CA1243946A

CA1243946A - Production of immunogens by antigen conjugation to liposomes

Info

Publication number: CA1243946A
Application number: CA000459707A
Authority: CA
Inventors: Demetrios Papahadjopoulos; Timothy D. Heath; Pang Shek
Original assignee: Minister of National Defence of Canada
Current assignee: Minister of National Defence of Canada
Priority date: 1983-08-03
Filing date: 1984-07-25
Publication date: 1988-11-01
Also published as: US4565696A

Abstract

ABSTRACT
The invention disclosed relates to antigen/liposome conjugates, wherein the antigen is covalently bound to lipid molecules in the surface membrane of liposome vesicles, and wherein at least about 25 g of antigen is provided per mole of lipid. Enhanced immunogenicity is achieved by injecting the antigen/liposome conjugate so formed into an appropriate vertebrate host to produce antibodies.

Description

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There is an increasing interest in improving and varying the capability of preparing antibodies to a wide variety of determinant sites~ The use of antibodies has been greatly expanded in diagnostics and therapy. The unique capability of antibodies to bind to a specific determinant site or chemical structure makes them peculiarly useful in directing drugs, radioisotopes, or markers to a particular site in a host. In addition, the ability of antibodies to distinguish a specific structure from similar structures has resulted in their wide use in diagnosis.
Regardless of whether one wishes monoclonal or poly-clonal antibodies, the initial step is the immunization of a host.
Usually, one hyperimmunizes the host by repeated injections of the immunogen in accordance with a predetermined schedule. Adjuvants are added to potentiate the immune response. Various adjuvants include aluminum and calcium salts, emulsifying adjuvants and bacteria, e.g., mycobacteria and corynebacteria.
In the case of monoclonal antibodies it is particularly desirable to enhance the immune response to specific epitopic sites. Since the preparation of monoclonal antibodies requires the detection of low population events, any technique which enhances the B-lymphocyte population of interest can prove to be important in the production of monoclonal antibodies.
Allison and Gregoriadis, Nature (London) (1974) 252:252 Heath et al, Biochem. Soc. Trans. (1976~ 4:129; and Shek and ____ ~ _ Sabiston, Immunology (1982) _ :349 describe potentiating the immune response by incorporating antigens in liposomes. Shek and Sabiston, Immunology (19~2) _:627; Shek, (1983) Applications of liposomes in immunopotentiationO In: Immunotoxicology, NAT0 Advanced Study Institute Series (POW. Mullen, ed.) Springer Verlag, ~eidelberg ar.d Van Rooijen and Van Nieuwmegen, Immunol. CommunO (1980) 9:243 describe the use of liposomes with immunogens bound to the membrane sur~ace. Leserman et al, Nature (1980) 228:602; E~eath et al, Biochem. Biophy . Acta. (1980) 5gg:42; Heath et al, ibido (1981) 6~0:66 and Martin and -Papahadjopoulos, J. ~iol. Chem. (1982) 257:286 describe the covalent bonding of proteins to lipid vesicles. See also U.S.
Patent Nos. 4,235,871 and 4,241,0~6, particularly columns 3-5 of '871.
~ ethods and compositions are provided for potentiating the immune response, whereby immunogens are covalently linked to vesicle surfaces. A minimum ratio of immunogen to lipid is provided for optimizing immune response.
In accordance with the subject invention, methods and compositions are provided for producing antibodies to antigenic materials. The compositions involved are liposomes to which the antigens are covalently bonded to the liposome membrane surface above a minimum ratio of protein to lipid. The proteins may be bonded to one or more lipid molecules which are involved in the liposome vesicle membrane~
The compositions of the subject invention can be conveniently prepared by preparing liposomes having active functionalities which can be used for linking by means of a ~2~3~

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convenient linking group -to the anitgen. The liposomes may be prepared from a s~i.de variety of lipid materials including phosphatidyl ethers and esters, e~g. phosp'natidylethanola~ine, phosphatidylcholine, etc.' glycerides, cerebros.i.des, gangliosides, sphingomyelin, steroids, e0g. cholesterol; ekc. See ~.S. Patent No. 4,235,871 for additi.onal lipid ma-terials for use in the preparation of liposomes. One or more of the lipid molecules wili be present in minor amount, generally ranging from about 1 to 15 mole percent, more usually ranging from about 2 to 12 mole percent, w~lich will have an ac-tive functionality which may be used for linkiny. Functionalities which ~la.y be present include activated olefins, par-ticularly olefins having from 1 to 2 carbonyl groups bonded to the olefin, e.g. acrylates and maleimide, aldehydes, carboxylic ac.icls, or the like. Preferably, the active functionality ~ill b~ an activated olefin.
The antigen will for the mos-t part be a poly-(amino acid), including peptldes and proteins, which may also include prosthe~ic groups. The antigen may or may no-~ r~quire modification in order to be linked to the active functionalilty of a liposome. For linking the activated olefin, thiol function-alities are:particularly useful. The resul-ting thioether is a stable llnk which provides for the stable retent.ion of the anti.gen to the vesicle surface~ Where the anitgen does not naturally have : available thiol groups, these can be introduced in a variety of ways with a variety of conventional reagen~s, such as 3-(2'-pyri-dylthio)-propionate, methyldithioacetic acid, dinitrophenylthio-acetic acid, ox the like. There Wl11 be et least one thio functionallty per antigen molecule, preferably at least two thio functionalities and usually not more than one thio functionality per 2000 daltons, more usually not more than one thio function-ality per 3000 daltons. Martin and PapahadjopoulosJ supra~
disclose an exemplary method for bonding proteins to liposomes employing a thioether link.
The aldehyde and carboxy functionalities can be linked to available amino groups of the antigen, in the former case ~ith reductive amination and in the latter case by employing carbodiimide or esters capable of forming peptide bonds in an aqueous medium.
The vesicles may be prepared in conventional ways by combining the lipids in appropriate ratios and vigorously agitating the mixture so as to produce the vesicles. Vesicle preparation may be achieved by the following techniques: See/ for example, Leserman et al., Nature (1980) 228:602; Heath et al., Biochem. Biophys. Acta. (1981) 640:66; and Martin and Papahadjopoulos, J. Biol. Chem. (1982) 257:286.
The antigen or modified antigen may be joined to a dispersion of the vesicles in an appropriate ratio under conditions where covalent bonds are formed between the vesicle and the antigen. Desirably, there should be at least about 25g of protein per mole of lipid, more preferably at least about 40g of protein per mole of lipid and preferably at least about 50g of protein per mole of lipid. There is generally no need to saturate the surface with protein to achieve the desired degree of immunogenicity~ so ~2~3~

that in most cases, the alnount of protein bound to the surface will be less than saturation.
Once the vesicle~protein conjugate has been prepared, it may be purified in accordance with conventiorlal techniques.
Conveniently, the conjugated liposomes may be separated from unbound pro-tein by flota-tion on an appropriate liquid gradient.
Of particular :interest is the presence of i~nuno modulators enclosed in the aqueous space or in the ~ilayer of the vesicle-antigen conjugate. The immunomodulators can serve to modulate the in~nune response by interac~ing preferentially or exclusively with certain subpopulations of cells, e.g. suppressor or helper T-cells, B-cells, macrophages, or the like. For the most part, immunomodulators will be compounds having a specific interaction or affinity for a particular subpopulation of cells which may be recogni~ed by one or more unique determinant sites.
The types of compounds which find use as immuno-modulators may be very diverse. The compounds may be irnmuno--stimulators, such as hydrophobic or hydrophilic derivatives of muramyldipep-tide, a derivative of bacterial cell walls, which acts ~0 as an immunostimulator and a potentiator of macrophage tumoricidal effects. Other bacterial isola-tes known to affect lymphocytes or macrophages may also Eind use.
Varlous drugs which act as immunopotentiators may be employed, such as levamisoIe, niridazole, oxysura~ or flagyl~
Another group of compounds which could ind use are cytotoxic agents or cell growth inhibitors, particularly where the liposome is specifically directed to interact with particular ~ ~3~

lymphocyte subpopulations, e.g. by the use of anitbodies or other specific receptor molecules. Such compounds may include enzyme inhibitors, such as methotrexa-'~:e and its derivatives; cortico-ster:oids, alky:La-ting aqents, such as chlorambucil, melphalan and the nitrosoureas; anthracyclines, such as adriamycin; vinca alkaloids, such as vincristine, antitumor anitbiotics, such as deoxycoformycin, ac-tinomycin D, mitomycin, bleomycin, cispla-tin, cy-tochalasin B, colchicine, etc; the ~ chain of -toxins, e.g. ricin and diphtheria; or the like.
The concentrations of the various immunomodulators will vary widely dependincJ on -the particular immunomodulator, its intended function, the host, the concen-tration of vesicles administrered -to tihe host, the solubility of the compound, and the like. Therefore, for -the most part, the concentration employed will be determined empirically.
It is believed that T-suppressor cells will bind to the liposomes of the present invention. To that extent the pro-liferation of the T-suppressor cells which bind to the antigen --antigenic or epitopic site---conjugated to the vesicle can be modulated. By suppressing the proliferation of such cells, antibody production may be enhanced. Of particular interest are compounds which inhibit prolifera-tion, such as enzyme inhibi-tors, e.g. me-thotrexate, antitumor antibio-tics, or the like.
The vesicle-anti~en conjugate may be administered -through a vertebrate host in accordance with conventional ways.
The veslcle-antigen conjugates may be administered intra-peritoneally, subcutaneously, intravenously or intramuscularly.

~3~6 The adrninis-tered dose will vary depending upor~ the antigen and the host. Usually, -total dosa~e~s administered a-t a single time ~ill be less than about .5mg/Xg of l-lOSt, usually less than about .25mg/kg oE ilOS~, and at least about 25ug/kg of host, more usually at leas~ abo~t 50ug~kg of host.
The vesicle-protein corljugates may be used for the production of monoclonal or polyclonal antibodies. In some instances, the vesicle-protein conjugates may be combined wi-th peripheral blood ce31s, transformed B-lymphocytes, or the like to provide for the production of antibodies.
EMPERIMENTAL
__ ~laterials ancl Methods Animals Male A/J mice, 6 to 8 weeks old, were purchasecl from the Jackson Labora-tories, Bar l-larbor, Maine. Animals were kept in plastic cages and wexe allowed free access to laboratory mouse chow and water.
Chemical and Biologicals _ _ _ Cltrated sheep's blood was supplied by Woodlyn Labora-tories, Guelph, Ontario. Phosphatidylcho:Li~e and cholesterol were purified as descrlbed by lleath et al., Biochem. Biophys. Acta.
(1981~ 640:66. N~[4-(p-ma]eimidophenyl)butyryl]phosphatidyl-ethanolamine (MPB-PE) was synthesized as descr:ibed in Martin and Papahad~opoulos, J. Biol. Chem. (1982) 257:~6.

Liposomes were prepared from phosphatidylcholine:choles-terol:MPB-PE at a molar ra-tio of 47:47:6 by the method of Szoka 39~6 and Papahadjopouls, Proc. Na-tl. Acacl. Sci. USA (1978) 75:4194.
_____ . _ The bufEer was 50ï~ morpholinoethanesulphonic acid (MES), -50mM
morpholinopropanesulphonic acid (MOPS), 80mM NaCl ~IES/MOPS), pI~ 6.7, 2~0mOsm.
CovalerI-t Conjugation of BSA -to Vesicle Surface Bovine serum albumin (BSA) was dissolved in O.lM
pho~pha-te, O.lM NaCl, pH 7.5 at 20mg/ml. SPDP (N-succinimidyl 3-(2'-pyridy:Lthio)propiona-te) was prepared a-t 20mM in e-thanol.
Sufficient SPDP solution was added to the BSA solution with stirring, to give a 20:1 molar ratio of SPDP:protein. After 30min the 3-(2'-pryidylthio~propionate-BSA (PDP-BSA) was separated from reac-tants by gel chromatography on a Sephadex G-75 column prepared in 50Ir~I citrate, 50mM phosphate, 50mM NaCl, pH 7Ø 'rhe PDY:protein ratio was determin~d by the method o~ Carlson et al.
B ochm. J. (1978) 173:723 and found to be 13.5 PDP groups per BSA
molecule. Preliminary experimen-ts showed that this number of thiols was necessary to achieve efficien-t conjugation. The PDP-PSA solution was adjusted to pH 4.5 and treated with dithiothreitol to a final concentration of 25mM. After 30mIn, the reduced protein was separated from dithiothreitol by gel chroma-tography on Sephadex G-75. The column was equilibrated with M~S/MOPS ~pH 6.7, 290mOsm) and p-IrcJed with argon. The thiol-BSA
peak was collected under argon and concentrated to 12mg/ml in a lOml Amicon concentrator cell with YM--10 membrane~
Conjugation was initia~ed by mi~ing liposomes with thiol-BSA at various concen-trations to control the final product.
(See Table 1.) Ater overnight conjuga~ion, the conjugated ~ 8 --3~

liposomes were separated f.rom unbound protein by flota~ion on a dlscontinuous metrizamide gradient (Heath et al. (1981), supraO).
The conjugated llposomes were analyzed for protein and lipid content as described in the immediately preceding reference.
Prepara-tion of Me-thotrexate Encap-tured BSA-vesicle Conjugates L.iposomes were prepared from phosphatidylcholine:
cholesterol: MPB-PE at a molar ratio of 47:47:5 by the method of Szoka and Papahadjopoulos, supra. The liposomes were prepared in a solution which contained 50l~ methotrexate (sodium salt), 50mM
morpholinoethanesulphonic acid, 50mM morpholinopropanesulphonic acid, pH 6.7, 29~nOsm. When the liposomes are prepared, a pro-portion of the solution, the methotrexate, is captured in the aqueous interstices of the liposomes. The non-encapsulated material is separ~ted chroma-tographically from -the liposomes on a column of Sephadex G-75 which is equilibrated with a buffer composed vf 50mM morpholinoethanesulfonic acid, 50mM morpholino-propanesulfonic ac.id, 80mM NaCl, pH 6.7, 290mOsm. The liposomes a.re then conjugated to the bovine serum albumin as described aboveO
When the final product is isolated from the gradient, it is analyzed for protein, lipid and drug content. The drug is measured spectophotometrically (~cm=7943 at 370n~) after extraction by the Bligh and Dyer method, which separates the dru~
from the lipid, thereby giving an optically clear suspension.
Haemolytic Pl.aque Assay The procedures used for the prepara-tion of spleen cells and for the determination of the BSA-specific plaque-forming cell (PFC) response were performed as described by Shek and Sabiston, lmmunology (1982) 45:349.

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able 1~ Effect of the ini-tial protein concentration on protein lipicl conjugate ratio in the coupling of ~SA to liposomes*

__ _ __ _ St.art g _ ac-tion Product Experiment Protein Conc. Lipld Conc. Protein:Lipid Number(m~/ml) (~mole/ml) ~g/~mole) __ _ l 8.80 6.9 174 1 4.45 6.9 156 l 2.18 6.9 13 l 0.53 6.9 36

2 13.64 6.0 2~5 2 2.0 6.0 135 2 1.0 6.0 57 2 0.5 6.~ 41 2 0.25 6.0 24 *Liposomes were prepared, conjugated, separated and analysed as described in Materials and Methods.

Res~lts As evidenced by the resul-ts reported in Table 1, vaxia-tion of the ini-tial protein concen-tratlon of the reaction mixture between 0.25 and 2mg/ml ca~lsed la~ge variations in the lipid:
protein ratio of the procluct. ~owever, when the initial protein concentration was increased above 2mg/ml, the Einal protein:lipid ratio of the procluct rose less rapidlyO This would indicate that at approximately 130~1g/~mole, the BSA may saturate the outer lipo-some surface, thereby inhibitina furt}ler significant attachment.
PFC Response -to BSA Antigen Conjugated to Liposome Surface Mice were given two intraperi-torleal injection, ~ weeks apart, of BSA conjuga-ted vesicles containing 30~1g of BSA. The ~SA-specific PFC response was assayed 3 to 5 days after the second injection of antigen. The peak anti-BSA PFC response occurrrecl on Day 4. At the peak of the response, essentially the same number of PFC was generated whe-ther the immunizing dose contains 7 oF
30yg of BSA. (See Table 2.) Different control groups of animals injected with native BSA, thiolated BSA, or unconjugated vesicles failed to elicit a detectable PFC response. The simultaneous injection of liposomes and thiolated BSA was also found ~o be lnefffective in engenderiny a significant response.
Lipososomal vesicles coated with BSA at different protein:lipid ratio (epitope density) were tested for their effec-tiveness in stimulating the PFC response to the protein antigen.
At an imm~nizing dose of 30 ~g, there was no difference in the PFC
elicited with antigen-coated vesicles con-taining approximately 60 to 250~g BSA/~mole lipid. Hc~ever, the magnitude of the response decreased signlEicantly at an epitope density oE 40~g BSA/~lmole lipid or less.

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In the ne~t st~dy, mice were immunized twice with BS~
attached to liposomes. Group A received 2x20~g BSA conjugated with liposomes (ratio 6g/mol)~ Group B received lipGsome BSA as in Group A, but with 0.05~unole o~ metho-trexate. Group C recei~ed 0.05umole o:E methotrexate in liposomes con3ugated with 20~g BSA.
Group D xeceived 20~1g BSA con~ugated to liposomes and 0~05~umo1e methotrexate in a separate population of liposomes. The results were determined as BSA-specific plaque-forming cell (PFC) re~pons~ The follo~ling are the results.

BS~-specific IgG
Group _'C/10 6 spleen cellsk *Assayed four days after antigenic challenge.

The extraordinary enhancement in response with the methotrexate in the vesicle conjugated with BSA is evident.
It is evident from the above results that by covalently conjugating protein to liposomes abo~e a minimum epitopic density, enhanced immunoyenic responses can be achieved. Furthermore, ~he liposomes can serve as vessels for various substances which may serve to further enhance the immune re~ponse. Ill accordance w:ith this invention, a simple effective procedure and compositions are provided for eliciting an~ibodies to one or mor~ epitopes of an antigenic matarial.

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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that ~ertain c~anges and modifications may be practi.ced within ~he scope of the appended claims.

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILIGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method of making an antigen/liposome conjugate, which comprises (a) reacting a predetermined protein antigen, with liposome vesicles having lipid molecules in the surface membrane thereof, said lipid molecules including active functionalities for linking to said antigen to covalently bond said predetermined protein antigen to said lipid molecules, wherein at least about 25 g of protein antigen is provided per mole of lipid in said liposome vesicles; and (b) separating the antigen/liposome conjugate so formed from unbound reactants.

2. A method according to claim 1, wherein at least about 40 g of protein antigen is provided per mole of lipid in said liposome vesicles.

3. A method according to claim 1, wherein at least about 50 g of protein antigen is provided per mole of lipid in said liposome vesicles.

4. A method according to claim 3, wherein the antigen is a poly(amino acid) including thiol functionalities.

5. A method according to claim 4, wherein the antigen is bound through a thioether group.

6. A method according to claim 5, wherein the active functionality of the lipid molecule is an activated olefin.

7. A method according to claim 5 which comprises the additional step of incorporating an immunomodulator within the liposome vesicles, prior to reaction with said predetermined antigen.

8. A method according to claim 6, which comprises the additional step of incorporating a cytotoxic agent within the liposome vesicles, prior to reaction with said predetermined antigen.

9. A method according to claim 6, which comprises the additional step of incorporating methotrexate within the liposome vesicles, prior to reaction with said predetermined antigen.

10. An antigen/liposome conjugate, comprising a predetermined protein antigen covalently bonded to lipid molecules in the surface membrane of liposome vesicles, wherein at least about 25 g of protein antigen is provided per mole of lipid in said liposome vesicles.

11. An antigen/liposome conjugate according to claim 10, wherein at least about 40 g of protein antigen is provided per mole of lipid in said liposome vesicles.

12. An antigen/liposome conjugate according to claim 10, wherein at least about 50 g of protein antigen is provided per mole of lipid in said liposome vesicles.

13. An antigen/liposome conjugate according to claim 10, wherein the antigen is a poly(amino acid) including thiol functionalities.

14. An antigen/liposome conjugate according to claim 13, wherein the antigen is bound through a thioether group.

15. An antigen/liposome conjugate according to claim 14, wherein the active functionality of the lipid molecule is an activated olefin.

16. An antigen/liposome conjugate according to claim 10, wherein the liposome vesicles inccorporate an immunomodulator.

17. An antigen/liposome conjugate according to claim 10, wherein the liposome vesicles incorporate a cytotoxic agent.

18. An antigen/liposome conjugate according to claim 17, wherein the cytotoxic agent is methotrexate.

19. An antigen/liposome conjugate according to claim 10.
wherein the lipid comprises a phospholipid.

20. An antigen/liposome conjugate according to claim 19, wherein the phospholipid is phosphatidylcholine.