monoclonal antibody protocols

The fusion product of the myeloma with the antibody-producing spleen cells will express both Balbk antigens from the MOPC-21 and those of the donor strain that provided the spleen cells.

CHAPTER 1

and J II Ulrich

1 Introduction The first step in preparing useful monoclonal antibodies (MAbs) is to immunize an animal with an appropriate “vaccine.” Animal and vaccine are both emphasized in the preceding sentence because this chapter describes how to generate satisfactory MAbs by maximizing interactions between the two The term vaccine was used purposefully to connote that not only antigens of interest may be contained in the immunizing product, but carriers and adjuvants may also be included These latter components can influence greatly the success of obtaining useful hybri- domas, which produce antibodies of the desired specificity and quality Immunization protocols for obtaining only murine MAbs are covered herein Although cross-species hybridizations can be made, they usually involve very specialized techniques Moreover, the lessons that can be derived from mouse immunization protocols can, in general, be extrapo- lated to other species as well

When considering which mouse strains to immunize, even though some initial advantage may be obtained by selecting a strain other than a Balb/c, there is an overriding consideration that must be taken into

From Methods m Molecular Bology, Vol 45’ Monoclonal Antrbody Protocols

Edlted by W C Davis Humana Press Inc., Totowa, NJ

1

2 Rudbach, Cantrell, and Ulrich

account The usual mouse myeloma used for fusion IS a HAT-sensitive variant of the Balbk-derived MOPC-2 1 myeloma The fusion product of the myeloma with the antibody-producing spleen cells will express both Balbk antigens (from the MOPC-21) and those of the donor strain that provided the spleen cells Therefore, any production of ascites as a source

of MAb must be performed in a histocompatible mouse strain This is easiest if the spleen cell donor is a syngeneic Balbk mouse If the spleen cell donor is an inbred strain other than Balbk, then the F, progeny of a Balbk-“spleen cell donor” cross, which contains both sets of histocompat- ibility antigens, must be used to grow the hybridoma for ascites production With these genetic restrictions, hybridomas generated from spleen cells donated by outbred mice would be allogeneic and precluded from growth m any recipient One way around this problem would be to generate MAbs only from cell-culture fluids, thus avoiding the histocompatibility problem However, this usually results in lower yields of antibodies Therefore, most investigators find it easier to manipulate the immunological responses of Balbk mice with adjuvants and/or carriers rather than reverting to the use of other inbred mouse strains for immunization

An antigen is a molecule that, when introduced into an appropriate animal, will stimulate an immunological (antibody) response in that ani- mal In basic terms, an epitope is the minimal chemical configuration in

an antigen that can be immunologically recognized as uniquely specific

by the immune system Inasmuch as a controlling reason for generating MAbs, instead of polyclonal antiserum, is to obtain a high degree of specificity, immunization with an epitopically restricted antigenic mate- rial is usually preferable to the use of a crude antigen Such antigens with restricted diversity can be obtained by blocking nondesired epitopes, by chemically conjugating purified chemical groupings to a carrier, or by syn- thesizing/cloning epitopically pure antigens However, regardless of the antigenic material used in the vaccine, the final selection of specificity will be made during screening of the hybridoma supernatant fluids for antibody The nature of the ligand attached to the solid support is of prime importance at this point

There is a paradigm in immunology that the first antibody developed after immunization is usually more specific than that produced later in the immune response On the other hand, a later antibody may have more

of the desired properties of affinity, class, and subclass (1) These con- siderations, as well as the desirability of generating sufficient numbers

Immunization to Enhance Immune Response 3

of antibody-secreting cells to yield a quantitatively satisfactory fusion run, require a well-designed immunization protocol It is thought that high-affinity antibody-producing cells can be selected by using minimal (suboptimal) amounts of antigen (I) In order to use this approach and not compromise some of the practical aspects of the procedure, immuno- logical adjuvants can be employed Appropriate adjuvants can be selected that will increase the number of antibody-forming cells and also can direct the response to yield a qualitatively desirable antibody Antigens, which are weakly immunogenic because they are functional molecules, related to tissue antigens of the mice, denatured or lack appropriate physi- cochemical properties, or too small, can have their immunogenicity increased through the use of adjuvants Furthermore, conjugation of antigens to carriers, with or without coadministration of adjuvants, can turn marginally immunogenic materials into useful antigens (2) Use of carriers and procedures for conjugating them have been described else- where (3), and are not covered in this chapter

Adjuvants are materials that are not (usually) themselves immunogenic, but which can be used in conjunction with antigens to alter an immune response quantitatively and/or qualitatively (4) Although many types of adjuvants are available, only those with proven utility for generating cells useful for MAb production in mice are covered These are commercially available and do not require extensive preparation or manipulation Com- plete Freund’s Adjuvant (CFA) is a potent adjuvant that has been used successfully for decades It can be used with weakly antigenic materials and has a reputation for stimulating the production of large amounts of high-quality antibody (5) CFA, however, suffers from its toxicity It has

a history of inducing necrotic lesions in animals even after a single use Moreover, many animal care committees have banned the use of CFA in their facilities

An alternative to CFA is the Ribi Adjuvant System (RAS), which has gained wide acceptance both by immunologists and animal care commit- tees (6) RAS is a ready-to-use product, two forms of which are recommended for use in mice to generate cells suitable for fusions lead- ing to MAb production One of these contains synthetic trehalose dicorynomycolate (S-TDCM) in a form that can be readily formulated into an oil-in-water emulsion The second form contains monophosphoryl lipid A (MLA) as a second immunostimulant, in addition to the S-TDCM The choice of which one to use is somewhat empirical, but can be

Rudbach, Cantrell, and Ulrich

directed by the nature of the antigen and the quality of the antibodies desired Our experience has shown that the use of S-TDCM only as the adjuvant produces predominantly IgGr isotype MAbs The use of S-TDCM + MLA increases the probability of a fusion yielding MAbs

of the IgG2 isotype

2 Materials

1, Antigen: Prepare or obtain antigen of choice

2 Adjuvant RAS (see Section 3.2 for details)

3 Phosphate-buffered salme (PBS): 0.15M NaCl and O.OlM

NaH2P04-Na2HP04, pH 7.4

4 Mouse: Use female mice (see Note 1)

1, Dry ice or a CO2 tank and regulator

2 Cotton, 500~mL beaker (or other container that can be covered)

3 Solution of sodium heparin (1500 U/mL) in saline

4 Pasteur pipets

5 Razor blade (single-edged)

6 1 mL Tuberculin syringes and 1/2-m., 27-gage needles

Because a mouse dose will be contained in 0.2 mL, this solution will be a lo-fold concentrate Store the PBS-soluble antigen preparation under con- ditions deemed appropriate for the material (-7O”C, 4OC, and so forth) This recommendation for preparation of an antigen solution is ideal, but is not absolutely necessary

2 Antigens soluble m detergent: Sometimes detergents are necessary to solu- bilize very hydrophobic proteins When possible, solubrlize antigen in

antigen concentration of five times a dose expected to be given to a mouse, the detergent concentration should be 0.2% or less

Immunization to Enhance Immune Response 5

3 Immobilized antigen: Another type of antigen preparation that is frequently encountered is a band cut from a polyacrylamide gel electrophoresis (PAGE) gel The slice of gel should be reduced to the smallest particles practical by suspendmg it in a small amount of salme and expressing it repeatedly through successively smaller hypodermic needles, beginning with an 18-gage and finishing with a 27-gage needle This suspension can

be treated as an antigen solution and prepared with the adjuvant as described in Section 3.2

It is recommended that the antigen under consideration be incorpo-

saline However, weak immunogens can be used at concentrations of up

to 1.0 mg/rnL If the amount of antigen available is very limited, the

has shown that it is better to give multiple doses of small amounts of antigen rather than to administer all of it in a single dose; this should be considered when deciding on formulations of a precious antigen

The RAS is available as an oil concentrate, which only requires recon- stitution with a solution of antigen Vaccines are formulated with RAS adjuvants as follows:

1 Each vial of lyophilized adjuvant emulsion contains 0.5 mg of each immunostimulant, 40 pL of oil (Squalene) and 4 uL of Tween-80 Vials should be stored at 2-8°C until used

at 40-45”C for 5-10 mm (alternatively, the vial can be warmed in a beaker

of hot tap water for 5-10 min)

3 Reconstitute each vial with 2.0 mL of sterile PBS containing the desired amount of antigen as follows:

a Inject the antigen-PBS solution (2 mL) directly into the veal through the rubber stopper, using a syringe fitted with a 20- or 21-gage needle (leave the cap seal in place)

b Vortex the vial vigorously for 2-3 mm to form emulsion, with rubber stopper in place

4 The final vaccine will contain 50 ug of each adjuvant/O.:! mL (a mouse dose) The final emulsion also contains 2% oil (Squalene) and 0.2% Tween-80

If the entire contents of the vial will not be used initially, reconstitute

to 1 mL with saline, and mix aliquots 1: 1 with antigen in saline imrnedi-

6 Rudbach, Cantrell, and Ulrich

lyophilized Do not store frozen Prior to animal inoculation, warm the vial to 37”C, and vortex briefly

When using a vaccine prepared with a RAS emulsion, it is recom- mended to inject mice with 0.2 rrL ip or SC (0.1 mL in each of two SC sites) Our experience suggests the SC route is the preferred route A minimum protocol for immunizing mice to generate cells for preparing hybridomas is as follows: immunize on d 0, boost on d 21, take a trial bleeding on d 26; if the antibody titers are satisfactory, boost on d 35 with antigen only, intravenously, and remove the spleen to obtain cells for fusion on d 38 (see Notes 3 and 4)

lo), if care is taken with handling of the mice Some institutional animal care committees stipulate that mouse bleedings be performed under car-

1 Either place a small piece of dry ice beneath cotton m a beaker or fill the covered beaker with CO, gas from a tank

2 Place mouse in beaker until It is anesthetized

3 Remove the mouse, and rapidly bleed by one of the followmg techniques:

a Retro-orbital: Insert the tip of a Pasteur pipet, which has been “wetted” with the heparin solution, into the retro-orbltal space, anterior to the eye Rotate gently to disrupt the vascular plexus, and collect by capil- lary action about 100 FL of blood

b Cardiac: With the mouse on its back, wet the chest with alcohol, and insert a 27-gage needle into the heart, between the ribs or under the sternum, through the diaphragm, Collect 100 PL of blood mto the

“heparm-wetted” syringe

c Tall vein: With the corner of a new, alcohol-wiped razor blade, nick a lateral vein, longitudinally, near the tip of the tad Collect, by capdlary action, 100 pL of blood into a “heparm-wetted” Pasteur pipet Compress

Immunization to Enhance Immune Response 7

with dry cotton to stop the blood flow Warming mice under a heat lamp for a few minutes immediately before bleeding will increase blood flow through the veins and speed the process of blood collection

4 Express the blood into a nncrocentrifuge tube that contains 10 pL of the hep- arm solution in its tip, vortex well, and centrifuge to separate the plasma

5 Remove the plasma to a second microcentrifuge tube, seal, and store m a freezer, if not tested immediately

4 Notes

1 It is recommended that female mice be used for immunization Male Balb/c mice fight; many times the tails are so damaged that inJections and bleedings are impaired

2 If sufficient antigen is available, mice should be immuruzed to prepare a pool of polyvalent antiserum The enzyme immunoassay (EIA) assay should be optimized with this antiserum pool (see Chapter 10) The short time between successful screenmg of the culture supernatant fluids for

ally is not sufficient for optimizmg the EIA assay

3 With most antigens, a good antibody titer can be achieved after a single booster injection If, however, the serum antibody titer is too low, a second

booster injection, with adJuvant, should be given, and another test bleed- ing taken to determine if satisfactory titers have been obtained

4 In order to increase the chances of obtainmg a hybridoma that will yield the desired quality of antibody, the immunization protocol should be

designed to yield the maxtmum number of antibody-forming cells from

the spleen Experience has shown that a mouse with a higher serum anti- body response yields splenic cells that result in proportionately greater numbers of specific MAb-producing hybridomas Therefore, the immuni-

zation protocol should be designed to maximize serum antrbody titers

References

1 Davis, B D., Dulbecco, R , Eisen, H N., Ginsberg, H S., Wood, W B., and McCarty, M (1973) Antibody formation, in Mzcrobiology, 2nd ed , Harper & Row, New York, pp 484,485

2 Benjamini, E and Leskowitz, S (1991) Immunology A Short Course, 2nd ed., Wiley-Liss, New York, pp 38-40

3 Kabat, E A and Mayer, M M (1961) Experimental Immunochemistry, 2nd ed , Thomas, Spnngfield, IL, pp 446-450,798-802,8 13-8 15

4 Hui, G S N., Chang, S P , Gibson, H., Hashimoto, A., Hashno, C , Barr, P J., and Kotam, S (1991) Influence of adJuvants on the antibody specificity to the

Plasmodmm falciparum maJor merozoite surface protein, gp195 J Immunol 147,

39353941

Rudbach, Cantrell, and Ulrich

5 Kabat, E A and Mayer, M M (1961) Experimental Immunochemuty, 2nd ed., Thomas, Springfield, IL, pp 309-310, 872

6 Rudbach, J A., Johnson, D A., and Ulrich, J T (1995) Ribi adJuvants: chemistry, biology and utility in vaccines for human and veterinary medicine, in Adjuvants Theory and Practical Applications (Stewart-Tull, D E S , ed.), Wiley, New York,

pp 287-313

CHAPTER 2

Margaret E SchelZing

1 Introduction

In vitro immunization involves the exposure of spleen cells to antigen

in tissue culture rather than the antigenic stimulation of spleen cells via immunization of mice The production of monoclonal antibodies (MAbs)

to highly conserved molecules, such as enzymes (1,2), is possible using

in vitro immunization MAbs to such “self’-antigens often are not pos- sible to make using traditional in vivo methods owing to immune sup- pression or tolerance Utilizing in vitro immunization, it is possible to elicit the formation of MAbs in response to picogram quantities of anti- gen (3-6) Although certain protocols (I, 7) indicate a minimum require- ment of from 30-100 yg antigen for in vitro immunization, we have found that the nanogram or picogram quantities of antigen available from blotted polyacrylamide gels provide sufficient antigen for the prepara- tion of MAbs by in vitro immunization (3,.5) Additional advantages of

in vitro immunization include shortening the immunization procedure from the 5 or 6 wk required for in vivo immunization to 4 d, allowing defined antigen concentrations, and controlling antigen degradation (3)

In vitro immunization is modulated by regulating the activation and maturation of antigen-specific B-lymphocytes using growth/differentia- tion factors An extensive literature describes interactions between the various lymphokines and cell types involved in the regulation of B-cell pro- liferation and differentiation, but these interactions are not sufficiently

From Methods m Molecular Brology, Vol 45 Monoclonal Antibody Protocols

Edtted by W C Davis Humana Press Inc , Totowa, NJ

9

Schelling

defined to provide a complete overview Factors that appear to increase the immune response to specific antigens in vitro include interleukin-2 (IL-2) When IL-2 was included in the in vitro immunization, Pollock and d’Apice (8) found that cultures produced a higher yield of hybrido- mas producing MAbs of the desired specificity Additionally, muramyl dipeptide (MDP) has been reported to increase the yield of specific anti-

attributed (8) to the ability of MDP to stimulate interleukin- 1 (IL- 1) pro- duction by monocytes/macrophages, which activates helper T-cells, and its adjuvant effect on immunizations MDP is not a polyclonal activator

of human lymphocytes, which may be important in limiting the number

of activated but irrelevant lymphocytes available for fusion following antigen stimulation The IL-2 effect is also possibly the result of its effect

on helper T-cells Jacot-Guillarmod (II) reported the use of 10% condi- tioned medium as a source of B-cell growth and differentiation factors This conditioned medium consisted of a 2-d-old supernatant from human spleen cells cultured in the presence of pokeweed mitogen The activity of the conditioned medium was replaced by 20 p,g/niL MDP and

200 UlmL IL-2 (I I) &helling (3) reported the addition of dextran sulfate

to thymocyte-conditioned medium (TCM) (12,13) for increased specific MAb formation for viral proteins Martin et al (14), however, reported that the addition of specific and nonspecific cell activators such as Sta-

sulfate, to the immunizing medium did not increase the in vitro secretion

The number of specific MAbs produced is higher when no more than 2% fetal bovine serum (FBS) is used in the in vitro unmunization system (3) Additionally, the number of specific MAbs produced is greater when the addition of FBS is delayed until 24 h following the addition of anti- gen to the in vitro immunization system, thus avoiding a competition

of the antigen with components of the FBS in the in vitro immunization system for the production of MAbs

In vitro immunization of B-lymphocytes frequently results in the pro- duction of IgM MAbs If IgG MAbs are preferred, it is possible to inject mice prior to harvest of the spleen cells for in vitro immunization according to in vivo technique immunization schedules It has been reported that sequential in vitro immunizations are possible, but given the short-lived existence of spleen cells in culture, it is difficult to

Methods of Immunization 11

maintain good condition of the spleen cells for a long enough duration to accommodate sequential in vitro immunizations Spleen cells that are cultured no longer than 4-5 d exhibit the highest fusion efficiency and yield of hybridomas Additionally, it is sometimes possible to obtain the

2 Materials

1 Tissue-culture medium (CDMEM):

a Add 5 mL of stock penicillin (10,000 U/n& concentratron of stock, 100 U/r& final concentratron) and 5 mL of streptomycin (10 mg/mL stock,

100 pg/mL final concentration) to Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 4.5 g/L glucose, available from the manufacturer in 500 ml/bottle

b Add 5 mL 100X L-glutamme (200 m&I) for a final concentratton of 2mM

c Add 5 mL 100X sodium pyruvate (100 rniI4) for a final concentration of 1mM

d Keep bottles tightly capped to prevent a change m pH of the medium

e Store at 4°C

2 Myeloma cell line: Obtain Balb/c nonsecreting myeloma cell lme SP2/0- AG14 from the Amerrcan Type Culture Collection (ATCC, 12301 Parklawn Drive, Rockvrlle, MD 20852), cat no CRL-1581 Other myeloma cell lures can be selected for use as a fusion partner rf desrred

3 Balb/c mace: Obtain healthy Balb/c mice from a reputable animal supplier

4 Glucose salt solution (GSS): 8 g NaCl, 0.4 g KCl, 1.77 g Na2HP04.2H,0, 0.69 g NaH,PO,aH,O, 2 g glucose, 0.10 g phenol red/L of distilled H,O Completely mtx Filter-sterthze Store at 4°C for up to 6 mo

5 Polyethylene glycol (PEG): Prepare a 50% mixture of Sigma mol-wt 1500 PEG in GSS

anthine (Sigma, St Louis, MO) 100X stock solution is 136 mg/lOO mL Thymidine 100X stock solution IS 76 mg/lOO mL Hypoxanthine and thy- midine (HT) can be prepared together To dissolve, add 1N NaOH until hypoxanthine is dissolved Add thymidine and readjust pH to 9.5 with ace- tic acid Filter-sterilize Store frozen at -20°C (15) Aminopterm (Sigma

or Lederle) 100X stock IS 1.8 mg/lOO mL Add NaOH to dissolve Adjust

to pH 7.8 Filter-sterilize Store frozen at -20°C (15) Both HAT and

HT can also be purchased rf desired (Sigma)

7 FBS: FBS lots vary m the ability to provide appropriate concentrations of necessary growth factors and m levels of endotoxm For best results, it is important to test several lots of FBS prror to purchase Myeloma lures can

supplemented with 2 mM L-glutamine, 2 mM sodium pyruvate, 10 rmJ4 MEM nonessential amtno acids (M A Bioproducts), and 2% FBS (see Note 1)

2 After 48 h of incubation at 37”C, 7% CO,, remove cells by centrifugation

(200g for 15 mm)

3 Store the TCM at -20” or -70°C until the day of the in vitro immuruzation

1 In sterile tissue culture hood, dip the frozen vial of cells in a clean 37°C water bath to quickly thaw the cells (see Note 2)

2 Dip the vial into ethyl alcohol (ETOH) to disinfect Open the vial and trans- fer the contents of the vial using a sterile, plugged Pasteur ptpet to a sterile 15-mL tissue culture tube Slowly, while gently shaking the tube, dropwtse add 10 mL of chilled (4’C) medium (CDMEM)

3 Transfer the cells and medium to a T-25 tissue-culture flask (Corning, Corning, NY) and incubate (with cap loosened to provide for gas exchange)

in the tissue culture incubator at 7% CO* for several hours

4 At the end of several hours, replace the medium by carefully withdrawing medium from the cells using a Pasteur pipet Replace medium (CDMEM + 20% FBS), and continue incubation

sufficient Increase the dilution until the cells are being passed daily at a 1: 10 dilution Continue passing daily at 1: 10 dilution until the day of the fusion The myeloma cells are now in log phase (between lo4 and 5 x lo5 cell/r&) and ready to be used for fusion

1 Kill mouse by cervtcal dislocation (see Note 3)

2 Remove spleen aseptically to a Petri dish containing 2 mL of GSS

3 Disperse clumps by pipeting gently up and down a few times with a plastic 10-n-L pipet

4 Transfer cells to a 15-mL tissue-culture tube Allow any clumps to settle out for 2 min

Methods of Immunization 13

5 Decant into another 15-r& tube

6 Centrifuge for 15 mm at 17Og, room temperature

7 Decant supernatant

8 Resuspend pellet of spleen cells in l-2 mL of GSS

9 Count spleen cells (20 p,L spleen cell mixture in 1 mL trypan blue)

10 Viability of spleen cells should be 95% From lo* to 2 x lo8 spleen cells (1 or 2 spleens) are needed for fusion

3.4 In Vitro Immunization

of 50% TCM and 50% CDMEM in a T-25 tissue culture flask (Corning) Add antigen Incubate overnight at 37°C 7% COZ

2 On the next day, add 2% FBS to the flask

3 Continue to incubate for 4 d The fusion should be on the 4th or 5th d, depending on the appearance of the spleen cells The spleen cells should look healthy with entrre membranes, and enlarged “blast” cells should appear

3.5 Fusion Protocol Fuse spleen and myeloma cells as outlmed in Chapter 3 (18)

4 Notes

1 Preparation of conditioned medium can be performed up to 3 mo prior to the in vitro immunization

2 Myeloma cells are placed in culture 1-2 wk prior to the fusion

3 In vitro immunization is performed 4 d prior to the fusion

4 Use of Balb/c mice 1s highly recommended Use of other strains of mice will introduce an unneeded problem of histoincompatibility when there is

a need to produce antibody in ascites form However, this problem can be obviated by preparing MAbs in a bioreactor (see Chapter 17)

5 Healthy, infection-free mice are necessary Certain virus-infected mice (e.g., mouse hepatitis virus) are not suitable donor mice for the spleen cells used in fusions

6 Dextran sulfate (3) is sometimes used to increase hybridoma yield Not all lots of dextran sulfate are suitable for this purpose Some lots are toxic, and lots must be pretested prior to use

7 In vitro immunization typically elicits a mixture of IgM and IgG antibod- ies It is possible to obtain a higher percentage of IgG antibodies by prior immunization of mice Although there are reports in the literature of sequential immunization in vitro, it is our experience that spleen cells do not fare well enough in culture for in vitro sequential immunization

8 Low concentrations of dimethyl sulfoxide (DMSO) are sometimes added

to the PEG mixture to improve fusion efficiency

14 Schelling

9 Other fusion Balb/c-derived cell lures have been developed for productron

of hybridomas (19) Such lines should be examined for their potential to Improve the yield of hybrldomas from m vitro cultures of antigen-stimu- lated spleen cells

10 It is possible to use fungtzone at a final concentration of 2.5 l.tg/mL However, it is difficult to “cure” hybrtdoma cultures when heavily Infested with fungus

11 Fungizone should not be used in medra durmg the first few days following fusion Fungizone radically reduces the outgrowth of hybrids

12 Lots of PEG differ in toxicity and fusion efficiency 1000 Baker PEG has also been reported as a suitable PEG for fusion Tissue culture medium suppliers frequently sell PEG suitable for fusion

References

1 Pardue, R L., Brady, R C , Perry, G W , and Dedman, J R (1983) Production of MAbs agamst calmodulin by in vitro immunizatton of spleen cells J Cell Biol 96,

1149-l 154

2 Glad, C , Wennerstrom, G., and Fredlund, B M (1988) Optimization of murine zn

vitro immunization against different antigens, in rn vitro immunization, in Hybn- doma Technology (Borrebaeck, C A K., ed ), Elsevier, Amsterdam, pp 295-301

3 Schelling, M E (1986) Increase of hybridoma formation by addition of dextran sulphate to in vitro immunization system Hybridoma 5(2), 159-161

4 Schelling, M E., Hawker, J R., and Granger, H (1987) Immunochemical com- parison of peptide angtogenic factors Tissue & Cell 19(4), 463-467

5 Venkateswaran, S , Blanckaert, V , and Schelling, M E (1992) Production of anti-fibroblast growth factor receptor monoclonal antibodies by in wtro immuni- zation Hybridoma 11(6), 729-739

6 Luben, R , Brazeau, P , Bohlen, P , and Guillemm, R (1982) Monoclonal antibod- ies to hypothalamic growth hormone-releasing factor with picomoles of antigen Science 218,887-898

7 Jonak, Z L and Kennett, R H (1984) In vitro immunization of mouse spleen cells, in Monoclonal Antibodies and Functional Cell Lines (Kennett, R H , Bechtol,

K B , and McKearn, T J., eds ), Plenum, New York, pp 368-370

8 Pollock, B J and d’Aptce, A J F (1988) Productton of human monoclonal antt- bodies against specific antigens by in vitro immunization, in In Vitro Immunization

m HybrrZomu Technology (Borrebaeck, C A K , ed.), Elsevier, Amsterdam, pp 277-284

9 Dinarello, C A and Krueger, J M (1986) Inductton of IL-l by synthetic and natu- rally occurmg muramyl peptides Fed Proc 45,2545-2548

10 Bahr, G M and Chedid, L (1986) Immunological activtties of muramyl peptides

J Immunol 136,3710-3715

14 Martin, D., Brodeur, B R., Larose, Y., Faucher, S., and Hamel, J (1988) Produc- tion of human monoclonal antibodies against Haemophilus influenzae type B usmg

a heteromyeloma, m In Vitro Immumzation in Hybrldoma Technology (Borrebaeck,

C A K., ed.), Elsevier, Amsterdam, pp 295-301

15 Kennett, R (1980) Fusion protocols by centrifugation of cells suspended in poly- ethylene glycol, in MonoclonaZAntibodies (Kennett, R , McKearn, T, and Bechtol, K., eds.), Plenum, New York, pp 365-367

16 01, T and Herzenberg, L (1980) Immunoglobulin-producing hybrid cell lines, in

Selected Methods in Cellular Immununology (Mishell, B and Shiigi, S., eds.), Free- man, San Francisco, CA, pp 351-372

17 McKearn, T (1980) Cloning of hybridoma cells by hmitmg dilution m fluid phase,

m Monoclonal Antlbodles (Kennett, R , McKearn, T., and K Bechtol, K , eds.), Plenum, New York, pp 374,375

18 Davis, W C (1988) Enhancement of myeloma-B-cell hybridoma outgrowth in primary cultures with B cell mitogens Periodicum Biologorum 90(3), 367-374

19 Harris, J F., Hawley, R G., Hawley, T S., and Crawford-Sharpe, G C (1992) Increased frequency of both total and specific monoclonal antibody producing hybridomas usmg a fusion partner that constrtutively expresses recombinant IL-6 J Immunol Methods 148,199-207

CHAPTER 3

Mary J Hamilton and William C Davis

1 Introduction Efforts to refine the methods of producing monoclonal antibodies (MAbs) of known specificity (I) have revealed there are many variables that affect the growth of hybridomas generated by the fusion of myeloma cell lines with spleen cells (reviewed in refs 2 and 3) These include the cell-cycle status of B-cells in immunized mice at the time of cell isola- tion, the myeloma or hybridoma used as a fusion partner, the composition of the reagent used for cell fusion, the presence of contaminating fibro- blasts and macrophages in the primary culture, the concentration of fused and unfused cells present in primary cultures, the presence or absence of essential growth factors in fetal bovine serum, coculturing of fresh hybrids with thymocytes, spleen cells, or peritoneal macrophages, the presence or absence of 2-mercaptoethanol, and importantly, the presence

or absence of inhibitory substances in culture medium Efforts to detail which factors exert the most prominent effect on hybridoma survival and outgrowth have verified the importance of culture medium and culture medium supplements, and in addition, have shown that B-cell mitogens have a profound effect on the outgrowth of hybridomas in primary cul- tures (2) The studies have shown that it is possible, on a routine basis, to obtain 2000-3000 hybridomas from 5 x lo7 spleen cells when B-cell mitogens are used as one of the culture supplements (see Notes 1 and 2 for further comment) Equally good results have been obtained with fusions of mouse (inter-species) and rat (cross-species) spleen cells with

From* Methods m Molecular B/ology, Vol 45’ Monoclonal Antibody Protocols

Edlted by: W C Davts Humana Press Inc , Totowa, NJ

17

18 Hamilton and Davis

a mouse myeloma fusion partner (2) The following is a description of the procedures that we have developed to optimize the yield of hybrido- mas The process, starting with immunized mice and ending with newly fused cells plated in 96-well plates, should take about 4 h

2 Materials

1 Medium for fusion: The basal medium used for preparation of tumor cells and immune spleen cells for the fusion protocol IS Dulbecco’s Modified Eagle Medium (high-glucose DMEM, Gibco/BBL, Grand Island, NY) Penicillm- streptomycin solution (P/S, penicillin [Base] 10,000 U/mL; streptomycm [Base] 10,000 u.g/mL; Gibco) is added at a concentration of 1 mL/lOO mL medium HEPES is added at a final concentration of 10 mA4 to mmimize radi- cal shifts m pH when cultures are taken out of the incubator for examination,

2 Medium for culture: Medium for plating the fused cells is full DMEM Full DMEM is prepared by adding to DMEM and P/S (as in step 1)

a 200 mM TC Glutamine (Gibco) at a concentration of 1 mL/lOO mL medium

b 75 mL Fetal bovine serum (FBS) or iron-supplemented calf bovine serum (CBS) (HyClone, Logan, UT) to 500 mL of medium (approx 13%)

c 2-Mercaptoethanol at a concentration of 0.1 mL stock 2-ME/100 mL medmm

Stock 2-ME is prepared by adding 0.035 mL 2-mercaptoethanol (Sigma,

St Lotus, MO) to 10 mL DMEM (5 x 10m5iV) (see Note 1)

3 Medium for selective growth: To prepare selective growth medium con- taining hypoxanthine, ammopterm, and thymidine (HAT), add 2 mL of 50X HAT to 100 mL of full DMEM 50X HAT and 50X HT can be pur- chased commercially (e.g., Sigma)

4 Preparation of polyethylene glycol (PEG) for fusion: Cell fusion is induced with autoclaved PEG 1500 (e.g., PEG 1540, Baker, Philipsburg, NJ) diluted with an equal volume of DMEM (50%) For convenience, prepare 0.5-mL aliquots of PEG m 4 mL screw-cap glass vials, auto- clave to sterilize, and then store at room temperature At the time of fusion, melt a vial of sterile PEG in a 60°C water bath or over a gas flame, and dilute with an equal volume of warm protein-free DMEM The resulting mixture will be yellow and will not resolidify Some investigators prefer to bring the pH back to 7.2-7.4

3 Methods 3.1 Animals Mice to be used as immune spleen donors should be immunized, allowed to rest for a period of 5-10 d, and then boosted by iv injection

Culture Conditions 19

a naive or immunized animal can be stimulated in vitro and then prepared for fusion (see Chapter 2) Because the most commonly used fusion part- ners were derived from Balb/c mice, Balb/c mice are used as the primary strain for immunization

If for some reason the cells cannot be used for fusion when planned,

The cells will then be ready for fusion whenever myeloma cells are avail- able (see Note 3)

and debris is present in the culture medium, centrifuge the cells over a 1.086 density cushion (e.g., Accu-paque, Accurate Chemical and Scien- tific, Westbury, NY) and collect live cells from the interface One day

medium at a concentration of 5 x lo6 cells/dish Cells should increase

cells at a ratio of 2.5 spleen cells to 1 myeloma or hybridoma cell The myeloma cell line we are using is X63 Ag8.653 (4)

3.3.1 Thymocytes

1 Remove the thymus from five 4- to 6-wk-old mice using sterile conditions (see Note 1) A thymus will yield approx 2 x lo8 nucleated cells (3+wk- old mice often yield 3 x lo8 cells/thymus)

2 Transfer the thymuses to a loo-mesh sieve in a sterile Petri dish containing approx 20 mL of full DMEM

3 Clip the thymus open with sterile scissors, Gently tease cells from the thy- mus on the screen with a rubber policeman, taking care to leave stroma intact (i.e., do not grind thymus on screen)

4 Pass the cells through a Pasteur pipet loosely packed with nylon wool (New England Nuclear) to remove debris Transfer the cells to a 50-rnL tube, and then determine the cell concentration and viability Keep the thymocytes

at 4OC until used

3.3.2 Spleen Cells

1 Remove the spleens from nnmunized mice using sterile conditions An immune spleen should yield between 1 and 2 x lo* nucleated cells

20 Hamilton and Davis

2 Place the spleens m 20 mL of serum-free DMEM in a loo-mesh sieve in a Petri dish Using a needle and syringe, inject the spleen with medium to distend and disrupt the spleen stroma and free the nucleated cells

3 Chp the splenic capsule open with sterile scissors, and gently tease the remaining cells from the splemc capsule with a rubber policeman

4 Flush the cell suspensron with a Pasteur prpet to disperse clumps of cells and then pass the cells through a pipet loosely packed with nylon wool to remove debris

5 Centrifuge the spleen cell suspension at 250s for 10 min Resuspend the pellet in serum-free DMEM (0.5 mL)

6 Add approx 1.5 mL of sterile distilled Hz0 to the cell suspensron, mix to lyse erythrocytes, and then dilute with 30 mL of serum-free DMEM All this must be accomplished m the absolute mimmum of time-not to exceed

4 s Filter through a sterile Pasteur pipet containing a plug of loosely packed nylon wool (New England Nuclear) to remove debris and DNA from drs- rupted cells

7 Again centrifuge the cells, resuspend m serum-free DMEM, filter d any debris is present, and determine the cell concentratron and viability Keep the cell suspension at 4°C until time of fusion

3.3.3 Myeloma Cells

1 Transfer the myeloma cells from the freshly seeded Petri dishes to 50-mL tubes

2 Subject the cells to two cycles of washing and centrifugation at 250g for

10 min in serum-free medium Resuspend in serum-free DMEM, and determine the cell concentration and viability Substantial cell debris or poor viability at this step is an indication of poor culture conditions, incor- rect medium, or an infected myeloma cell line Stop the fusion process, and grow fresh myeloma cells Cryopreserve the spleen cells for later use

as described m Section 3.1 (see Note 3)

3.4 Cell Fusion

1 Mix the myeloma cells and spleen cells in a conical 50-mL tube m serum-

when usmg X63 Ag8.653 myeloma cells Centrifuge the mixture of cells

at 300g for 10 min While the cells are centrifuging, set aside 30 mL of serum-free DMEM in another 50-mL tube Prepare the 50% PEG and place the timer in the hood

2 Remove all the supernatant from the cell pellet Overlay the pellet of cells with 1 mL of 50% PEG with a Pasteur pipet Set the timer for 3 min and then, wrth the tip of the prpet, gently loosen and disperse the pellet of cells in the

Culture Conditions 21

PEG The pellet should be uniformly dispersed mto fine aggregates of cells (0.2-S mm) over the 3-min period Gently draw the cell mixture into the pipet

to observe clumps The fusion process is performed at room temperature

3 At the end of 3 min, discard the Pasteur pipet and fill a lo-mL pipet with serum-free DMEM Reset the timer for 10 min, put the pipet to the bottom

of the tube, and add the 10 mL of medium m OS-mL aliquots over a lo-min period with gentle mixing Then add 20 mL DMEM over the next

5 min The cell aggregates should disperse with mixing If excessive debris and clumpmg are evident, it may mean there was high cell death

4 Centrifuge at 250g for 8 min Gently resuspend the fused cells in full DMEM Add 8 x lo*-lo9 feeder cells to the cell suspension, and adjust the volume to 170 mL Distribute the cell suspension into 96-well flat-bottom tissue-culture plates, using a lo-mL pipet, 2 drops/well The first plate should be prepared without addition of mitogen This plate serves as a standard control to document the activity of the mitogen To the remainder

of the preparation of fused cells, add 900 p.g of Salmonella typhimurium

mitogen (STM) (a B-cell mitogen, Ribi Immunochemical Corp., Hamilton, MA), and dispense the cells into 9-12 additional 96-well tissue-culture

and 1 pg of STM m approx 0.15 mL of medium (see Note 2)

5 Incubate the plates at 37°C in 5% CO2 for 24 h and then add one drop of selective growth medium containing HAT to each well (see Notes 1 and 2) Colonies should become visible at 3-4 d Half the medium should be replaced with medium containing HT at 3-d intervals Colonies should reach sufficient size to assay for antibody production by 7-10 d

6 Three to 4 d m HAT medium is sufficient for arresting the proliferation of unfused myeloma cells (see Section 4.1 for troubleshooting)

1 Identify antibody-producing hybrids in primary culture using the appro- priate assay (Chapters 10-14, 16)

2 Transfer hybrids of interest to 12-well plates, adding 3 x lo6 thymocytes and 4 mL of medium containmg HT to each well Replace half of the medium with fresh medium at 3-4 d mtervals Cryopreserve each culture

of cells when they have reached at least 3/4 confluency, 2 ampules/cell culture Save the supernatant for further analysis

Following selection of hybridomas producing antibodies of interest (see Chapters lo-14 and 16 for methods of assay), the hybridomas are

22 Hamilton and Davis

subjected to one or two cycles of cloning The purpose of the following procedure 1s to obtain colonies derived from a single hybridoma cell

1 Thymocytes: Select mice 4-6 wk old, and prepare a suspension of thymocytes

as described m Section 3.3.1 One thymus is needed for every two 96-well plates set up

2 Hybridoma cell lines:

a Remove one ampule of the desired hybridoma lure from hquid mtro- gen Quickly thaw by shaking the ampule m a 56°C water bath Keep the top of the ampule dry

b Transfer the cell suspension to a sterile 15mL centrifuge tube Add 6

mL of full DMEM dropwise with gentle agitation m order to dilute the DMSO m the cell suspension slowly

3 Centrifuge at 250g for 10 mm Resuspend the pellet rn 4 mL of full DMEM and then place the cells m one well of a 12-well culture plate Incubate for 3-4 h or overnight

1 Following incubation of cultures for 3-4 h or overnight, examine cultures for viability with a phase microscope If the cell concentration is too low

or viability is low, contmue culture for a longer time period If the cell concentration and viabihty are adequate, dilute 50 ltL of the well-mixed cell suspension with 50 jtL of 0.2% trypan blue and determine cell count and viability

2 Determine the volume contammg 800-1200 cells If this is a very small volume (~20 PL), make a 1: 10 dilution of the culture in an adjacent well of the 12-well plate, and take the ahquot for clonmg from the 1:lO dilution Place the volume containing 1200 cells (ideally, between 50 and 100 pL) mto a third well, and check under the phase microscope to assure that an adequate number of viable cells are present

3 Dilute thymocytes to the required volume (approx 65 ml/set of four 96-well plates) Add the 800-1200 hybridoma cells Distribute the cell mixture mto flat-bottom 96-well plates, 4 plates for each cell culture, 2 drops/well from a lo-mL pipet (approx 200 l.tL) Feed the cultures at 3-4 d intervals

by removing half the medium and replacmg with full DMEM Avoid dis- rupting the colonies of cells when feedmg, so that colonies derived from single cells can be easily identified

4 Single colomes should be readily visible by 3 d At 5-6 d, mark the wells with single colonies At 7-8 d, collect medium from wells contannng single

6 Continue expanding each of the three cloned cell lines to two wells m the 6-well plate When the cells have reached 3/4 confluency, cryopreserve the cells, 6 ampules/primary clone and 2 ampules/backup clone Continue the culture of each clone to prepare an antibody rich supernatant for further analysis If the cultures will not be used further, allow the cultures to grow for 7 d, and then collect the supernatants

7 If few or no single antibody-producing colonies are identified in the initial screening that are producing antibody, screen all the remaming wells of the 96-well plates that contain multiple colonies Select a positive cell cul- ture and reclone Cryopreserve cells from additional positive cultures, and mark the ampules for potential use in recloning If the second cloning is successful, discard the extra ampules and proceed to preserve three clones

If the cell line still appears to be unstable, subject it to additional cycles of clonmg (Make a notation in a log book that the cell line is unstable.)

8 Determine Ig isotype (see Chapter 9) Record m log book

9 Make ascites or, alternatively, prepare a large pool of culture medium

in a bioreactor (see Chapter 17) and test for activity with the appropri- ate assay (see Chapters 10-14, 16) Quantitate the antibody, and record

in the log book Mark stock tubes with information on immunoglobu- lin isotype and concentration

10 Record the summary mformation on the cloned cell lme in a final log book

or master record

4 Notes

1 Many variables affect outgrowth of hybrids following fusion As a conse- quence, it has been difficult to assess how many hybrids are actually pro- duced in a given fusion and how many are capable of forming stable clones Our studies have clearly shown that a large number of hybrids are usually formed and that the majority fail to grow under the culture conditions used

in many laboratories (Table 1) As noted in Table 2, by varying the culture conditions and the timing of exposure to HAT, we have been able to

Table 1 Comparrson of the Effect of B-Cell Mitogens on the Outgrowth of Hybridomas”

ONumbers in parentheses indicate the number of 96well culture plates/set

bNumber of spleen cells used in the fusion

Table 2 Comparison of the Effect of Time Delay in Adding HAT to STM-Treated Hybridomasa

Average number of wells Average number of wells Average number of wells Average number of wells

=Numbers m parentheses mdxate the number of g&well plates used

‘Number of spleen cells used m the fusion

26 Hamilton and Davis

demonstrate that treatment of primary cultures of fused cells during the first

24 h is critical (2) Medium containing only FBS or CBS and antibiotics does not support maximal outgrowth Addition of 2-ME increases the yield

of hybrids and reduces the variability m outgrowth associated with varia- tions in the capacity of serum supplements to support outgrowth How- ever, outgrowth remains variable, especially when fused cells are distributed at low density Addition of 2-ME and thymocytes increases the yield of hybrids, but does not provide conditions that support maximal out- growth, especially when fused cells are distributed at low densities Similar observations have been made by other investigators using various growth supplements in place of thymocytes or other types of cells Although some

of the studies have demonstrated slightly better results than those obtained with thymocytes, the overall yields have been low, mdicatmg that neither supplements, thymocytes, nor other types of cells alone provide optimal culture conditions for survival and outgrowth The fmdmg that there is a remarkable increase m outgrowth of hybrids when B-cell mitogens are added to the medium has clearly demonstrated the presence of hybrids far

in excess of what might be expected from previous mvesttgations

2 The mechanisms by which B-cell rmtogens mediate their effect remam to be elucidated However, the data suggest that the mitogens induce the production

of cytokines (including IL-6 [5,6fi critical to stabilization of newly formed hybrrds, and their eventual capacity to divide and form continuous colonies It

is clear that the effect can be mediated by several types of mitogenic prepara- tions Lipopoly-saccharide (LPS) or derivattves, diphosphoryl lipid A (DPL) and monophosphoryl lipid A (MPL), have effects srmilar to those obtained with STM (Table 1) (2) Further studies should reveal that additional mitogens are equally effective Also, analysis of supematants derived from cells stimulated with mitogens should reveal which components are regulat-

mg survival and outgrowth of hybndomas At this juncture, it is clear that other culture conditions are critical also When used alone, the mitogens increase outgrowth, but to a lesser extent than when used m conjunction with 2-ME and thymocytes It is also evident that the events transpiring during the first 24 h are Influenced by the presence of HAT If HAT is added during the first 24 h of culture, outgrowth is greatly diminished If HAT is added after 24 h, outgrowth is greatly enhanced, Delaying the addition of HAT for a longer time period has no apparent beneficial effect,

3 We have found that spleen cells can be cryopreserved and used in the pro- duction of hybridomas with yields comparable to those obtained with fresh spleen cells (Table 3) The actual yield of hybridomas is dependent on the status of the spleen cells at the time of collection

Culture Conditions 27

Table 3 Comparison of the Effect of STM on the Outgrowth of Hybrrdomas Made

with Fresh and Cryopreserved Spleen Cells Control-thymocytes only STM-thymocytes

Number of Average number

wells with Number of of wells with one or Average number Fusion colomeP colomes/plateb more colonies/plates of colonies/plateb AMGC 65 (1) 145 (I-11) 96 (9) 439 (1-15) (5 x 107)d

CACTBe 79 (1) 173 (1-13) 90 (9) 251 (1-16) (5 x 107)

BAQ 25 (1) 33 (l-3) 90 (9) 249 (l-11) (5 x 107)

BLVC 57 (1) 113 (l-27) 80 (9) 287 (1-16) (5 x 107)

OParentheses indicate number of 96-well culture plates/set

“Parentheses mdlcate range of colonies/well

cCryopreserved spleen cells used m the fusion

‘Number of spleen cells used m the fusion

eFresh spleen cells used in the fusion

4 Even when using optimal culture condmons, difficulties can be encoun- tered in obtaining maximal outgrowth of hybrids Thus can be attrrbutable

to a number of factors, including choice of fusion partner, presence of mycoplasma or other slow-growing microorganisms, source and purity of water used to prepare medium, glassware, pH of medium m incubator, temperature, and the quality of PEG used to induce fusion Experience in our laboratory has shown that exposure to a high or low pH within the first

24 h followmg fusion will radically reduce the outgrowth of hybridomas Also, comparative studies have shown ingredients, such as Fungizone (amphotericin B), often used to reduce the risk of fungus growth in cultures, interferes with outgrowth of hybrids

4.1 Troubleshooting

1 Outgrowth of hybrids IS low in both control and STM-treated cultures

a First verify that the cell counts for splenocytes and myeloma were cor- rect and that the cells were mixed in the correct ratio

b Check and verify that serum-free medium was used during the fusron process Fusion is greatly diminished or blocked completely in the pres- ence of protein

28 Hamilton and Davis

c Check whether the pH of the medium was correct durmg the first 24 h

of culture Low and high pH will arrest growth of hybrids Also, check for the presence of slow-growing microorganisms in cultures of myeloma cells used as fusion partner Mycoplusma is usually the prime contaminant However, other organisms can be introduced into the cul- tures through inadequate sterilization of glassware used m culture and handling of cells

d On occasion, lack of growth can be attributed to the source of medium and/or the presence of excess 2-ME in the culture medium

e Check the condition of the fusion partner at the time of fusion Poor outgrowth might be attributable to low viability of the myeloma cells or

to cells not m log phase of growth

2 No difference is evident in the number of hybrids growing m the control and ST&I-treated cultures Check the concentration of STM used Lack of

a clear difference in the percentage of hybrid outgrowth suggests the mito- gen was overdtluted or possibly that it has lost activity The primary diffi- culty that we have encountered has been associated with overdilution of the mitogen

ogy, vol 21, Academic, San Diego, CA, pp 3-947

4 Kearney, J F , Radbruch, A., Liesgang, B., and RaJewsky, K (1979) A new mouse myeloma cell line that has lost immunoglobulin expression but permits the con- struction of antibody-secreting hybrid cell lines J Zmmunol 123,1548-1550

5 Harris, J F., Hawley, R G., Hawley, T S., and Crawford-Sharpe, G C (1992) Increased frequency of both total and specific monoclonal antibody producing hybridomas using a fusion partner that constitutrvely expresses recombinant IL-6

J Immunol Methods 148, 199-207

6 Van Snck, J (1990) Interleukm-6 an overview Annual Rev lmmunol 8,253-278

CHAPTER 4

Production

1 Introduction

In many circumstances, it is advantageous to have a continuous source

of human antibody of a given specificity and immunoglobulin isotype Reliance on human volunteers as a source of such antibody is problem- atic Therefore, it has been a goal of investigators to establish immortal cell lines that produce the desired human antibody The methods described in this chapter are a way to achieve this objective

Construction of hybridomas producing human antibodies is generally not very satisfactory because of the paucity of available myeloma fusion partners that yield stable hybridomas, which produce homogenous anti- body at a high rate Mouse-human heterohybridomas are generally not difficult to produce and have about the same in vitro growth characteris- tics as the mouse-mouse hybridomas (1,2) However, a severe limitation

to the usefulness of mouse-human heterohybridomas is their genetic instability Underlying this instability is the presence of the K light-chain gene on the human chromosome 2, which is preferentially expelled The

h gene is located on chromosome 22 and is also quite frequently lost Hybridomas that produce the h light chain can be recovered, but in humans these antibodies constitute only about one-third of the serum repertoire and, thus, would restrict the characteristics of antibodies pro- duced by such hybridomas

From: Methods II) Molecular Brology, Vol 45 Monoclonal Antibody Protocols

Edlted by W C Davis Humana Press Inc , Totowa, NJ

29

30 Cooper and Kirkpatrick

We have found that by using a human-mouse hybrid as the fusion partner, stable hybrldomas producing human antibody can be produced (Fig 1) (3,4) These hybridomas appear to be stable for several years with repeated cryopreservatron without loss of the antibody production This technique provides an excellent way of immortalizing B-cells from humans that produce antibody of desired specificity

2 Materials All materials used are of the highest chemical purity, and deiomzed water is of 18-MQ resistance

1 L-glutamine (200 n-r&& Sigma, St Louis, MO)* Add 1 mL of a 100X con- centration to 99 mL of medium

2 Gentamicin (10 mg/mL, Grbco, Gaithersburg, MD): Add 100 pL to 100

mL of medium to achieve a final concentration of 1 pg/mL

3 Sodium pyruvate (100 rnJ4, Cellgro, Herndon, VA): Add 1 mL of a 100X solution to 99 mL of medium

4 RPMI-10: RPMI-1640 containmg 10% fetal bovine serum (PBS) and anttbt- otics (gentamtcin 1 pg/rnL), 5% human Al3 serum, L-glutamine, and sodium pyruvate The serum 1s stable for 1 mo, but usually used wtthtn a week

glutamine, Na pyruvate, and 100 ~.LL of gentamtcm

6 Phosphate-buffered saline (PBS): NaCl (6.8 g/L), Na,HP04 (1.585 g/L), RI&PO, (0.3 15 g/L) Dissolve m 1 L of deionized water The pH will be 7.4

7 Htstopaque (Sigma): Polysucrose (5.7 g/100 mL) and sodium dratrizoate (9.0 g/mL) Use undiluted

cially m a 10X stock solution (e.g., Gtbco) To prepare working soluttons, dilute 10 mL of the 10X stock with 90 mL of stertle, high-purity deton- ized water

commercially in a 10X stock solution from multiple companies (e.g., Bio- Whittaker, Walkersvrlle, MD) MEM contains Earle’s balanced salt solu- tion without L-glutamine and sodium bicarbonate To prepare working solutions, dilute 10 mL of the 10X stock with 90 mL of sterile, high-purity deionized water

Production of Stable Heterohybridomas

Naive enrched

0 -+14-n + I Days 1 8 3 -j 0 -

Check for human Igs

CLones producing

no human Ig’s Feed with medtum contamlng 8-azagun,ne

31

8-azaguanme reslstatt heterohybrldomas

8-azaguame reststant Immumzed enrlched

Ftg 1 Flow chart for the production of stable mouse-human heterohybridomas

12 Sheep red blood cells (SRBC): Obtain sheep blood from a commercial source or directly from animals maintained for research Collect blood in

an equal volume of Alsever’s solution (e.g., Mtcropure Medical Inc., Stillwater, MN, or local supplier) The SRBC are stable in Alsever’s solu- tion for about 2 wk if maintained at 4OC

13 Immunization medmm: RPMI-1640 contaming 10% FBS, 40% thy- mocyte-conditioned medium (TCM), human IL-l (5 U/n.& obtain through commercial sources), L-glutamine, Na-pyruvate, gentamicin, Staphylococ- cus aureus Cowan 1 (5 x lo4 CFU/mL), and whole killed bacteria (our anttgen preparatton) 1 x 1 O6 CFU/mL

32 Cooper and Kirkpatrick

14 50% Polyethylene glycol (PEG 4000, mol wt 3000-4000, Gibco): This is a 50% solution of PEG made in Dulbecco’s PBS without Ca++ or Mg++ This solution is used undiluted

15, Medium for selective growth:

a HAT (hypoxanthme, aminopterin, and thymidine) supplement (Gibco, 100X): 10 mM sodium hypoxanthine, 40 @4 ammopterin, 1.6 n&f thy- midine Rehydrate with 10 mL of sterile deionized water, and dilute 1: 100 to prepare a working solution

b HAT medium: Add 1 mL of HAT supplement to 99 mL of RPMI-10 medmm to prepare a working solution

c HT (hypoxanthine and thymidine) supplement (Gibco, 100X): 10 mA4 sodium hypoxanthine, 1.6 mA4 thymidine

d HT medium: Add 1 mL of HT supplement to 99 mL of RPMI-10 medmm to prepare a working solution

e Selective medium, 8-azaguanme (Sigma, 50X): The stock solution is 6.6 rnJ4 when reconstituted with 10 mL of RPMI A few drops of 1M NaOH can be added to the solution if the 8-azaguanme does not go into solution easily

f Selective medmm: Add 2 mL of the 8-azaguanine to 98 mL of RPM1 medium to prepare a working solution

16 Clonmg medium: RPMI-10 containing 40% TCM

3 Methods

As an alternative to feeder cells, we use TCM to avoid potential con- tamination (see Note 2) (1,5-7)

1 Remove the thymus from 10 Balb/c mice, and place in RPMI-10 medium

2 Free thymocytes from the thymus by teasing the lobes apart with forceps and passing the tissue fragments progressively through a 16-gage cannula, an 18-gage needle, and then a 20-gage needle to obtain a single-cell suspension

3 Pool the thymocytes, and wash once m culture medium

4 Resuspend the thymocytes to a concentration of 3-5 x lo6 cells/ml in RPMI-

10 medium containing 20% FBS, and culture for 48 h in 7% CO, at 37OC

5 Remove the thymocytes from the culture medium by centrifugation (5OOg)

6 Prepare aliquots of the culture medium, now considered as TCM, at desired concentration, and store at -70°C

1 Collect fresh heparinized blood in either a 15- or 50-n& conical centrifuge tube, and dilute it with an equal volume of PBS at ambient temperature Mix the diluted blood

Production of Stable Heterohybridomas 33

2 Slowly underlay the blood/PBS mixture with H&opaque (Sigma) by pass- ing it through a sterile pipet placed in the test tube The working ratio is 3 mL

of Histopaque/lO mL of the blood/PBS mixture

3 Centrifuge the blood preparation for 30 min at 500g with no brake

4 Carefully remove the upper layer (containing plasma and most of the plate- lets) with a pipet Using a new pipet, collect the mononuclear cell layer at the interface, and transfer it to a new centrifuge tube

5 Wash the mononuclear cells m HBSS by centrifugation for 10 min at 300g

at ambient temperature Repeat the cycle to remove platelets

6 After the final wash resuspend the mononuclear cells in RPMI-10, and count the cells with a hemacytometer

1 Have the lymphoid tissue removed aseptically from the human donor, and either cut into small pieces or mmce m HEPES-MEM containing antibiotics

2 To prepare a single-cell suspension, press the tissue fragments through the cup of a cell selector fitted with a loo-mesh sterile wire screen Use a glass pestle (Bellco-Cell Selector) to disrupt the tissue Rinse the wire screen with RPMI-1640, and bring total volume to 45 mL

3 Centrifuge the cells at 250g for 10 mm

4 Remove the supematant, and resuspend the cell pellet in 5 mL of RPMI-1640

5 Underlay cell suspension with Histopaque as described in Section 3.2., step 2 Centrifuge for 30 min at 500g without using the brake

6 Remove the mononuclear cell layer at the interface, wash twice in HBSS

by centrifugation 300g for 10 min at ambient temperature, and resuspend the pellet m RPMI-10 Count the cells

1 Concentrate the lymphocytes by centrifugation at 300g for 10 min Resus-

40% FBS

2 Wash SRBCs m HBSS twice, and make a 3% suspension of SRBCs in HBSS containing 40% FBS This is done by adding 3 mL of the packed cells to 97 mL of the HBSS containing 40% FBS

3 Mix equal volumes of the lymphocytes and SRBC suspension, and mcu- bate the mixture over ice for 1 h

4 After incubation, place the suspension over 4 mL of Histopaque, and cen- trifuge at 500g for 30 min to separate E-rosetted T-cells from the nonrosettmg B-cells

34 Cooper and Kirkpatrick

5 Remove the cells remaining at the interface (B-cells) of the Histo-

paque/medium and wash twice by centrlfugatlon (300g) in warmed (37°C)

serum-free RPMI-10

6 Count the cells with a hemocytometer The cell preparation IS considered

to be predominantly B-cells This can be confirmed by flow cytometry (Chapters 15 and 16)

Our laboratory has been primarily interested in producing monoclonal antibodies (MAbs) against surface-expressed antigens of whole bacteria

most antigens

1 Use B-cells isolated and enriched according to the protocols described m

Sections 3.3 and 3.4

2 Place the total population of B-cells in 20 mL of immunization medium

containing the antigen of interest B-cells are amplified by the addition of the Staphylococcus aureu~ Cowan I (SAC), which serves as a super antl-

3 Incubate the B-cell populations for 6 d with the addition of 1 mL of medrum without SAC every other day

4 At the end of the 6-d immunization period, test the culture medium for the presence of antibody by enzyme-linked lmmunosorbent assay

ELISPOT (Chapter 1 l), or other assays to determine If there is a spe- cific antibody present against the immunizing antigen We prefer to

use ELISPOT assays because they can be set up to determine both the

specificity of the antibody, Its isotype, and the relative number of spe- cific antibody-producing cells

5 If antibodies are being produced, the cells can be used as a fusion partner

for the heterohybrldomas

To obtain stable heterohybridomas producing human MAbs, we devel-

method for producing the fusion partner 1s outlined here as part of the

MAbs (Fig 1)

Production of Stable Heterohybridomas 35

1 Grow the P63 cells in RPMI- 10 to log phase by splitting the culture the day before the fusion is to take place

2 Wash 6 x lo6 P63 cells twice by centrifugation (3OOg), resuspend in serum-

in 10 mL of serum-free medium in a conical centrifuge tube

3 Centrifuge the cell mixture at 350g for 10 min at ambient temperature to form a tight cell pellet

4 Remove all the supernatant from the pellet

5 Using a 1-mL pipet, add 1 mL of warm (37OC) 50% PEG over a 1-min period

6 Stir the mixture for an additional minute

7 Using the same pipet, add another 1 mL of warm serum-free medium to the mixture, and stir for an additional minute

11 Transfer aliquots of the fused cells (400 uL/well) mto each well of a 24-well cluster plate

the fusion

13 Feed the cells with HT-containing medium every 2-3 d until macroscopic growth is seen This usually takes about 3 wk Change to regular medium when growth is evident

14 After 3 wk of growth (macroscopic growth of colonies is evident), add

resistant hybridomas

15 Grow the cells in the presence of 8-azaguanine for 3 wk There will be a large die off of the cells, but some wells will have rapidly growing cells These hybridoma cells are now HAT-sensitive because of the lack of HGPRTase enzyme activity

16 These 8-azaguanine-resistant hybridoma cells can be propagated for long periods of time as well as frozen for use in the future as fusion part- ner cells

ner should be well established and in log-phase growth when harvested

36 Cooper and Kirkpatrick

for the fusion We normally grow the myelomas to a density of around

1 x lo7 cells/ml and have a viability in excess of 95%

1 Mix the 8-azaguanme-resistant mouse-human heterohybndoma cells with the immunized human B-cells in a ratio of 1:4 heterohybridomas to B-cells, respectively

2 Grow the heterohybridoma fusion partner cells to log phase in RPMI-10

by splitting the culture the day before the fusion is to take place

3 Wash 1 x lo6 myeloma cells twtce (350g) in serum-free RPMI, and mix with approx 4 x lo6 immune human B-cells in serum-free RPMI-10 medium

4 Centrifuge the cell mixture at 35Og for 10 min at ambient temperature to form a tight cell pellet

5 Remove all the supernatant from the pellet by careful pipeting

6 Using a I-mL pipet, add 1 mL of warm (37OC) 50% PEG over a l-mm period

7 Stir the mixture for an additional minute

8 Using the same pipet, add an addrtionall mL of warm serum-free medium and stir for another minute

9 Repeat step 8

10 Add an additional 7 mL of serum-free medium over a 2-3 min period with stirring

11 Centrifuge the cell mixture at 35Og for 10 min and remove the supernatant

12 Count the total cell population, and add sufficient medium (RPMI-10) to give a final concentration of 1 x lo6 cells/O.1 mL

13 Add the medium directly to the pellet

14 Plate aliquots of the cell mixture (400 pL/well) into each well of a 24-well cluster plate

15 Feed the cell cultures with HAT-contaming medium on d 1 and 3 following the fusion

16 Feed the cell cultures with HT-contammg medium every 2-3 d until mac- roscopic growth is seen This usually takes about 3 wk

17 After 3 wk of growth in HT, collect medium from wells containing colo- nies of cells, and test for antibody production Set up dilutions of cultures

of cells producing antibody using a limiting dilution format

18 At this time, screen each well with visible colonies, using an ELISPOT assay (see Chapter 11) This assay allows the determination of the isotype and specificity of antibodies reactive with the immunizing anttgen, and an estimate of the number of specific antibody-producing cells We observe some multiple-isotype expression on individual cells within these uncloned cultures of heterohybridomas At this stage, it is imperative that limiting dilutions be done on the wells containing antibody-producing cells, This phenomenon has been confirmed by flow cytometry,

Production of Stable Heterohybridomas 37

1 Suspend cells in wells of interest, and then take a 1-mL aliquot, count the cells, and check cell viability

2 Dilute the cells to a concentration of 230 live heterohybridoma cells in 4.6

mL of cloning medium This dilution yields a concentration of 50 cells/ml

of medium

3 Plate 36 wells of a g-well plate with 0.1 mL of the cell suspension, which will give approx 5 cells/well

4 In the remaining 1 mL of cell suspension, there are about 50 cells Add 4

mL of cloning medium to the 1-mL suspension, and mix well

5 Plate 0.1 n&/well m 36 additional wells of the 96-well plate This dilution and plating should yield about 1 cell/well

6 To the remaining 1.4 mL of cell suspension, add 1.4 mL of cloning medium and mix Plate 0.1 mL in each of the remaining 24 wells of the 96-well plate This dilution should give about 0.5 cells/well

7 Examme the cultures after 48 h using an inverted phase microscope to identify wells containing single colonies of cells

8 At d 5, and again at d 12, feed the cells with 200 p.L of cloning medium

9 Following identification of hybrids producing antibody, transfer cells from wells containing a single colony to 24-well plates to expand the culture, and then transfer to 25-cm2 tissue-culture flasks

10 Following expansion of the cell line, cryopreserve ahquots of cells, and then grow the cells in large tissue-culture flasks using RPMI-10 culture medium to prepare stocks of antibody Alternatively, transfer cells to bioreactor casette for production of antibody (Chapter 17)

4 Notes

1 In our laboratory, the direct fusion between mouse myeloma cells (P63 or NS-1) and immune human cells results in heterohybridomas that produce specific human antibody directed against surface-exposed bacterial antigens However these hybridomas are very unstable, and after 8-10 passages, the cells cease to produce antibody and ultimately die This problem has been overcome by the described procedures

2 Many workers have reported that the use of feeder cells (i.e., peritoneal macrophages, thymocytes, splenocytes, and medium conditioned by these cell types) in the hybridoma culture increases the yield of viable hybndomas

It has been shown that active conditioned media contain soluble hybndoma growth factor (HGF), which is active on newly formed hybridomas This molecule is similar to the plasmacytoma growth factor and has been identified as interleukm-6 (IL-6) This finding has suggested that, in addition to its role in cell proliferation, it also influences the expres-

38 Cooper and Kirkpatrick

sion of the immunoglobulin genes in the newly formed murine B-cell hybridomas We consistently use TCM instead of coculturing with thy- mocytes This works well in our hands and avoids having to maintain a ready source of mice The medium can be maintained at -70°C for at least 9-12 mo

3 For the determination of antibody production by in vitro immunization, the ELISPOT assay is our method of choice (see Chapter 11) We find that

it is more sensitive than other assays and has the ability to detect antigen- specific antibodies Further, the system can be arranged so that one can determme the isotype of the antigen-specific antibody One can also obtain

an estimate of the relative number of the antibody-producing cells The system does have some disadvantages in that sterility must be maintained when working with the cells Also, some cells are lost durmg the manipu- lations when overlaying the cells with the mtrocellulose The immunoblot and ELISA assays work well if there is an adequate concentration of anti- body An advantage they have is that they can be worked with under nonsterile conditions Flow cytometry can determine antibody-producing cells There are two disadvantages in using this system as a screening tool First, if only analysis is performed, the cells are lost Second, determining antibody specificity is not possible The first problem can

be overcome if the flow cytometer has sorting capabilities, but only

the antibody

References

1 Teng, N N H., Reyes, G R., Bieber, M., Fry, K E., Lam, K S., and Herbert, J M (198.5) Strategies for stable human monoclonal antibody production, in Human Hybridomas and Monoclonal Antibodies (Engleman, E G., Foung, S K H.,

Larrick, J , and Raubitschek A., eds.), Plenum, New York, pp 71-91

2 Ostberg, L and Pursch, E (1983) Human x (mouse x human) hybridomas stably producing human antibodies Hybridoma 2,361-367

the construction of antibody-secreting hybrid cell lines J Zmmunol 123,

1548-1550

4 01, V T and Herzenberg, L A (1980) Immunoglobulm-producing hybrid cell lines, in Selected Methods in Cellular Immunology (Mishell, B and Shiigi, S M., eds.), Freeman, San Francisco, pp 351-372

5 Bazin, R and Lemieux, R (1989) Increased proportion of B-cell hybridomas secreting monoclonal antibodies of desired specificity in cultures contammg mac- rophagederived hybridoma growth factor (IL6) J Imm~rwl Methods 116,245-249

Production of Stable Heterohybridomas 39

6 Norden, R P and Potter, M (1986) A macrophage-derived factor required by plasmacytomas for survival and proliferation in vttro Science 233,566-569

7 Yokoyama, W M (1994) Preparation of cloning expansion medium, in Current Protocols in Immunology (Coligan, J E., Kruisbeck, A M., Marguhes, D H., Shevach, E M., and Strober, W., eds.), Wiley, New York, pp 2.5.11-2.5.12