage eluted after each round of panning is shown in Table

3.7. Generally the numbers of transforming units eluted increases after each round

of panning. However, if the numbers do not increase the panning may still be

successful (Nissim et al. 1996). After each round of panning, phage antibodies

from 100-30 enriched clones were screened by ELISA for their ability to bind

VT- 1. In the first two rounds of panning 160 clones were tested and 300 clones

were tested in the third round of panning. Of al1 of the clones tested none were

able to bind VT- 1.

3.5 Construction of scFv library #2

After identiQing a population of EBV-irnmortalized B cells that were

producing anti-VTI IgG, total RNA was isolated from these cells and reverse

transcribed into cDNA. The cDNA was used to amplify the VH and VL genes.

All 7 amplification reactions were successful in that they resulted in single bands

of the correct size. See Figure 3.4. The VH and VL genes were linked in 6

separate polymerase chah reactions followed by attachment of restriction sites

ont0 the linked fragments. See Figure 3.5.

Table 3.7 Phage eluted after panning with VT1

Figure 3.4 Amplification of VH and VL genes for Iibrary #2. PCR products separated on an agarose gel. Amplification of VL-K and VH genes produced 325bp and 350bp fragments respectively.

MW rnarker VL-IC genes VHl genes VH2 genes VH3 genes VH4 genes VH5 genes VH6 genes

Figure 3.4 Amplitlcation of VH and VL genes

Figure 3.5 Linking MI and VL genes. PCR products were separated on an agarose gel. Linked fragments are labelled as 750bp bands.

VH 1-K V H ~ - K VH3-K VH~-K VUS-K m6-1~ W6-K MW marker

Figure 3.5 Linking VH and VL genes

A B C D E F G H

15 ng of scFv DNA ligated with 25ng of pCANTABSE vector yielded a

scFv library of 1. i x 10' clones. Therefore, 1 pg of scFv DNA would have yielded

a library of 4 . 4 ~ 16 clones.

3.5.1 Screening library #2

Phage particles were panned on a solid phase support using purified VT- 1.

The number of bound phage eluted after each round of panning is shown in Table

3.8. Once again, the number of transfonning units did not increase substantially

after each round of panning. The phage antibodies from 300 enriched clones were

screened by ELISA for their ability to bind VT-1 after each round of panning.

None were able to bind VT- 1.

Table 3-8 Phage eluted after panning library #2 against VTI

1st pan

I .4x 16

2nd pan

1 . 2 ~ 1d

3rd pan

8.0~ 105

3 -6 Screening Nmven Iibrarv

A library of 108 scFv clones was donated by Dr. Hozumi's lab for the

purpose of screening for VTl binden. This library was made from Hu-PBLs

retneved from the spleen of S C I D rnice which had been engrafted with Hu-PBLs

and immunized with RSV. The library was panned 3 times, using punfied VTl

and enriched clones were tested by ELISA for their ability to bind VTl. See

Table 3.9. No VT-1 binden were detected by ELISA.

Table 3.9 Panning of Nguyen library with VTl

1st pan

freq. of O/ 1 0 0 binders

2nd pan

1 . 9 ~ 1 0 '

0/40

3rd pan

I .2x lo4 0/140

3.7 Construction of an Fab library

Total RNA was isolated from PBLs collected from a non-immune

individual. Heavy and light chah immunoglobulin genes were PCR amplified

using the appropriate primers, PCR products were separated on agarose gels and

the 750 bp PCR products were purified from the agarose. See figure 3.6.

Although the purified chains were in the process of being subcloned into the pCR-

script vector, this strategy was aborted. The reasons for this decision will be

presented in the discussion.

Figure 3.6 Amplification of heavy and light chains for an Fab library. PCR products were separated on an agarose gel. Both K and h light chains were amplified, as well as both p and y heavy chains. They are labelled as 750bp fragments.

Lane A B C D E F G H I J K L M N O P

--ci --P =-cr W C - p w-cr K genes h genes MW marker VHA-p vH'Ew WC-p =-CL VHA-y m-Y WC-y VHD-y

Figure 3.6 Amplification of heavy and light chains

I J K L M N O P Q

4.1 DISCUSSION

The first objective of this project was to find a VTI seropositive

individual. In order to do this, an ELISA was developed which could detect VTI

specific IgM, IgG, or IgA molecules. It was difficult to find an individual who

was VTI seropositive, which is not surprising considering it is estimated that only

approximately 7% of the population have neutralizing antibodies against VTI

(Caprioli et al. 1994). Person 1, who had had an E.coli 0 157:H7 infection many

years ago was found to be VTI seropositive and supplied PBLs for this project.

Another individual, person 2, was found who had recently been exposed to VTl

but was not VT1 seropositive. Person 2 also supplied PBLs for this project.

With sources of B lymphocytes specific for VTI it was possible to move

ont0 the next stage: enriching the VT I specific B lymphocytes. Our rationale

was that by enriching this ce11 population it would be easier to clone the VH and

VL genes of interest and to get a combination of VH and VL genes that code for

an antibody binding molecule with high affinity for VTI.

4.2 Engrafting NOD-SCID Mice

The first method used to enrich these B lymphocytes was through the

production and irnmunization of Hu-PBL-NOD-SCID rnice with VTI. It has been

shown that specific populations of B lymphocytes can be enriched by engrafting

SCID mice with human PBLs from individuals who are sempositive against the

antigen of interest and immunizing the mice with that antigen (Mosier et ai. 1988;

Carlsson et al. 1992). NOD-SCID mice were used instead of SCID mice in this

study, because NOD-SCID mice have very few NK cells and as a result they do

not require pretreatrnent with anti-asialo GM 1 before engraftment with Hu-PBLs

(Shultz et ai. 1995).

There was no detectable antibody response to VTI or TT in the Hu-PBL-

NOD-SCID mice. Upon reviewing the data and analysing new information it

appears that one reason for the Iack of any antibody response rnay be due to the

use of NOD-SCID instead of SCID mice. It has recently been demonstrated that

NOD-SCID mice lack complement (Shultz et al. 1995). Complement may be

necessary for development of a secondary immune response because it binds to

immune complexes which can then associate with the CR2 complement receptor

on follicular dendntic cells in the germinal centres of the spleen or Iymph nodes.

This allows antigenic epitopes to be presented on the surface of dendritic cells and

drives the activation of germinal B lymphocytes. Without complement, dendntic

cells cannot present antigen and there can be no B ce11 or antibody maturation

(Travers et al. 1994). It is important to note that the NOD-SCID rnice were

successfully engrafted as assessed by irnrnunohistology. In addition. the Hu-PBL-

NOD-SCID mice did not develop graft versus host disease, GVHD, over the

period of the experiment. This is in contrast with SCID mice, most of which

develop GVHD within 21-28 days of engraftment (Sandhu et ai. 1994). Taking

these facts into consideration, it transpires that engraftment and imrnunization of

NOD-SCID mice cannot be used for the purpose of enriching a specific B

lymphocyte population.

4.3 EBV Immortdization of Human B Lym~hocytes

The second method used to enrich VTI specific B lymphocytes employed

EBV-immortaiization of the cells. As a pilot for this procedure B lymphocytes

from a tetanus toxoid boosted individuai were EBV infected, cultured and tested

for TT specific antibodies. The success of this preliminary study led to the EBV

immortdization of B lymphocytes from person 1. the VT1 seropositive individual.

niese B lymphocytes were successfully immortalized with EBV and wells

containing anti-VTI IgG in the supernatant were detected. In addition, PBLs from

person 2 were EBV imrnortaiized and wells containing anti-VTI IgM were

de tec ted,

It was observed that the amount of detectable VTI specific antibody

decreased the longer the cells were in culture. This suggests that sorne cells may

have ceased production of the antibodies, or they may have died, as it is known

that EBV transformed cells are unstable and spontaneously terminate antibody

production (Roder et al. 1986; Winter and Milstein. 1991). As well, mouse-

human heteromyelomas are just as unstable (Roder et ai. 1986). It is for these

reasons that EBV immortdization of B lymphocytes and heteromyeloma

production are not extremely usefui for producing monoclonal antibodies and why

the antibody genes must be genetically cloned.

A larger nurnber of immortaiized VTI positive B lymphocytes were

anticipated from peson 1 as this individual's antibody titre was high. However,

this may not have occurred since it is likely that resting B lymphocytes are

preferentiaily immortalized over antibody secreting B lymphocytes (Aman et al.

1984). In addition, antibody producing B lymphocytes are located in the bone

marrow and memory B lymphocytes are found in the peripherai blood.

4.3 CIoning the Variable Heavv and Light Genes

With a source of mRNA from an ennched population of B lymphocytes

producing anti-VT I antibodies, it was possible to begin immortalizing the specific

antibodies by cloning their genes. The fint approach taken to clone the VH and

VL genes was to construct a combinatorid library of Fab fragments, using a

filamentous phage expression system, pComb3. This system was chosen because

i t was being sucessfully used in the lab of Our collaborator. Dr. Hozumi.

This system proved ineficient for subcloning the V H and VL genes,

because the cDNA fragments had to first be subcloned into a PCR-Script vector

before cloning into the pComb3 vector. The additional cloning steps likely

resulted in the loss of cDNA fragments , greater than 80% loss, and a lowered

cloning efficiency. This system was aborted after the VH and VL genes had been

PCR amplified since our collaboraton had optimized a more efficient system of

cloning these genes, constructing single chah variable fragment libraries.

4.5 Construction of a Single Chain Variable Fragment Librarv

The first scFv library, constructed with mRNA from a clonally mixed

population of EBV-irnrnortalized human B lymphocytes, consisted of 3x 1@ clones.

The clones generated were too few to expect any positive VT1 binding fragments

since anti-VT antibodies are rare. After analysing the procedures used for

constmcting this library some areas were identified which needed improvement.

In order to improve the size of the next scFv library polymerase chah reactions

needed to be optirnized to increase the arnount of cDNA being produced. As well,

to increase the size of the library the efficiency of cloning scFv fragments into the

pCANTABSE vector had to be improved by digesting a larger proportion of

fragments at both ends and by optimizing the vector to insert ratio.

The second scFv Iibrary, constructed with mRNA from another clonally

mixed population of EBV-immortalized human B lymphocytes consisted. of

1 .1 x 10' clones. Polymerase chah reactions were optimized pnor to amplifying

the VH and VL pnes, which resulted in more cDNA being produced and more

scFv fia-gnents being subcloned. As well, the efficiency of purifying the smdl

DNA fragments was increased from 10% to 30% recovery. Al1 of the VH and VL

linking reactions were successful, unlike for the fint library, which improved the

diversity of this library. The bacteria used for transformation were highly

electrocompetent (10' transforrnantdpg pUCl8) and the ligation reaction was

improved by digesting the scFv fragments with higher concentrations of enzymes

in order to increase the number of fully digested fragments for optimal ligation

with the vector.

4.6 Comparison of scFv Librarv #2 to Winter's scFv Library

In order to assess the utility of the second scFv library it is important to

compare it to a successfully constnicted scFv library, herein designated the Winter

library (Winter et ai. 1991). The Winter library was prepared using 5 0 M of

peripheral blood from a non-immune individual. This represents approximately

109 PBLs. In contrast, Our Iibrary was prepared from l O d of peripheral blood

from an individual with a detectable igG antibody response to the antigen. This

represents approximately 1 . 5 ~ IO6 PBLs. Winter assembled four different first

strand cDNA synthesis reactions for y, p, K, and h chains. Each reaction

contained 400ng of total RNA. We focused on y and K chains alone. since the

antibodies to be cloned were specific IgG molecules and it is estimated that K

chains are more prevalent in secondary immune responses. Each reaction

contained 900ng of total RNA. The amplification of heavy and light chah genes

were sirnilar, in that we both used 1/10 of the first strand cDNA reaction products

in each PCR reaction tube. We had a yield of approximately 1 pg of PCR product

per reaction tube. However, only 30% of these products were recovered during

purification. It is accepted that purifying small fragments (350bp and 325bp) such

as these is notably difficult. A maximum of 200ng of DNA from each of the

seven amplification reactions was recovered. Data are not available for the Winter

library. Winter's linking of the heavy and light chah genes utilized lpg of VH

aenes. Ipg of VL genes and 25ûng of linker DNA. We employed lûng of VH b

genes, long of VL genes and 2ng of linker DNA. The proportions were alike,

however, We used 11100 of the amount of input DNA which yielded lûûng of

linked scFV product afier purification. Winter digested 1-4 pg of scFv and ligated

it with 6pg of vector. After appending the resmction sites. 60ng of scFv was left.

15ng of digested scFv was ligated with 25ng of vector. This was one tenth the

size of the suggested ligation reaction (Pharmacia, RPAS system). The ratio of

insertvector was accurate at 3.7: 1 with the suggested ratio being in the range of

3-5: 1 (Pharmacia, RPAS system).

After transformation there were 1.1 x 10' clones. Extrapolating these data,

the full size ligation reaction would have yielded 1.1 x ld and Ipg of vector would

have yielded 4 . 4 ~ 1 6 clones. In contrast, Winter's library yielded 2 . 9 ~ 1 0 ~ IgM

clones and 1 . 6 ~ 108 IgG clones. These results show a marked improvement over

the first scFv library constmcted. However, more refinement is desirable.

Specific improvements will require an increase in the arnount of input

mRNA. Polymerase chain reactions will have to be repeated multiple times in

order to accumulate a larger arnount of purified cDNA. The amount of linked

fragments will have to be increased at least 10 fold and the arnount of vector and

fragment used for the ligation should be increased 10-20 fold.

One other area of interest is the panning results. After multiple pannings

of Our scFv library against purified VTI, there was no marked increase in the

number of clones k ing isolated, even though the numbers of non-specific binders

was repeatedly low, that is approximately 10% of the specific binders. Typically,

the number of phage eluted after each round of panning increases. However, it

has been reported that enrichment may still occur in the absence of increasing

numbers of eluted phage (Harrison et al. 1996).

4.7 Screening of N-uyen Librarv

This library consisted of 1x10~ clones and was made using splenocytes

retrieved from a Hu-PBL-SCID mouse irnmunized with respiratory syncytial virus

(Nguyen et al. 1997). Since the repertoire of this library was quite large it was

screened for VT1 binding scFv fragments as well as ïT binding fragments. No

binders were found for either antigen, which is not surprising, considering that the

population of splenocytes was enriched for RSV specific B lymphocytes through

immunization and therefore there would not have been any stimulation of VTI

or TT specific B lymphocytes.

4.8 Conclusion

Recently, it has been dernonstrated that scFv Iibraries constructed with

splenocytes from immunized Hu-PBL-SCID mice are better sources of high

affinity scFv compared with those libraries constructed directly from donor's PBLs

(Nguyen et al. 1997). These results suggest that imrnunization of Hu-PBL-SCID

mice is an effective method for inducing human antibody afinity maturation and

a means to increase the number of specific phages in a scFv library conshucted

with these splenocytes.

Since the report by Winter (Winter et al. 1991) few other groups have

produced scFv libraries. Nissim and colleagues produced a scFv library of IgG

and IgM variable chain consisting of 10' ciones (Nissim et al. 1994). They pooled

their library with Winter's library and used it to select irnmunochemical reagents.

Phage were isolated with binding activities to 18 antigens inciuding, intracellular

proteins p53. elongation factor EF-la, rhornbotin-2 oncogene protein and sex

determining region Y protein (Nissim et al. 1994). More recently Finnem and

colleagues produced a scFv IgG library of 106 clones using splenocytes from a

patient with systemic autoimmunity (Finnem et al. 1997). They detected phage

with binding activities to the autoantigen proteinase 3. A number of research

groups have used the various libraries. Carnemolla and colleagues used the

Nissim library to locate a scFv that can be used as a marker of angiogenesis in

animal models, that is, a scFv that has binding activity to a specific isoform of

fibronectin (Carnemolla et al. 1996). Schier and colleagues used the Winter

library to detect scFv with binding activity to a glycoprotein tumor antigen, c-

erbB-2 (Schier et al. 1996).

4.9 Future Considerations

Once a scFv that binds to VT1 is found it will have to be demonstrated

that it is specific for VTl and that it c m neuaalize VTI. This is done by

performing ELISAs. coating the rnicrotiter plates with antigens such as, tetanus

toxin or diphthena toxin. As well, ELISAs can determine if the scFv binds the

VTl A or B subunit and whether it recognizes VT2. Neutralization assays using

ver0 cells will determine whether the scFv has VTl neutralizing activity.

Neutralizing scFvs would bind VTI, thereby inhibiting its cytotoxic effect on the

ver0 cells.

With these parameters met. it would then be possible to subclone the

specific VH and VL genes into a vector that allows the full size antibody

molecule to be expressed while retaining its antigen binding specificity (Ames et

al. 1995: Tsui et al. 1996) as the therapeutic effectiveness of the antibody depends

on the effec tor functions provided by imrnunoglobulin constant domains. Using

rnutator strains, such as E. coli: mutD5, the affinity of the VTl specific scFv

could be increased if desired (Winter et al. 1996). Point mutations accumulate

dunng multiple rounds of growth using the mutator strain, possibly resulting in

improved binding affinities after increasingly stringent selection of phage.

It would also be valuable to produce a scFv that recognizes VT2 to be used

in conjunction with a VTI antibody in an immunotherapy. since many E-coli

0 157:H7 isolates produce both VT 1 and VT2 during infections in humans (Pierard

et al. 1994). As mentioned earlier, it may also be possible that in vivo antibodies

directed against the VT A subunit may be cross-protective with both VTI and

VT2, as reticulo endothelid sequestration of immune complexes formed with such

antibodies and both VTI and VT2 has been demonstrated (Bielaszewska et al.

1997). In any case, if immune response to VTs are MHC restricted. then the

development of an immunotherapy will become more important than producing

a VT vaccine.

Using the improvements described above, the construction of another scFv

library should result in a phage with VT-1 binding activity. This will put us one

step closer to realizing our goal of an irnmunotherapy for E. coli 0157:H7

infections.

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