AU663144B2 - In vitro antibody production - Google Patents

Description

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i I I- OPI DATE 19/07/93 APPLN. ID AOJP DATE 16/09/93 PCT NUMBER 31643/93 PCT/GB92/02285 1111111111111 III I I !l IiII 111111 1111ll111 AU9331643 fY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 93/12247 C12P 21/08 A l (43) International Publication Date: 24 June 1993 (24.06.93) (21) International Application Number: PCT/GB92/02285 (81) Designated States: AU, BR, CA, JP, RU, US. European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, (22) International Filing Date: 9 December 1992 (09.12.92) LU, MC, NL, PT, SE).

Priority data: Published 9126094.3 9 December 1991 (09.12.91) GB With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of (71) Applicant (for all designated States except US): IMMUNE amend ts.

SYSTEMS LIMITED [GB/GB]; 571 Fishponds Road, Bristol BS16 3AF (GB).

(72) Inventors; and 6 Inventors/Applicants (for US only) BOURNE, Stephen, Paul [GB/GB]; POPHAM, Christopher, John [GB/GB]; 571 Fishponds Road, Bristol BS16 3AF (GB).

(74) Agent: DEAN, Withers Rogers, 4 Dyer's Buildings, Holborn, London ECIN 2JT (GB).

(54)Title: IN VITRO ANTIBODY PRODUCTION (1 (57) Abstract (3) This invention provides a method of in vitro immunization of cells with an antigen to produce antibodies against that antigen, the method comprising incubating the cells with the antigen and support cells in which the support cells have previously been Il (4) primed with the antigen prior to incubation with the cells to be immunized. (2) S (51) (7) Z' ANTIGEN (9) (111 WO 93/12247 PCT/GB92/02285 1 IN VITRO ANTIBODY PRODUCTION This invention relates to in vitro antibody production, and in particular monoclonal antibody production.

Since the work of Milstein K6hler the priming of rodent B cells with antigens has largely been accomplished by animal immunization. In vitro immunization, that is to say a tissue culture-based primary system which permits rapid primary activation of antigen-specific cells has hitherto seldom been used because the resulting hybridomas produce antibodies which are predominately of the IgM structural type or class. IgM antibodies are of relatively low affinity and are of limited general application compared to antibodies of the IgG or IgA structural type or class.

Human monoclonal antibodies offer the advantage of minimizing the adverse reactions which can result from the administration of foreign (i.e rodent) antibody protein to the human body.

However, routine human monoclonal antibody production is not common because of the dangers and ethical problems of immunizing humans with certain antigens and the difficulty of collecting suitable primed B cells. Recent efforts with human monoclonal antibodies produced by in vivo immunisation have focused on the use of chemical molecular modification to cange specificities.

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WO 93/12247 PCT/GB92/02285 2 Therefore there is a need for an efficient method for in vitro immunization which results in an increased yield of IgG/IgA antibodies.

Further advantages of in vitro immunization include the ability to maintain specific concentrations of antigen during the immunization, the effect of various monokines and lymphokines (cytokines) at various concentrations can be tested, periodic sampling of the same culture is possible during the course of a culture, it is possible to produce antibodies to "self" antigens which are not produced in vitro due to suppression or tolerance, use of very small quantities (compared to in vivo) of antigen, potential for use of antigens toxic in vivo, short time course with respect to in vivo; (8)enhanced yield of IgA with respect to in vivo immunisation;(9) antigens which are very small, such as some peptides, can be successfully used for immunization; and (10) the undesirable use of laboratory animals can be reduced.

Reading Immunological Methods 53 (1982) 261-291) reviews in vitro immunization and describes a method o£ iA vitro immunization in which spleen cells from an unprimed mouse are mixed with thymocyte conditioned medium. Thymocyte conditioned medium contains monokines and lymphokines and other factors.

A disadvantage of this technique is that the antibodies produced are predominantly IgM and low affinity IgG.

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WO 93/12247 P~7rGB92/02285 3 W091/17769 discloses a method of in vitro immunisation using human lymphocytes and essential numbers of autologous accessory cells which are immunized with a specific antigen under specific culture conditions. Relatively large quantities of IgG antibodies with improved affinities are said to be produced by this method. The use of human cells for immunization only,however, is disclosed. In a preferred embodiment the cells to be immunized and the accessory cells are primed together with the antigen of interest prior to immunization proper.

WO 89/05308 discloses a method of in vitro immunisation based on the Reading paper referred to above, in which mouse spleen cells, for example, Balb/c- mouse spleen cells, are isolated without separating the B and T lymphocytes, an oligopeptide with a particular sequence or a hapten is immobilized and contacted with the mouse spleen cells, in the presence of Ewing Sarcoma Growth Factor and anti-L3/T4 monoclonal antibodies.

Ii4 PCT/SE89/00486 discloses a method of in vitro immunisation in which a lymphocyte containing cell population is contacted with a lysomotropic agent to remove cells having a negative effect on immunisation and subsequently the lymphocytes are immunized with an antigen in the presence of a polyclonal activator.

Increased yield of IgG and antigen-sensitive hybridomae are claimed.

SSUBSTITUTE SHEET_ WO 93/12247 PCT/GB92/02285 4 According to one aspect of the invention there is provided a method of in vitro immunization of cells with an antigen to produce antibodies against that antigen, the method comprising incubating the cells in vitro with the antigen and support cells which have previously been contacted with the antigen.

Preferably the cells to be immunized are human cells but cells from other species ,such as mice may also be used. An advantage of non-human systems is that syngeneic support cells can be used.

It has been found that the method of the invention results in the production of as much as 67% IgG (using Keyhole Limpet Haemocyanin as the antigen),but typically about 55%, according to the antigen used. IgA proportions of as high as 16% have been obtained and are typically in the range of 5-15%. It will be appreciated that a person of ordinary skill in the art may be able to obtain higher proportions of IgG or IgA antibodies on the basis of the present disclosure.

Whilst not wishing to be bound by the theory, it is believed that the support cells express antigen on the cell surface membrane in a form which facilitates presentation of the antigen to the cells to be immunized. Support cells may also secrete cytokines active on the cells to be immunized and contact between the cells may also be important.

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WO 93/12247 PCT/GB92/02285 The method of the invention is simple to operate; is sensitive I to nanograms of antigen and can generate responses to weak or Kj conserved antigens within 1 week from priming of support cells to yield screened hybridoma lines within 3-4 weeks.Fven Sbetter response times may be obtained by use of extended Simmunization and/or repeated immunizations.

The method of the invention has been used to generate i antibodies to snake venom toxin with improved results compared Sto in vivo techniques.

The method of the invention is further advantageous in that it does not require the use of purified adjuvants or polyclonal activators,cytokines or monokines, such as interleukins, required by prior art methods.

The support cells are preferably incubated in vitro with the antigen. Typically the support cells will be antigenpresenting cells, preferably macrophages, dendritic cells or other cells of the granulocyte macrophage lineage or derivatives. Other support cells include Glial cells such as astrocytes,microglial cells, or epithelial cells,erythrocytes,B and T lymphocytes, Muller cells, Kupffer cells, fibroblasts, neoplastic cells. Derivatives, such as drug-resistant variants or hybrids of the above cells may also be used as support cells. Such support cells may be manipulated to yield a stable reproducible ,immortal line which is in addition susceptible to selection a-ents or treatment such as radiation which may WO93/12247 PCT/GB92/02285 6 be used to select during the immunization process and/or kill these support cells after complete immunization. Such a system for cell toxicity may be for example selection of cells deficient in the enzyme hypoxanthine-guanine phosphoribosyltransferase (Lloyd Mayer M: Human Hybridoma and Monoclonal antibodies, ed. Engleman et al Plenum Press, New York 1985.) The cells to be immunized may be any cell capable of antibody production and are preferably splenocytes or derivatives, bone marrow-derived or peripheral blood lymphocytes or derivatives.

As noted above the cells may be human or other species derived.

Thus the method of the invention is not limited to any tissuespecific lymphocyte.

In a murine system the cells to be immunized may be incubated in vitro with the primed support cells in growth factor, preferably thymocyte, conditioned medium.

It has been surprisingly found that the method of the invention produces relatively large quantities of IgA antibodies compared to conventional in vivo immunisation methods. Thus the method of the invention provides a simple process for IgA production.

According to another aspect of the inventi.on there are provided antibodies produced by a method in accordance with the invention.

i- 'L WO 93/12247 PCT/GB92/02285 7 According to another aspect of the invention there is provided 'I a kit for the use in a method in accordance with the invention for in vitro immunization of cells with an antigen, the kit comprising support cells, and support cell growth medium, and growth medium (immunization medium) for cells to be immunized.

An in vitro immunization method in accordance with the invention and antibodies produced using the method will now be described, by way of example only, with reference to the accompanying drawing which is a flow diagram illustrating the method.

There are typically 4 stages in the in vitro immunization process. First, the antigen iz prepared. Second, a monolayer or suspension of support cells is established and primed with the chosen antigen. Third, a suspension of the cells to be immunized preferably spleen cells (splenocytes), is prepared. Fourth, the cells to be immunized are incubated in the immunization medium with the chosen antigen and preprimed support cells. The procedure is summarised on the accompanying flow chart.

As shown in the flow chart, support cells are prepared grown in a monolayer for about 24 hours. Separately antigen responsive cells, eg spleen cells, are prepared suspended and pelleted p i I -mr :i WO 93/12247 PCT/GB92/02285 8 The medium of the support cells is replaced and antigen is added and the support cells are incubated with the antigen, typically for two days prior to combing the primed support cells with the antigen responsive cells and optionally adding further antigen The cells are incubated undisturbed, typically for 3 days prior to use in hybridoma production.

1) PREPARATION OF ANTIGEN a. The antigen must first be prepared in a suitable form.

If the antigen is possibly cytotoxic, a cell toxicity test is advised. The effect of addition of the preparation at the recommended concentration for in vitro immunization (30 ig in 30 ml) to a healthy culture of myeloma cells is a suitable test.

b. i) Soluble antigens As a general guide-line, a maximum of 30 pg of antigen in a volume of 0.3 ml is required for in vitro immunization, with a similar amount required to prime the support cells. Antigen concentrations of as little as Ing per ml medium have been successfully used, and the preferred concentrations are in the range 50 ng to 1 ig per ml in the immunization medium. The optimum concentration varies according to the nature of SWO 93/12247 PCT/GB92/02285 JJ 9 the antigen. The ant:,gen should ideally be Ij prepared in a sterile form. Workin from a sterile lyophilised preparation or a solid, the 'I antigen can be dissolved directly in 0.2 ml of phosphate buffered saline or tissue culture medium in aseptic conditions.

If the antigen is already in solution, for example if it has been prepared biochemically, then it should be tested for sterility. Working in aseptic conditions, 0.2 ml of the antigen solution is added to a vial of sterility test broth. The vial is sealed tightly and shaken to mix and then incubated for 48 hours at 37°C. After this time, solutions which are contaminated will have a cloudy suspension. A clear solution indicates a sterile antigen preparation.

ii) Complex, surfaceborne antigen systems.

If whole cell preparations such as entire microorganisms, including bacteria or fungi, are used for immunization, the preparations must be treated to ensure they will not continue to grow in vitro. This can be achieved by gamma irradiation or fixation.

As a guide for cell based systems, 107 cells will be required for immunization. Irradiation of the WO 93/12247 PCT/GB92/02285 41 preparation to 50 Gy is necessary to prevent in 11 vitro growth.

To fix the cells a suspension of the antigen is i prepared, and pelleted by centrifugation and washed free of growth medium components as necessary. The pellet is L~tuspended in iml of 0.15% glutaraldehyde in phosphate buffered saline (PBS) and incubated at room temperature for minutes. Using aseptic procedure the suspension is re-pelleted by centrifugation and resuspended in 5ml of a sterile solution of bovine serum albumin in PBS. This blocking step is necessary to prevent other non-specific proteins from binding to the fixed material. The suspension is incubated for 90 minutes at room temperature and pelleted and resuspended in 0.2ml sterile splenocyte medium.

t iii)Antigens, particularly relatively small antigens, or antigens which ai; immiscible with the cell culture medium, may be bound to a carrier substance or a solid matrix to enhance presentation to support cells. Suitable carriers include molecules such as Bovine Serum Albumin(BSA) or Keyhole Limpet Haemocyanin (KLH) or macroscopic carriers such as latex beads or nitrocellulose.

I ~I WO 93/12247 PCT/GB92/02285 11 2. ESTABLISHMENT AND PRIMING OF SUPPORT CELLS Using aseptic technique, 5ml of support cell medium is pipetted into a 25 cm 2 flask. Sterile support cells are obtained and transferred to a laminar flow cabinet.

Preferred support cells are from a Balb/c murine macrophagederived cell line, J774A.1, A.T.C.C accession No T1B67.

Alternatively, a non-renewable source of peritoneal macrophages may be used. The cells are pipetted into the flask. Vented flasks are preferred. The support cell medium, Eagle's minimal essential medium, alpha modification with ribonucleosides and deoxyribonucleosides (Gibco Life Technologies Cat. No. 072-01900) and 10% fetal calf serum (Gibco Life Technologies Cat. No. 013-06290) is typically used.

The flask is capped and placed in an incubator and left undisturbed for 24 hours. Using aseptic technique,, the medium is removed by pipette from the 25 cm 2 flask and discarded. 5ml of fresh support cell medium is gently added to the flask and 20-30 pg of the sterile antigen is added.

3. PREPARATION OF SPLENOCYTES Thymocyte conditioned media is prepared in advance as set out below.

Homogenise the thymuses typically from 4 x C57 Black 6 and 4 x BALB/c strains of mice in Dulbecco's Modified Minimal i .e I, i-i i WO 93/12247 PCT/GB92/02285 12 Essential Medium (Gibco Life Technologies Cat No. 074-02100 Appendix 2) with the recommended concentration of sodium hydrogen carbonate, (DMEM) 10% rabbit serum, (Sigma R4505) keeping cell strains separate. Centrifuge and resuspend in DMEM 2% Rabbit serum 1% Non-essential amino acid solution (Sigma Cat. No. M7145) and 1% sodium pyruvate (Sigma Cat. No. S8636).

Mix cell strains in 1:1 ratio after counting. Adjust cell numbers to 4.6 x 106 cells /ml and add P Mercapto ethanol (BMe) to a final concentration of 1 piM. Aliquot the cell suspension into 75 cm 2 growth area tissue culture flask, ml per flask. Incubate 48 hours undisturbed in an atmosphere of 6% Co 2 at a temperature 37.2 "C in a humidified atmosphere.

Collect media and centrifuge out cells. Filter media using 0.2|im pore size membrane (typically Sartorius Minisart unit Cat. No. SM16534K).

To prepare immunization medium; Mix in ratio 12.5 ml Thymocyte conditioned medium 6 ml fetal calf serum 6 ml Feeder Supplement for Hybridoma cells (Immune Systems Cat. No. ISP1/FS) 8 ml tissue culture medium (typically Eagles MEM alpha modification without ribonucleosides, deoxyribonucleosides, (Gibco Life Technologies Cat. No. 072-02000 with NaHCO 3 as per manufacturer's recommendations) or fetal calf serum.

WO 93/12247 PCT/GB92/02285 13 Add A mercaptoethanol to final concentration of 1iM, and aliquot into sterile bottles. Freeze at 20 0

C.

For use The immunization medium is thawed at room temperature.

The spleen is removed from a 10-12 week old mouse (typically j balb\c). The spleen capsule is ruptured and the spleen is teased apart into small pieces, forming a crude suspension.

This is then disrupted to form a single cell suspension.

The cell suspension is pipetted into a centrifuge tube and spun at 400xg approximately for 5 minutes.

4. IMMUNIZATION The flask of support cells is removed from the incubator to a laminar flow cabinet. The medium is aspirated from the flask. 5ml of fresh support cell medium is used to rinse the support cells. The medium is aspirated from the support cells. The supernatant from the spleen cell pellet is discarded by pipetting, and the pellet is resuspended in immunization medium and transferred to the flask containing the support cells. The remaining immunization medium is added to the flask. The sterile antigen preparation is added to the flask. The cap of the flask is fully tightened and the flask is placed with its 25 cm 2 growing surface horizontal in the incubator undisturbed for up to five days.

Immunization is not recommended to extend beyond 4 days as there is a rapid decline in cell viability due to medium exhaustion and cell fusion efficiency is reduced. After a WO 93/12247 PCT/GB92/02285 14 this time the primed splenocyte culture is ready for further processing as required. For preparation of antibody secreting hybridomas by fusion to a myeloma cell line or other form of immortalization is recommended.

The results of in vitro immunizations and subsequent hybridoma production in accordance with the invention compared to a protocol in which pre-primed supplementary support cells are not introduced are set out below.

1. Comparative Example Isotype analysis of reactive clones: Expected Result IgM 75-83% IgG 17-25% 2. in vitro immunization with use of pre-primed support cells: Isotype analysis of reactive clones: Typical results Test No.1 2 3 4 5 6 7 8 9 IgM(%) 45 33 45 47 42 ND 33 29 43 IgG(%) IgA(%) N

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55 67 55 53 47 ND 66 59 53 ND ND ND ND 10 16 ND 12 3 WO93/12247 PCT/GB92/02285 It will be seen that the method of the invention provides enhanced presentation of antigen via support cells which may also secrete factors active on the cells being immunized resulting in an increased proportion of antibodies of the IgG and/or IgA type or class.

Claims (11)

1. A method of in vitro immunization of cells with an antigen to produce antibodies against that antigen, the method comprising incubating the cells with the antigen and support cells in which the support cells have previously been primed with the antigen.

2. A method according to claim 1 in which the cells to be immunized are splenocytes or lymphocytes or derivatives thereof.

3. A method according to claim 1 or claim 2 in which the support cells are antigen presenting cells.

4. A method according to claim 3 in which the antigen presenting cells are selected from macrophages, dendritic cells or other cells of the granulocyte macrophage lineage, Glial cells such as astrocytes, microglial cells, or epithelial cells, erythrocytes, B and T lymphocytes, Muller cells, Kupffer cells, fibroblasts, neoplastic cells; or derivatives thereof. p

5. A method according to any preceding claim in which the support cells are primed in vitro with the antigen.

6. A method according to any preceding claim in which the cells to be immunized are incubated with the primed support cells and antigen in immunization medium. I t WO 93/12247 PCT/GB92/02285 17

7. A method according to any preceding claim in which of the antibodies produced by the cells more than 50% are IgG antibodies.

8. A method according to any preceding claim in which of the antibodies produced by the cells more than 10% are IgA antibodies.

9. A method according to any preceding claim in which antibodies are produced within 5 days of priming the support cells.

Antibodies produced according to a method of any preceding claim.

11. A kit for the use in a method for in vitro immunization of cells with an antigen according to any one of claims 1 to 9, the kit comprising support cells, and support cell growth medium, and growth medium (immunization medium) for cells to be immunized. i