56

Chapter - 4

Generation of Mouse

Monoclonal Antibodies

Attributes: Bioinformatics and peptide designing for LAMB3 and LAD1 were performed

by Dr. Craig Bencsics. Animal work including maintainance of mice population and

peptide immunizations were performed by Dr. Matthew Holsti.

57

4.1 INTRODUCTION

For the generation of mAbs a mammalian host, preferably a rodent, has to be

immunized with the antigen. Since mice are relatively easy to handle and inexpensive to

maintain, inbred strains of BALB/c mice were used in this study for generating mAbs

(Potter, 1985). Synthetic peptides that represent a specific portion of the protein were

used as antigens. Immunization was followed by fusing the immunized host spleen cells

(splenocytes) with myeloma cell line using Polyethylene glycol (PEG) which is the most

widely used agent for fusing the mammalian cells (Pontecorvo, 1975). PEG fuses the

plasma membranes of adjacent myeloma and antibody-secreting B-cells, forming

immortal somatic hybrid cells, known as “hybridomas”. They have the ability to produce

antibodies, raised against the target, under standard mammalian tissue culture

conditions. (KÖHLER and MILSTEIN, 1975). As antibodies are secreted into the culture

medium, hybridoma cell culture supernatants were tested for the presence of antibody

of interest. The fusion techniques are based on the technique developed by Galfre et al.

(Galfre G, 1977; Galfrè G, 1981) and Gefter et al (Gefter ML, 1977). Post-fusion cell

culture was carried out in special type of selection medium, commonly known as HAT

medium. (Ed Harlow, 1988).

There were no effective mAbs available for LAD1 and only one mAb was

available for LAMB3. Even though another mAb was available for LAMB3, it did not

yield successful results in the initial validation experiments for swELISA. Therefore, two

mouse mAbs were developed for LAD1 and one for LAMB3. The current chapter is

focused on the antibody generation for these two targets. A brief outline of the antibody

generation is shown in Figure 4.1.

58

Figure – 4.1: Outline of antibody generation: Female BALB/c mice were immunized with LAD1 and LAMB3 peptides. Sera were evaluated for antibody titer and then spleens were fused with mouse myeloma cells. Resulting hybridomas were tested for LAD1 and LAMB3 antibodies and selected clones were scaled up and antibodies were purified by FPLC. Quality control (QC) was performed at each step by various assays.

Selection of the epitopes on the protein and peptide designing

Immunizations

Sera screening against the antigen by BSA-peptide ELISA and WB and selection of the serum with best antibody titer

Fusion of the spleen with mouse myloma partner (P3 x 63 Ag

8.653 or Sp2/0-Ag14

Initial characterization of the hybridomas by ELISA and WB for productivity and specificity

Subcloning and scale-up

Antibody purification and final QC.

Bioinformatics. QC: Mass Spec

Immunizations and Sera screening QC: ELISA and

WB

Fusion and hybridoma generation

QC: Testing the cultures for mycoplasma; ELISA

and WB

Antibody production

Collection of spleens

59

4.2 MATERIALS AND METHODS

4.2.1 Peptide designing

Comprehensive bioinformatics analysis was performed to examine the

immunological potential of the target. Peptides were designed for each target for which

neither ELISA kits nor antibodies were available. Two peptides, predicted to be unique

in the proteome and that could distinguish the target from any closely related

homologues, were selected for each target. Each peptide corresponds to a specific

epitope on the protein and the antibodies raised against each epitope were unique and

were expected to be non-cross reactive. Two different kinds of peptides were generated

for each target: (1) KLH cross-linked peptides for immunization and (2) BSA cross-

linked peptides for screening by ELISA and WB. Once the final peptide sequences were

selected, they were commercially synthesized by the New England Peptide LLC,

Gardner, MA.

Peptides were designed for immunization based on the following criteria:

1. Regions with lowest possible homology to the protein sequence of the mouse were

selected as potential epitopes to avoid immunological tolerance.

2. Regions with lowest possible homology to other proteins in the human proteome

were chosen to design peptides to avoid potential cross reactivity of the antibodies

with other human proteins.

3. A hydropathy plot, which is used to predict antigenicity, was reviewed for the epitope

regions selected and peptides whose sequences had a hydropathy score < -1 were

selected.

60

4. Signal sequence (a short peptide chain, usually 3-60 amino acids long, that directs

the post-translational transport of a protein and is immediately cleaved from the

polypeptide once it has been translocated into the ER by signal peptidase in

secretary proteins) was avoided during the peptide design for the secreted proteins.

A complete list of the peptides used in the present study is shown in Table-4.1.

Table-4.1: Peptides selected for each target for immunization. Peptides were cross linked to KLH by Cysteine or amine (H2N) residues.

Target Region Peptide

LAMB3 AA 516-530 H2N-IRQCPDRTYGDVATG-amide

LAD1-peptide 1 AA 416-428 Ac-RRSESVKSRGLPC-amide

LAD1- peptide 2 AA 160-176 Ac-SLVGREPEERKKGVPEKC-amide

4.2.2 Immunization of mice

Female BALB/c mice were used for all immunizations. At least two mice were

used for the immunization of each peptide. All mice were housed at the animal facility

located at Harvard School of Public Health. All studies involving animals were

conducted in compliance with state and institutional animal care guidelines. Peptides

were injected into the mice intra-peritoneally once every two weeks for a total of 5

doses. Each dose contained 300 µg of immunization antigen (target-specific peptide)

diluted in 1x PBS (Media Tech Inc) in a total volume of 900 µL. This was mixed

thoroughly, using a 3-way stopcock (Mallinckrodt), with equal volume (900 µL) of

Complete or Incomplete Freund‟s adjuvant (Sigma, St. Louis, MO). Complete adjuvant

was used only for the first immunization and incomplete adjuvant was used for all the

61

following booster doses. Five hundred µL of peptide-adjuvant was injected to the

mouse‟s left, lower abdomen between the midline and nipple. After fifth immunization,

sera were collected from the mice and tested for the antibody titers. Final booster dose

was given to the mice with best antibody titers. For the final boost, mice were

immunized with 50 µg of the peptide diluted in 180 µL of PBS through the tail vein

intravenously using an insulin syringe (BD Biosciences).

4.2.3 Sera screening by indirect ELISA

Sera were collected from pre- and post-immunized mice and tested on target-

specific peptide antigen conjugated to bovine serum albumin (BSA) by indirect ELISA.

Ninety six-well EIA-RIA plates (Costar, Corning, NY) were coated with 1 µg/mL of BSA-

peptide and incubated for 16-20 hours at 4°C. Plates were blocked with TBS-Tween 20

supplemented with 0.5% of BSA for 1 hour at RT. Various dilutions of serum samples

were prepared in PBS (1:1,000, 1:3,000, 1:10,000, 1:30,000 and 1:100,000) and added

to the plate at 100 µL/well of each sample. After two hours of incubation, plates were

washed with TBS-Tween 20 wash buffer and then incubated with goat anti-mouse

detection antibody conjugated with AP. Plates were again washed with wash buffer and

developed using p-Nitrophenyl phosphate disodium hexahydrate (Sigma) dissolved in

diethanol amine substrate buffer (Thermo Fisher Scientific). Plates were incubated at

RT for 30-60 minutes and read at 405 nm using a spectrophotometer. Sample was

considered positive if the Optical density (OD) value was atleast 2-3 fold higher than

that of the background.

62

4.2.4 Sera screening by WB

Pre- and post-immunized mice sera were tested on cell lysates derived from

293T cells that were transfected with or without target-specific expression vector.

Proteins in the whole cell lysates were separated by SDS-PAGE on 2D-prep gels as

mentioned in Section 3.2.13. Serum dilutions were prepared as mentioned above.

Approximately 70 µL each of diluted serum was loaded on to the nitro-cellulose

membrane using Miniblotter® system (Immunetics, Boston, MA) and incubated for 16-

20 hours at 4°C. Blots were washed with TBS-Tween20 buffer and probed with HRP-

conjugated goat anti-mouse secondary antibody and then developed as mentioned in

the WB technique (Section 3.2.13).

4.2.5 Spleen-myeloma cell fusion

Based on sera screening by indirect ELISA and WB, serum with highest antibody

titer was selected and spleen from the corresponding mouse was harvested aseptically.

A single cell suspension of the spleen was prepared in serum free DMEM medium and

the spleen cells were used immediately for fusion or frozen down in FBS with 10%

DMSO and stored in liquid nitrogen until further use.

Mouse myeloma cell lines, P3 x 63 Ag 8.653 or Sp2/0-Ag14 were used for

spleen-myeloma fusions. Fresh batch of myeloma cells was started every 30-45 days to

make sure that there were no phenotype changes due to long term cultures. Myeloma

and spleen cells were washed (centrifuged for 5 minutes at 1000 rpm and 1400 rpm

respectively) separately in serum-free DMEM medium. Cells were counted using

automated Countess Cell counter (Invitrogen). Myeloma cells were mixed with spleen

63

cells in 1:2 to 1:4 ratios and then centrifuged at 1400 rpm for 7 minutes. Excess medium

was completely aspirated and. One mL of polyethylene glycol (PEG) was added to the

cell pellet slowly at a rate of ~0.1 mL/6 seconds. The suspension was incubated at RT

for 1 minute and then was re-hydrated with sterile PBS which was added at a gradually

increasing rate to a final volume of 50 mL in 7 minutes. The suspension was incubated

in 37°C water bath for 10 minutes and then centrifuged at 1400 rpm for 7 minutes.

Excess PBS was aspirated and the cell pellet was gently re-suspended with HAT

medium (DMEM medium supplemented with 20% FBS, 1x glutamine, 1x

penicillin/streptomycin, 50 µg/mL of gentamycin, 3% hybridoma cloning factor and a

mixture containing hypoxanthine-aminopterin and thymidine). Cells were then seeded in

96-well, flat-bottom tissue culture plates at a concentration of 20 to 30 million cells/plate.

4.2.6 Fusion screen by indirect ELISA

EIA/RIA plates were coated with target-specific BSA-conjugated peptide and

blocked with TBS-Tween 20/0.5% BSA as mentioned in the “sera screening by indirect

ELISA” technique (Section 4.2.3). After blocking, culture supernatants from fusion plates

(50 µL from each well of the fusion plate) were added to the ELISA plates and the total

volume per well was brought up to 100 µL using the blocking buffer. Remaining steps of

the screen were same as in Section 4.2.3.

4.2.7 Parental screen by WB

All hybridomas that were positive in the primary indirect ELISA screen were

tested for their ability to recognize the denatured protein in the whole cell lysates

64

derived from 293T cells that were transfected with or without target-specific expression

vector. Approximately 70 µL of culture supernatant was used for screening and the

method was exactly same as mentioned in the “sera screening by WB” technique

(Section 4.2.4).

4.2.8 Parental screen by rabbit anti-FLAG swELISA

EIR/RIA microtiter plates were coated with 250 ng of rabbit-anti FLAG

polyclonal antibody (Genscript, NJ) and incubated overnight at 4°C. Plates were

blocked for 1 hour with 300 µL/well of blocking buffer. Thirty µg/well of LAD1-transfected

293T whole cell lysate, diluted in blocking buffer, was added to the plate and incubated

for 90 minutes at RT. Plate was washed three times with 300 µL/well of wash buffer and

then 100 µL/well of hybridoma supernatant was added and incubated for 90 minutes at

RT. Plate was washed thrice and AP- conjugated-goat anti-mouse detection antibody

(Jackson ImmunoResearch Inc., West Grove, PA) with minimal cross reactivity to rabbit

immunoglobulins was added. The plate was washed again and developed as mentioned

in the indirect ELISA technique (Section 4.2.3).

4.2.9 Subcloning

Parental hybridomas that had best signal by both WB and indirect ELISA or

indirect ELISA alone were selected for subcloning by limiting dilution protocol. Cell

suspension was diluted 1000-fold in D10 medium (5 µL of culture added to 5 mL of

medium) supplemented with 3% hybridoma cloning factor. Two hundred µL of this

diluted cell suspension was added to the top row of the 96-well, flat-bottom tissue

65

culture plates and then serially diluted by 2-fold until the last row assuming that the

serial dilution would result in formation of one colony per well. To make sure that the

hybridomas are clonal, culture supernatants from 1 colony/well were selected after 10-

12 days of culture and tested by indirect ELISA and WB assays as mentioned above.

4.2.10 Isotyping ELISA

Isotype of the purified antibodies was determined by using Rapid ELISA Mouse

mAb Isotyping Kit (Thermo Fisher Scientific) as per the manufacturer‟s instructions.

Briefly, culture supernatants or purified antibodies were diluted in Tris Buffered Saline

and 50 µL of this was added to the 8-well strips. Fifty µL of goat anti-mouse

IgG+IgA+IgM HRP conjugate was added to each well and incubated for one hour at RT.

Strips were washed with wash buffer and 75 µL of TMB substrate was added to each

well of the strip and incubated until the development of blue color. The reaction was

stopped using 75 µL of 2N sulfuric acid and read at 450nm in an ELISA plate reader.

4.2.11 Scale up and purification of antibodies

All hybridomas were scaled up to ~150 mL in D10 medium with 10% low IgG

FBS instead of regular FBS. After 150 mL culture was started in a 162 cm2 tissue

culture flask, hybridomas were cultured for 7-10 days or until viability of the culture was

dropped down to ~70% as assessed by trypan blue counting. Culture supernatants

were then collected by separating the cells by centrifugation at 1400 rpm for 5 minutes.

Antibodies in the hybridoma culture supernatants were purified by passing the latter

66

through HiTrap-mAb Select Sure Protein G columns by (Fast Performance Liquid

Chromatography) FPLC. In brief, culture supernatant was applied to the Protein G

column of the FPLC machine, AKTAxpress, (GE Healthcare) at 4°C. Antibody bound to

the Protein G column was eluted using low pH (3.0) sodium citrate buffer and

immediately neutralized using high pH (9.0) Tris-HCl buffer. Antibody present in the

Tris-HCl buffer was then transferred to PBS by dialyzing over night at 4°C using Slide-

A-Lyzer dialysis cassette (Thermo Fisher Scientific). Final concentration of the antibody

was adjusted to 1 mg/mL by concentrating the antibody using Amicon Ultra-15

centrifugal filter unit (Millipore, Bedford, MA). Aliquots of antibodies were prepared and

stored at -80°C.

4.3 RESULTS AND DISCUSSION

4.3.1 Bioinformatics: peptide designing

In an effort to generate mAbs, two peptides were designed for LAD1 and one for

LAMB3. A detailed description of peptide designing performed for LAD1 is explained

below. A similar procedure was followed for LAMB3 as well (data not shown).

Amino acid sequence of the protein: Complete protein sequence and

structural information for each target were obtained from online databases including

http://ca.expasy.org/ and http://www.ncbi.nlm.nih.gov. These databases provided

information on the sub-cellular location of the protein (cytoplasmic, extra-cellular or

transmembrane), presence and location of any signal sequence, potential glycosylation

or phosphorylation sites in the protein, cycteine-rich domains and any possible isoforms.

67

All this information played a key role in selecting the appropriate regions on the target

protein for peptides. As shown in Figure 4.2a, LAD1 had a total of 517 amino acids with

two phosphorylation sites at AA 177 and AA 512 and one polyarginine domain at AA 28

and 8 SEK (Serine, Glutamic acid and Lysine) repeats. As reported in

http://ca.expasy.org/, LAD1 is a secreted protein and the subcellular location is ECM

and basement membrane zone. However, no signal sequence has been reported for

LAD1 (Fig 4.2a). No isoforms have been reported in the database.

Figure 4.2a: Sequence and predicted structure of human Ladinin-1 (extracted from

http://ca.expasy.org/)

68

Sequence homology: The protein sequence thus obtained for each target was

compared with that of the mouse using online sequence alignment tool,

http://ca.expasy.org/tools/sim-prot.html. The most non-homologous regions in the

human protein sequence were selected to design the peptides, avoiding the most

conserved regions. This is critical because the peptides behave as antigens when

injected into the mice. If the sequence of the peptide chosen for a particular target is

homologous to that of the mouse protein, the mouse immune system recognizes the

peptide as “self” and would not raise antibodies against the peptide, an important

phenomenon, known as, “immunological tolerance”. As shown in Figure 4.2b, protein

sequence of LAD1 had almost 63% homology to that of the mouse and only the non-

homologous regions were selected for designing peptides.

Figure 4.2b: Sequence homology of LAD1 between mouse and human species. Regions with asterisks represent the homologous and those without represent non-homologous regions.

69

Peptides from MS analysis: Peptides found in the MS analysis of the sera from

patients with PDAC were then reviewed. Based on the sequence homology and the

number of hits by MS analysis, a list of peptides was generated for each target. In some

cases, even if a peptide was well represented in the MS data set, the peptide was not

selected because of its poor antigenicity or high homology to the mouse protein

sequence. MS analysis of patient sera for LAD1 protein showed 6 peptides as shown in

Figure 4.2c. The rationale behind MS analysis of patient sera was that if peptides were

found in sera for any particular target, it indicates that the target is expressed in cancer

patients and might serve as a potential biomarker. Furthermore, detection of serum

LAD1 using antibodies raised against these peptides would be more appropriate since

they were found in PDAC patient sera.

Figure 4.2c: Peptides found for LAD1 by MS analysis of PDAC patient sera. Bold letters in the protein represent the peptides sequences found.

70

Hydropathy plots and antigenicity: Using online software from

http://gcat.davidson.edu/rakarnik/kyte-doolittle.htm, Kyte-Doolittle Hydropathy plots

which indicate potential transmembrane or surface regions in proteins, were generated

for the full length protein (Kyte and Doolittle, 1982). Hydropathy plots were used to find

clusters of hydrophobic amino acids, which could indicate that the polypeptide in

question is a transmembrane protein. These plots allowed us for the visualization of

hydrophobicity over the length of a peptide sequence and were useful for locating the

hydrophobic interior portions of globular proteins as well as membrane spanning

regions of membrane bound proteins. These plots also identified regions that are likely

exposed on the protein's surface and therefore may by antigenic. A hydropathy score

(which ranges from +4.6 to -4.6) of 4.6 is the most hydrophobic and a score of -4.6 is

the most hydrophilic. When looking for surface regions in a globular protein, a window

size of 9 amino acid residues was found to give the best results. Surface regions can be

identified as peaks below the mid line. A hydropathy plot for full length LAD1 is shown in

Figure 4.2d.

Figure-4.2d: Hydropathy plot of full length human LAD1. Peaks with negative values represent hydrophilic regions that could be accessible to monoclonal antibodies.

71

Figure 4.2e: Hydropathy plots of the peptides selected for human LAD1

As hydrophobic regions with a hydropathy score greater than zero are likely to be

located in the interior of a globular protein, these cannot be exposed to the antibodies

which bind to the surface in solution and are predicted to be poorly antigenic in nature.

On the other hand the hydrophilic portions, with a hydropathy score less than zero, are

generally located outside of the protein. Hence, these surfaces could be exposed to the

Hydropathy Plot of Human LAD1 (AA 160-176: SLVGREPEERKKGVPEKC) - Region of interest is highlighted. - Predicted to be antigenic. - Has minimal identity between mouse and human LAD-1. - Does not share identity with other proteins

Hydropathy Plot of Human LAD1 (AA 416-428: RRSESVKSRGLPC) - Region of interest is highlighted. - Predicted to be antigenic. - Has minimal identity between mouse and human LAD-1. - Does not share identity with other proteins - Would have a different epitope from other antibody.

72

antibodies and are therefore predicted to be highly antigenic in nature. Hydropathy

scores of the peptides found in the sera analysis by MS were compared with those of

the regions of the protein which are highly hydrophilic (Hydropathy score, less than -1).

The antigenicity of the peptides was determined by mapping them to the Kyte-Doolittle

plots and the peptides with best hydropathy index were selected (Figures 4.2e).

For LAD1, peptides were selected such that they were separated by 240 amino

acids to reduce the likelyhood of any steric hindrance in antigen-antibody reactions. The

rationale for using peptides over recombinant protein for immunizations was that they

are easy to synthesize and particular regions of a protein can be targeted specifically for

antibody production. With recombinant proteins, it is difficult to predict which epitopes

the antibodies recognize without additional evaluation which makes it difficult for the

selection of optimum capture-detection antibody combinations for the development of

swELISA.

4.3.2 Immunizations and evaluation of anti-sera

For each antibody, two mice were immunized separately with KLH-conjugated

peptide. Together, for LAD1, two peptide immunizations and for LAMB3, one peptide

immunization was performed. No post- immunization inflammatory reactions were

observed in any of the mice. All mice were healthy and behaved normally throughout

the immunization course.

73

In an effort to select the mice with best antibody titer, anti-sera were obtained

from mice immunized with target-specific peptides. They were evaluated in a dilution

series by indirect ELISA against the immunizing antigens (BSA-conjugated peptides).

Serum samples from pre-immunized mice served as negative controls for these

experiments. Positive signals, readily distinguished above background, were observed

only in the post-immune but not pre-immune sera for all three peptides (Figure 4.3).

Decreased signals were observed at higher dilutions suggesting that the antibody

concentration was reduced when the sera were diluted. Immuno-reactivity of all mice

was almost similar to their corresponding peptides. These data suggest that the mice

raised antibodies against the immunogens (peptides).

74

LAMB3

0

0.5

1

1.5

2

2.5

Pre-bleed 1:1000 1:3000 1:10000 1:30000 1:100000

Serum dilution

OD

@ 4

05

nm

Mouse #1

Mouse #2

LAD1 -peptide 1

0

0.5

1

1.5

2

2.5

Pre-bleed 1:1000 1:3000 1:10000 1:30000 1:100000

Serum dilutions

OD

@ 4

05

nm

Mouse #1

Mouse #2

LAD1 - peptide 2

0

0.5

1

1.5

2

2.5

Pre-bleed 1:1000 1:3000 1:10000 1:30000 1:100000

Serum dilutions

OD

@ 4

05n

m

Mouse #1

Mouse #2

4.3a

4.3b 4.3c

Figure-4.3: Evaluation of anti-sera by Indirect ELISA. Pre and post-immune anti-sera were tested against BSA-peptides coated on EIA plates. ELISA plates were developed using anti-mouse secondary antibody conjugated to Alkaline-phosphatase. All samples were analyzed in duplicates. For all three targets that include LAMB3 (4.3a), LAD1 peptide-1 (4.3b) and LAD1 peptide-2, mouse #1 showed relatively higher antibody titer.

75

In addition to indirect ELISA, anti-sera were also evaluated by WB assay to

determine if they could recognize full length proteins. Pre-and post-immune sera were

tested against whole cell lysates derived from target- or mock-transfected 293T cells.

Same lysates were used to test the sera for both peptides of LAD1. Signals were

observed at the expected molecular weight for each target only in the target-transfected

but not in the mock-transfected 293T cell lysates. Mouse #1 for LAMB3, mouse #1 for

LAD1 peptide-1 and mouse #2 for LAD1 peptide-2 showed relatively stronger signals

(Figure 4.4). Therefore these mice were selected for fusion to generate mAbs. Anti-

FLAG or anti-HA antibodies and pre-immune sera were used as positive and negative

controls respectively in this assay. For LAMB3, anti-HA antibody was used instead of

anti-FLAG antibody. A summary of sera screening results is shown in Table 4.2.

Figure-4.4: Evaluation of anti-sera by WB assay. Pre and post-immune anti-sera were tested against target-transfected or mock-transfected 293T whole cell lysates using 2D-PAGE gels. Post-immune sera were diluted serially up to 100,000 folds. Anti-HA and FLAG antibodies were used as positive controls to confirm the expression of proteins. Expected size of the protein is indicated with a black arrow.

Lane Serum dilution

1 Pre-bleed 2 1:1000 3 1:3000 4 1:10,000 5 1:30,000 6 1:100,000 A anti-HA antibody B anti-FLAG

antibody

LAMB3

LAD1 Peptide-1

LAD1 Peptide-2

Target-transfected 293T whole cell lysates

Mock-transfected 293T whole cell lysates

Mouse #1 Mouse #2 Mouse #1 Mouse #2

Molecular weight in

kDa 1 2 3 4 5 6 1 2 3 4 5 6 A 1 2 3 4 5 6 1 2 3 4 5 6 A

1 2 3 4 5 6 1 2 3 4 5 6 A B 1 2 3 4 5 6 1 2 3 4 5 6 A B

1 2 3 4 5 6 1 2 3 4 5 6 A B 1 2 3 4 5 6 1 2 3 4 5 6 A B

130

95

72

55

43

34

130

95

72

170

130

95

72

55

170

43

1 2 3 4 5 6 1 2 3 4 5 6 A 1 2 3 4 5 6 1 2 3 4 5 6 A

1 2 3 4 5 6 1 2 3 4 5 6 A B 1 2 3 4 5 6 1 2 3 4 5 6 A B

1 2 3 4 5 6 1 2 3 4 5 6 A B 1 2 3 4 5 6 1 2 3 4 5 6 A B

130

95

72

55

43

34

130

95

72

55

43

34

130

95

72

170

130

95

72

170

130

95

72

55

170

43

130

95

72

55

170

43

130

95

72

55

43

34

130

95

72

170

130

95

72

55

170

43

76

Table 4.2: Summary of sera screening results

Target Peptide Assay Screened against

Results Mouse selected

for fusion

Expected molecular weight (native protein)

Mouse #1

Mouse #2

LAMB3 Peptide-1

Indirect ELISA

BSA-peptide 1:100k 1:30k 1 129

WB Trasnfected 293T lysate

1:100k 1:10k

LAD1

Peptide-1 Indirect ELISA

BSA-peptide-1 1:30k 1:10k

1

57

WB Trasnfected 293T lysate

1:100k 1:100k

Peptide-2 Indirect ELISA

BSA-peptide-2 1:100k 1:10k

2 WB

Trasnfected 293T lysate

No signal

1:3k

Keyhole Limpet Hemocyanin (KLH) is a carrier protein extensively used in

immunizations for antibody production. As peptides are small molecules and are

relatively poorly immunogenic, they were conjugated to KLH and this stimulated a

strong immune response. However, immunized mice develop antibodies against both

KLH and the target-specific peptides. In order to exclude anti-KLH antibodies, BSA-

conjugated peptides were used for testing the anti-sera. This eliminated all anti-KLH

antibodies and facilitated the selection of only target (peptide)-specific antibodies.

Peptides represent only a small portion of the protein and indirect ELISA

doesn‟t prove whether the anti-sera contain antibodies that can recognize full length

proteins. In order to address this challenge, the anti-sera were also tested by WB

against target- or mock-transfected 293T cell lysates. Positive signals in the lanes

where anti-FLAG and anti-HA antibodies were used confirmed the expression of full

length protein with both N- and C-terminal tags. As LAMB3 did not contain FLAG tag,

anti-HA antibody was used.

77

In summary, these data suggest that the anti-sera were able to recognize

immunogens (peptides) as well as full length proteins indicating that the immunizations

were successful.

4.3.3 Myeloma - spleen fusions

Separate fusions were performed for each peptide using mouse myeloma cells

and spleen cells harvested from the selected immunized mice. Fusion plates were

evaluated for fusion efficiency (number of wells with colonies/total number of wells) 10-

14 days after cell fusion. Live hybridoma colonies were observed in >80% of the culture

wells (>80% fusion efficiency) indicating that the fusions were successful.

Approximately, the average number of hybridoma colonies per each well ranged

between two and three. Apart from live hybridoma colonies, many dead cells were also

observed in the wells as expected. This indicates that all un-fused myeloma and spleen

cells failed to survive in the selective HAT medium (Figure 4.5).

Figure-4.5: Hybridoma colony in HAT medium. After spleen-myeloma fusion was performed using PEG, cells were cultured in selective HAT medium for 10-14 days. A representative well in a 96-well fusion plate with live hybridoma colony surrounded by dead myeloma and spleen cells in HAT medium is shown. Picture was taken on Day-10.

78

4.3.4 Fusion screening

Culture supernatants from fusion plates were tested for the presence of target-

specific antibody by three independent assays: indirect ELISA using target-specific

peptide, WB using target-transfected 293T cell lysates and a unique swELISA using the

293T cell lysates that over-expressed the target protein. The latter assay is described

separately in the next section.

For indirect ELISA, total number of hybridoma culture supernatants screened for

each fusion was as follows: 960 for LAMB3 (from 10 plates), 576 for LAD1-peptide 1

(from 6 plates) and 864 for LAD1-peptide 2 (from 9 plates). Serum samples from the

mice selected for fusion were used as positive controls. HAT medium used for

hybridoma cell culture served as negative control for this assay. Only those

supernatants whose signal was at least 5 times higher than that of the negative control

were considered positive. Positive signals were readily observed in some wells

indicating that some hybridomas produced antibodies that were able to recognize the

peptide. However, the majority of the hybridoma supernatants tested in all three fusions

did not yield a positive signal (Figure 4.6). This suggests that not all hybridomas

produced antibody. All hybridomas that yielded a positive signal were selected and

scaled up to a 24-well plate. They were re-tested again by indirect ELISA in order to

determine if they retained the antibody producing ability. Interestingly, only few

hybridomas turned out to be positive in the re-screen. Total number of positive

hybridomas identified for each fusion after re-screen was as follows: 9 for LAMB3, 6 for

LAD1-peptide 1 and 25 for LAD1-peptide 2.

79

Parental hybridoma supernatants that were positive by indirect ELISA were also

tested by WB against whole cell lysates at 24-well stage in parallel with indirect ELISA

re-test. This was to determine if the antibodies present in the supernatants are able to

recognize full length proteins. As evident from Figure 4.7, bands were observed at the

expected molecular weight only in some hybridoma supernatants for each target.

Positive signals in target-transfected 293T lysates suggest that the antibodies were able

to recognize full length proteins. For LAMB3, 5 out of 34 supernatants tested were

positive by WB; for LAD1-peptide 1 this number was 3 out of 6; and for LAD1-peptide 2

it was 8 out of 25. This data shows that only some hybridomas produced antibodies that

were able to detect full length proteins.

Figure-4.6: Fusion screen by indirect ELISA. Hybridoma culture supernants were added to the ELISA plate coated with target-specific peptide (4.6a, LAMB3; 4.6b, LAD1-peptide 1; 4.6c, LAD1-peptide 2). Plates were then developed using goat-anti-mouse secondary antibody conjugated to AP. Each bar in the graph represents one well of a 96-well plate.

4.6a

4.6b 4.6c

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FIGURE 4.7 WB analyses of parental hybridomas. Parental hybridoma supernatants of LAMB3 (4.7a), LAD1-peptide 1 (4.7b) and LAD1-peptide 2 (4.7c) were tested against cell lysates derived from target-transfected 293T cells. Black arrows represent positive controls (probed with anti-HA antibody) and yellow arrows represent negative controls (culture medium). White arrows represent hybridomas that were selected and persued further. Expected size of the protein is indicated with a black arrow.

When myeloma cells were fused with spleen cells, not only B-cells but also other

splenocytes were involved in the fusion resulting in non-B-cell hybridomas that did not

produce any antibodies. Furthermore, B-cells constitute only a small proportion of the

spleen cells. Therefore, only a small portion of the total hybridomas screened produced

antibodies as evident from the indirect ELISA data. Not all hybridomas produced

antibodies that were able to recognize both purified peptides and full length target

proteins. This could be due the complexity of the whole cell lysates, where the target

protein is present in a mixture of various cytoplasmic and nuclear proteins, compared to

peptides which are purified antigens without any contaminants. Primary hybridomas

derived from a fusion plate are known as parental hybridomas. Interestingly some

hybridomas have lost the ability of producing antibodies as it was evident from the

4.7a. LAMB3

4.7c. LAD1-p2

4.7b. LAD1-p1

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indirect ELISA re-test. This could be due the instability of hybridomas at early stages of

fusion. Hybridoma stability issues primarily include mutations and chromosomal losses

(Castillo FJ, 1994).

4.3.5 Sandwich ELISA for early screening of hybridomas

Hybridomas that were positive in the initial indirect ELISA and WB screens were

further tested to determine if the supernatants could recognize soluble, non-denatured

target proteins in whole cell lysates. Over-expressed, FLAG- and HA-tagged LAD1 in

293T cell lysates was captured using rabbit anti-FLAG antibody immobilized on the

microtiter plate. A diagrammatic representation of this assay is shown in Figure 4.8a.

When hybridoma culture supernatants were added to the ELISA plate, signal was

observed only in some of the wells. As expected, no signal was observed in the wells

where culture medium was used (Figure 4.8b). Serum from pre- and post-immunized

mice served as negative and positive controls respectively. As an additional control,

biotinylated anti-HA antibody was used in place of hybridoma culture supernatant. This

data suggests that some hybridomas produced antibodies that were able to detect

soluble, denatured proteins. Together these findings suggest that some hybridomas

produced antibodies that were able to detect only denatured protein and some were

able to detect only non-denatured, soluble proteins. Antibodies produced by only a few

hybridomas were able to perform both of the above mentioned tasks. A summary of

fusion results obtained from all three assays is shown in Table-4.3.

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4.8a

4.8b

Figure-4.8: Early screening of hybridomas by rabbit-FLAG sandwich ELISA. 4.8a. Rabbit anti-FLAG antibody acts as capture antibody, transfected 293T lysates serves as target and hybridoma supernatant serves as detection antibody. 4.8b. EIA plate was coated with rabbit-anti-FLAG antibody. Whole cell lysates over-expressing LAD1 were added followed by hybridoma supernatants. Plate was then developed using anti-mouse antibody conjugated to AP. Supernatants for which the Optical Density (OD) value was at least 3 times or higher than that of the culture medium were considered positive and these are shown with arrows in the graph. Mouse-anti-HA antibody served as positive control for the assay.

FLAG and HA-tagged LAD1 protein in transfected 293T lysate

Rabbit-anti-FLAG Antibody

LAD1-specific antibody in the hybridoma culture supernatant

goat-anti-mouse 20 antibody

conjugated to Alkaline Phophatase

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Table 4.3: Summary of fusion results (p1 and p2 represent peptide 1 and 2)

Target Mouse used

Total number

of fusion plates

Fusion Efficiency

Fusion Results Info (# of positives) Clones picked for

scale up ELISA WB

R-FLAG swELISA

LAMB3 #1 10 85.6% 9 6 L8.P20.D10

LAD1-p1 #1 6 99.0% 6 4 3 L13A.P4.1.4

LAD1-p2 #2 9 88.5% 25 8 4 L13B.F2.P18.1G7

In this study, a unique swELISA was developed to identify the hybridomas

producing antibody that can detect soluble protein in its native conformation without

antigen immobilization early in the antibody screening process. Antigen-specific mAbs

present in crude hybridoma supernatants are normally screened by ELISA on plates

coated with the relevant antigen. One of the major disadvantages with these assays is

that it is difficult to determine if the crude hybridoma supernatant can detect protein in

solution. Some advanced methods such as Biacore, and flow based immunoassays are

available but they are time consuming and expensive.

Recent studies showed that antibody immobilization has distinct advantages over

the ELISA which uses antigen immobilization (Harvey Lodish 2008). However, none of

the assays employ whole cell lysates that contain the native target protein in a pool of

other cytoplasmic and nuclear proteins for the identification of antibody producing

hybridoma clones. The assay developed in this study circumvented the purification of

antibody in order to determine if they can detect soluble proteins. As this assay was

performed using parental hybridoma supernatants, it served as a powerful tool for early

screening of the mAbs. Furthermore, sera from immunized mice also showed strong

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signal, indicating that this assay could be used for selection of mice with best

immunoreactivity for cell fusions. This strategy proved useful for the rapid establishment

of sensitive swELISAs compared with the traditional assays in which the best condition

is determined by trial and error. LAMB3 could not be tested by this approach because it

did not express FLAG tag.

4.3.6 Hybridoma subcloning and scale up

Only those parental hybridomas that were positive by at least two of the three

above mentioned assays (indirect ELISA, WB and swELISA) were expanded and

cloned by limiting dilution to obtain clonal hybridoma lines. All subclones were evaluated

in the same way as parental hybridomas. Subcloning was performed until more than

90% of the clones tested (from a total of 20) were positive by both WB and peptide

ELISA. A representative of one subclone screen by WB for each target is shown in

Figure 4.9. Bands were found at the expected molecular weight in the WB assay

suggesting that the hybridomas retained antibody producing ability. After extensive

evaluation, final hybridoma lines for each target were scaled up to 100 mL for antibody

purification. Table 4.4 shows a list of final hybridomas selected for each target.

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Figure 4.9 Subclone screening by WB assay. All subclones were initially screened by indirect ELISA and selected subclones were screened by WB against transfected 293T cell lysates. Arrows represent positive controls. Expected size of the protein is indicated with a black arrow. White arrows represent positive control where anti-HA antibody was used.

Table 4.4 Final hybridoma cell lines selected for each target

Target Parental hybridoma

selected Total number of

subclonings performed Final clone name

LAMB3 L8.P20 1 L8.P20.D10

LAD1-p1 L13A.P4 2 L13A.P4.1.4

LAD1-p2 L13B.P18 1 L13B.P18.1G7

Subcloning procedure involves the selection of clones that produce antibody. It

ultimately results in the formation of a cell line that stably produces antibody of interest.

The chromosomal number of the parental hybridoma cells is 4n. Therefore the cells are

highly unstable in this stage and they tend to lose chromosomes. The loss of

chromosomes containing the antibody producing genes results in the formation of a

hybridoma that is unable to make antibody. Therefore, hybridoma colonies derived from

single cell were subcloned multiple times until the cells stably produced antibody.

4.9a. L8.P20 (LAMB3)

4.9c. L13B.P18 (LAD1-p2)

4.9b. L13A.P4.1 (LAD1-p1)

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4.3.7 Antibody characterization and purification

Binding of the Fc portion of antibodies to the chromatography column depends

on the isotype of the antibody. Therefore, isotype of the antibodies was determined prior

to purification by ELISA. When hybridoma culture supernatants were added to the

ELISA plate pre-coated with anti-isotype-specific antibodies, positive signals (at least 5-

fold higher than the back ground) were readily detected only in the anti-IgG1 antibody

coated wells for each sample. Similarly, positive signals were detected only in the anti-

kappa light chain antibody coated wells. All antibodies generated for LAMB3 and LAD1

were found to have IgG heavy chains and κ light chains (Figure 4.10). Results show that

all antibodies belonged to IgG1 subclass containing kappa light chains.

Figure-4.10: Isotyping ELISA for antibody characterization. EIA plate was coated with different types of anti-Fc heavy chain antibodies and anti-light chain antibodies. Hybridoma supernatants were tested against all these antibodies.

LAMB3 LAD1-p1 LAD1-p2

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Once the isotype of the antibodies was determined, hybridoma culture

supernatants were run in a Protein G sepharose column in a high-throughput FPLC

machine. FPLC data shows that a total of 2.5 mg of antibody was obtained for LAMB3

from a 100 mL hybridoma culture supernatant. The same for LAD1-p1 and LAD1-2 were

15 mg and 5.9 mg respectively (Table 4.5). Antibody purification graphs

(chromatograms) for LAMB3 and LAD1 antibodies are shown in Figure 4.11. This data

indicates that hybridomas significantly vary in their antibody production abilities.

Table 4.5. Summary of antibody characterization and purification

Target Clones picked

for scale up Name of the

mAb Isotype

Amount of antibody (from 100 mL culture)

LAMB3 L8.P20.D10 LAMB3-1 IgG1 kappa 2.5mg

LAD1-p1 L13A.P4.1.4 LAD1-1 IgG1 kappa 15mg

LAD1-p2 L13B.F2.P18.1G7 LAD1-2 IgG1 kappa 5.9mg

Figure 4.11. Antibody purifications: Chromatograms of LAMB3 (4.11a), LAD1-peptide 1 (4.11c) and LAD1-peptide 2 (4.11c) are shown. UV absorbance is shown on the Y-axis. Various fractions collected in the 96-well plate during the run are shown on the X-axis. Amount of antibody in the sample was calculated based on the UV-absorbance. Fractions where the peak was observed were collected and pooled from the 96-well sample collector. Antibodies were concentrated to a final concentration of 1 mg/mL after dialysis was performed.

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Immunoglobulins are made up of two heavy chains and two light chains held

together by disulfide linkages (Silverton EW, 1977). They contain two fragments that

include Fab (fragment antigen binding) and Fc (fragment crystallizable). Based on the

structural features of Fc heavy chain, there are five different subclasses of

immunoglobulins: IgG, IgM, IgA, IgE and IgD and there are two different types of Fab

light chains, kappa and lambda. It is important to determine the subclass of the

antibodies because the efficiency of the antibody binding to the Protein A or G in the

chromatography column during purification mainly depends on the type and species of

heavy chain of the antibody (Akerstrom, 1985; Frank, 2001; Richman, 1982).

4.3.8 Evaluation of purified antibodies

To determine if the purified antibodies can be used for any assays other than

swELISA, they were tested initially by WB against whole cell lysates derived from

various PDAC cell lines. LAMB3 mAb recognized the target in at least two cell lines,

SW1990 and Capan2 (Figure 4.12a). Similar approach for LAD1 antibodies was not

successful (data not shown). Therefore LAD1 antibodies were tested by

immunoprecipitation (IP) to determine if they can pull down endogenous LAD1 from

PDAC cell lines. Initially they were tested on transfected 293T cell lysates. Strong signal

was observed only when LAD1-1 or LAD1-2 mAbs were used for both IP and probing

the blot (lanes 1 and 4 in Figure 4.12b). However, the signal was weak when the lysates

were immunoprecipitated with LAD1- and probed with LAD1-2 or vice-versa (lanes 2

and 3 in Figure 4.12b). These findings strongly suggest that the antibodies are specific

for their corresponding epitopes. Similar findings were observed when the antibodies

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were used for IP on endogenous protein (Figure 4.12c) in PDAC cell lines. Interestingly,

LAD1-1 antibody was unable to detect native LAD1 when the protein was

immunoprecipitated with LAD1-2 antibody (data not shown). Further more, additional

band between 95 KDa and 130 KDa could be variant of endogenous LAD1 (Marinkovich

et al., 1996; Uitto and Pulkkinen, 1996; Zillikens D, 1999). These data suggest that at

least one of the two LAD1 mAbs could IP native LAD1 from PDAC cells.

Sera and hybridoma screens performed by epitope-tag swELISA demonstrated

that the antibodies can recognize soluble, non-denatured LAD1. Screening by WB

provided valuable information on the specificity and ability of the antibody to recognize

denatured protein. Furthermore, the LAD1 antibodies reported here were also able to

immuoprecipitate LAD1 from transfected 293T cells as well as native LAD1 expressed

in PDAC cell lines. However, signal variations in the WB when the protein was

immunoprecipitated separately with LAD1-1 and LAD1-2 antibodies suggest that both

antibodies differ in their antibody binding efficiencies and that they are different in their

affinities for these distinct epitopes. This assay also demonstrated that both antibodies

were specific for their corresponding epitopes against which they were raised.

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Figure 4.12 Evaluation of purified antibodies by WB and Immunoprecipitation. 4.12a LAMB3- or mock-transfected 293T cell lysates and various PDAC cell lysates were tested by WB. Purified LAMB3 antibody was used as primary antibody. 4.12b Sepharose beads were coated with LAD1 antibodies (LAD1-1 and LAD1-2) and these were used to pull down the recombinant LAD1 protein in transfected 293T cell lysates. 4.12c Similar approach was employed to pull native LAD1 protein in lysates from PDAC cell lines. Data suggests that these antibodies can immuoprecipitate both recombinant and native LAD1 protein.

1. LAMB3-293T

2. Mock-293T

3. SW1990

4. Miapaca

5. 8988s

6. Capan2

1 2 3 4 5 6

LAMB3

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1. IPed with LAD1-1 and probed with LAD1-1

2. IPed with LAD1-2 and probed with LAD1-1

3. IPed with LAD1-1 and probed with LAD1-2

4. IPed with LAD1-2 and probed with LAD1-2

LAD1

1 2 3 4

Blot probed with LAD1-2 (13B.P18.1G7), 1:500 dilution

LAD1

1. SW1990 - LAD1--1 Immunoprecipitated

2. 8988s - LAD1-1 Immunoprecipitated

3. Miapaca - LAD1-1 Immunoprecipitated

4. SW1990 - LAD1-2 Immunoprecipitated

5. 8988s - LAD1-2 Immunoprecipitated

6. Miapaca - LAD1-2 Immunoprecipitated

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In summary, novel mouse mAbs were generated for LAD1 and LAMB3. These

antibodies were able to recognize denatured as well as native, non-denatured soluble

proteins both in the cell lysates over-expressing the target protein and also in the cell

lysates derived from PDAC cell lines. A novel rabbit-anti-FLAG swELISA assay was

also developed. This assay could be used to identify hybridomas that produce

antibodies that are capable of recognizing proteins in their native conformation at an

early stage during the antibody generation process.