HK1008226B - Induction of cytotoxic t-lymphocyte responses - Google Patents

Description

Induction of cytotoxic T lymphocyte responses

Background

The entire contents of pending U.S. sequence No. 08/919,787 (filed 24.7.1992) and U.S. sequence No. 07/735,069 (filed 25.7.1991), both entitled "induction of cytotoxic T lymphocyte response," and William h. The present invention relates to methods and compositions for inducing a cytotoxic T cell-mediated response in a human, domestic animal or agricultural animal.

Cytotoxic T Lymphocytes (CTLs) are believed to be the main defense mechanism of the host in response to various viral infections and tumor or cancer growth. These cells are depleted of infected or transformed cells by recognizing antigen fragments associated with different molecules (called MHC class I molecules) on the infected or transformed cells. Experimental induction of CTLs can be achieved using cytoplasm that carries a soluble antigen in a particular cell. The use of soluble antigens alone is generally insufficient to induce specific cytotoxic T lymphocytes.

One method by which CTL responses can be induced involves the use of recombinant engineering techniques to incorporate the key components of the above antigens into the genome of benign infectious agents. The goal of this strategy is to generate antigen-specific cytotoxic T lymphocytes to the desired epitope by allowing the host to produce a modest, self-limiting infection. Vaccinia, poliovirus, adenovirus and retrovirus, and bacteria (such as listeria and BCG) can be used as the chimeric vector. For example, Takahashi et al are inUSA Journal of national academy of sciences85, 3105, 1988, the use of recombinant vaccinia expressing the HIV gp160 envelope gene as a powerful means of inducing cytotoxic T lymphocytes is indicated.

A second method by which cell-mediated responses can be induced involves the use of adjuvants. Although the use of adjuvants is well discussed in the art, it is unclear whether the use of adjuvants induces cell-mediated immunity and whether such cell-mediated immunity includes a cytotoxic T lymphocyte response. Several representative publications in the art are listed below.

Stover et al are inNature of nature351, 456: 1991 (not prior art to the present application) describes the induction of CTL responses to beta-galactosidase with recombinant BCG containing the beta-galactosidase gene. This response was not induced with incomplete freund's adjuvant and beta-galactosidase.

Mitchell et al inJournal for clinical oncology8,856, 1990 (not prior art to the present application) described treatment of metastatic melanoma patients with five administrations of an adjuvant called "DETOX" and of the same melanoma lysate within six weeks, with a small number of patients finding an increase in cytotoxic T lymphocytes. The authors indicated a need to increase the production levels of cytotoxic T lymphocytes and suggested a combination treatment with adjuvant and IL-2 and pre-treatment with cyclophosphamide to reduce the levels of tumor specific T suppressor cells that may be present. DETOX includes derivatives derived fromSalmonella minnesotaAttenuated endotoxins (monophosphoryl lipid A),Mycobacterium phleiThe cell wall skeleton of (a), squalene oil and an emulsifier.

Allison and Gregoriadix in Immunology Today 11, 427, 1990 (not as prior art to the present invention) indicated that the only adjuvant "allowed" in human vaccines is aluminium salt (alum), which does not continuously elicit cell-mediated immunity. Allison and Gregoriadis teach "therefore, there is a need to develop an adjuvant that has the efficacy of freund's complete adjuvant but does not have its various side effects, such as granuloma. They continue to indicate that there are three possible scenarios, for example: using liposomes; the use of adjuvants known as immunostimulating complexes (ISCMs, including saponins or Quil a (triterpenoids with two sugar chains), cholesterol and phosphatidylcholine), which are allowed for equine influenza Vaccines (Morein et al, immunologicals addivants and Vaccines, Plenum Press, 153); squalene or squalane (with or without Pluronic agents) and Muramyl Dipeptide (MDP) emulsion (SAF) are utilized. Although "it has long been thought that antigen subunits are unable to elicit a Cytotoxic T Lymphocyte (CTL) response, SAP is said to elicit cellular immunity".

Takahashi et al inNature of nature344, 873: a single subcutaneous immunization of mice with ISCOMs was described in 1990 as inducing MHC II restricted helper T lymphocyte and cytotoxic T lymphocyte production. They indicated that freund's adjuvant, incomplete freund's adjuvant and phosphate buffer did not induce cytotoxic T lymphocyte activity against their target of interest. In contrast to the results for other exogenous soluble protein antigens, immunization with intact exogenous proteins using ISCOMs elicits antigen-specific MHC I restricted CD8⁺CD4^-And (3) CTL. They also indicated that the experiments described showed that human CTLs could be induced with ISCOMs containing HIV proteins, and that ISCOM-based vaccines could achieve the long sought goal of inducing both CTL and antibody production by purifying the proteins.

Byars and Allison inVaccine5: 223, 1987 describe the use of SAF-1, including tween 80, PLURONIC L121 and squalene or squalane, with or without muramyl dipeptide, and their data indicate that formulations with muramyl dipeptide can be used in human and veterinary vaccines. Booster injections (boost shot) of adjuvant did not have muramyl dipeptide. Muramyl dipeptide is said to significantly enhance antibody production, so it is not present in the adjuvant. Cell-mediated immunity is measured by skin tests as a delayed-type hypersensitivity reaction to determine the production of helper T cells. This hypersensitivity reaction becomes more intense and more persistent when muramyl dipeptide is present in the adjuvant. The same adjuvant was used by Allison et al, us patent 4770874 (which states that muramyl dipeptide and pluronic polyol are the essence of generating a strong cellular and humoral immune response to albumin); allison et al, U.S. patent 4772466; Murphy-Corb, etcScience of246: 1293, 1989 (wherein it is indicated that the combination of an adjuvant and a muramyl dipeptide enhances both cellular and humoral immunity in the immune response); allison and Byars et alVaccine87: 56, 1987 (which indicate induction by SAF (containing muramyl dipeptide)Cellular immunity can be shown by these: delayed hypersensitivity, proliferative responses of T cells to antigen, production of IL-2, specific genetic restricted lysis of target cells bearing immune antigens); the combination of Allison and Byars,immunopharmacology of infectious diseases: non-specifically limited vaccine adjuvants and modulators191-201, 1987; morgan et al, J. medical virology 29: 74, 1989; kenney et al, journal of immunology 121: 157, 1989, Allison and Byars,immunological method95: 157, 1986 (in which aluminum salts and mineral oil emulsions are shown to enhance antibody production without enhancing cellular immunity; muramyl dipeptide preparations induce cellular immunity); the Byars et al, and the like,vaccine8: 49, 1990 (not prior art to the present invention, where it was indicated that their adjuvants significantly enhanced the humoral response to influenza serum antigens, with a low degree of enhancement of cell-mediated responses); the combination of Allison and Byars,molecular immunization28: 279, 1991 (not prior art to the present invention, in which it is indicated that muramyl dipeptide functions to induce cytokine expression and enhance expression of Major Histocompatibility (MHC) genes; better antibody and cellular responses can be obtained compared to other adjuvants, and it is desirable to determine whether the same protocol is effective in humans); the combination of Allison and Byars,advanced technology in vaccine development401, 1988 (in which SAF mediated cellular immunity is described); an acid addition salt of Epstein and the like,review of new drugs(Advance drug delivery reviews) 4: 223, 1990 (in which different adjuvants used in the preparation of vaccines are reviewed); allison and Byars, journal of immunological methods 95: 157, 1986 (wherein it is indicated that the addition of muramyl dipeptide to an adjuvant significantly enhances the cellular immune response to different antigens, including monoclonal immunoglobulins and viral antigens); the method of Morgan et al,medical virology magazine29: 74, 1989 (which describes the preparation of vaccines for Epstein-Barr virus with SAF-1).

A Kawk, etc. of the general formula,individual networks in biology and medicineElsevier science publishers, p163, 1990 (not as prior art to the present invention) indicated the use of SAF without muramyl dipeptide as an adjuvant for human B cell lymphoma genotypesIn particular, an emulsifier of pluronic L121, squalane and phosphate buffer containing 0.4% tween 80 was applied with the individualized form. They indicated that "the addition of adjuvants should further enhance … the humoral response, and also help to induce cellular responses".

Other immunological formulations include liposomes (Allison et al, U.S. Pat. nos. 4053585 and 4117113); cyclic peptides (Dressman et al, U.S. Pat. No. 4778784); complete Freund's adjuvant (Asherson et al,immunology22: 465, 1972; the Berman et al, in the prior art,international journal of cancer2: 539, 1967; allison, immune enhancement 18: 73, 1973; allisonInfluence host resistance Non-specific factors of sex247, 1973); ISCOMs (Letvin et al, vaccine 87: 209, 1987); adjuvants comprising a non-ionic block polymer of mineral oil, surfactant and tween 80 (Hunter and Bennett,journal of immunology133: 3167, 1984; the Hunter et al, the number of the patents,journal of immunology127: 1244, 1981); adjuvants consisting of mineral oil and emulsifiers, with or without inactivated mycobacteria (Sanchez-Pescador et al,journal of immunology141: 1720, 1988); and other adjuvants, such as lipophilic derivatives of muramyl dipeptide, muramyl dipeptides covalently bound to recombinant proteins: (Same as before)。

Summary of The Invention

Applicants have discovered a safe and convenient method and composition for inducing CTL responses in humans and domestic animals or important agricultural animals. The method employs an antigen preparation that is less or non-toxic to the animal and lacks an immunostimulatory peptide (e.g., muramyl dipeptide) whose presence reduces the desired cellular response. Furthermore, the methodology is simple to use and does not require extensive in vivo work to alter cells to be more antigenic using recombinant DNA techniques. This finding is surprising because antigenic agents such as these which do not contain immunostimulatory peptides or analogues thereof are expected to induce CTL responses. Applicants have found that such antigenic preparations can be used in a wide variety of disease states or as prophylactic agents. For example, such antigenic preparations may be used in the treatment of viral diseases such as HIV infection or influenza, where CTL responses are important; also has extended application in the treatment of bacterial infections, cancer, parasitic infections, and the like. As a prophylactic agent, the antigenic preparation in combination with a suitable antigen can be used for the prophylaxis of infections with viruses causing the above-mentioned viral diseases, in particular HIV infections, and also for the prophylaxis of such tumor-prone persons as those after resection of the primary tumor.

Accordingly, the present invention relates firstly to a method of inducing CTL responses in humans, domestic animals (e.g. cats and dogs) or agricultural animals of interest (e.g. horses, cattle or pigs) to antigens other than B-cell lymphoma antigens or ovalbumin. The method comprises the steps of providing an antigen for which a CTL response is desired, providing a non-toxic antigenic preparation comprising, consisting of or consisting essentially of a stabilizing detergent, a micelle-forming agent and a biodegradable and compatible oil. The antigenic preparation preferably lacks any immunostimulatory peptide or contains a sufficiently low level of peptide moieties so as not to reduce the desired cellular response. Such formulations are preferably stable oil-in-water emulsions. That is, the time for each of the different components to be selected to maintain the emulsion in an emulsified state without phase separation is at least one month, preferably more than one year. In this method, the antigen and the antigenic preparation are mixed to form a mixture (preferably microfluidised) and administered to the animal in an amount sufficient to induce a CTL response in the animal, only 1 administration.

"stabilizing detergent" means a detergent that maintains the emulsion in a stable state. Such detergents include polysorbate 80 (tween) (sorbitan-mono-9-octadecanoic acid-poly (oxy-1, 2-ethanediyl); manufactured by ICI Americas, wilmington de), tween 40, tween 20, tween 60, amphoteric detergents (Zwittergent)3-12, tebol HB7, Span 85. These detergents are generally used in amounts of about 0.05% to 0.5%, preferably 0.2%.

"micelle-forming agent" means a compound capable of reacting with other componentsThe emulsion formed is stable, thereby forming a micelle structured agent. Such agents preferably cause stimulation at the injection site to accumulate macrophages that enhance the cellular response. Examples of such agents include polymeric surfactants described in the BASF Wyandotte publication, such as Schmolka,J.Am.Oil.Chem.Soc.54: 110, 1977 and Hunter et al,journal of immunology129: 1244, 1981, incorporated herein by reference, PLURONIC (PLURONIC) L62LF, PLURONIC L101, PLURONIC L64, polyethylene glycol 1000 and quaternary ketone (TETRONIC)1501, quaternary ketone 150R1, quaternary ketone 701, quaternary ketone 901, quaternary ketone 1301, quaternary ketone 130R 1. The chemical structures of these agents are well known in the art. As taught by Hunter and Bennett, preferred are agents having a hydrophilic-lipophilic (HLB) balance of from 0 to 2,journal of immunology133: 3167, 1984. The amount of agent is preferably between 0.5% and 10%, most preferably between 1.25% and 5%.

The oil is selected to retain the antigen in an oil-in-water emulsion, i.e., to provide a vehicle for the desired antigen, and preferably has a melting point below 65℃, so that an emulsion can be formed at room temperature (about 20℃ -25℃) or below. Such oils include squalene, squalane, EICOSANE (EICOSANE), tetratetracontanes (tetratetracontans), glycerol, peanut oil and other vegetable oils. The amount of oil is preferably in the range of 1-10%, most preferably 2.5-5%. The biodegradability and biocompatibility of the oil are important so that the body can degrade the oil over a period of time to ensure that side effects such as granuloma are not caused.

It is important that the above formulation is free of peptide components, especially free of Muramyl Dipeptide (MDP). Such peptides interfere with the induction of CTL responses if the amount contained in the ordinary preparation administered to each person exceeds 20. mu.g. Despite their marked stimulatory effect on the humoral part of the immune system, it is still preferred that these peptides be completely absent from the antigen preparation. That is, applicants have found that although these peptides can enhance humoral responses, they are disadvantageous when a cytotoxic T lymphocyte response is desired.

In other related aspects, such an antigenic preparation consists of only two of the three components described above, and the use of the antigenic preparation with a desired antigen (including proteins, polypeptides and immunogenic fragments thereof) other than any one of the egg albumin (or other albumins, such as HSA, BSA and ovalbumin) can induce CTL responses in animals and humans.

The applicant believes that the above formulations have significant advantages over previous formulations (including ISCOM, DEToX and SAF) when used in humans. Unlike other preparations, the preparation contains both the micelle-forming agent and no peptide, cell wall scaffold or bacterial cellular components. The agent induces CTL responses that were not induced by previous agents or induced significantly more strongly than they.

By "non-toxic" is meant that the antigenic preparation is found to be non-toxic or minimally toxic in animal and human therapy. Those skilled in the art will recognize that the meaning of this term is broad, for example, only mild toxicity to a substantially healthy animal or human, and substantial toxicity to a person suffering from an immune disorder.

In a preferred embodiment, the antigen preparation consists essentially of two or three of a detergent, a medicament and an oil; the method essentially consists of administering the mixture (antigen and antigen preparation) once to a human or animal; the human or animal being infected with the virus and suffering from one or more symptoms caused by the virus infection (usually diagnosed by a physician in the relevant field); the antigenic preparation is non-toxic to humans or animals.

Other preferred embodiments are those where the antigen is selected from antigenic portions of: HIV antigen: gp160, gag, pol, Nef, Tat and Rev; malaria antigen: CS protein and sporozoite surface protein 2; hepatitis b surface antigen: Pre-S1, Pre-S2, HBc antigen and HBe antigen; influenza antigen: HA. NP and NA; hepatitis a surface antigen; herpes virus antigens EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV early protein products, human papillomavirus antigens (e.g. HPV antigens such as L1, E4, E6, E7 antigens, especially E6 and E7 from HPV16 and 18 which are the two most common types of HPv associated with cervical cancer, E4 and L1 from HPV6 and 11 which are the two most common types of HPV associated with condyloma acuminatum; Prostate Specific Antigen (PSA), cytomegalovirus gB, cytomegalovirus gH and IE protein gp 72; respiratory syncytial virus; F protein, G protein and N protein; tumour antigen CEA, cancer associated mucin, carcinoma P21, P53, melanoma MPG, melanoma P97 and carcinoma Neu oncogene products, the gene product of carcinoma P53 and melanoma antigen of MAGE and the P78 mutant proteins present in different malignant tumours and ras 21.

In a related aspect, the invention relates to a composition comprising, consisting of or consisting essentially of an antigen selected from the antigenic moieties listed above in admixture with an antigenic preparation as described above.

In other related aspects, the invention relates to a method of treating a patient infected with an HIV virus, suffering from malaria, influenza, hepatitis, cancer, herpes virus, suffering from cervical cancer, condyloma acuminatum (genital warts) or infected with respiratory syncytial virus by administering a composition comprising a suitable antigen (e.g., selected from the antigens listed above) in admixture with a preparation of the above-described antigen. These antigens and therapeutic methods are only a few examples of the antigens that may be used in an antigen formulation.

Other features and advantages of the invention will be further described in the following preferred embodiments and claims.

Description of the preferred embodiments

The drawings are first briefly described.

Drawings

FIGS. 1A-1C and 4A-4C are graphs of data comparing the induction of CTL responses by different ovalbumin preparations; in all figures E: T represents the effective target ratio.

FIGS. 2A and 2B are graphs comparing CTL responses induced by different β -galactosidase preparations;

FIG. 3 is a graph of data comparing the induction of CTL responses by ovalbumin in liposomes and ovalbumin in antigen preparations;

FIGS. 5 and 6 are graphs of data on the observation of the effect of CD4, CD8 cell depletion on the induction of CTL responses;

FIG. 7 is a graph of data for gp120 induced CTL;

FIG. 8 is a graph showing data obtained when CTL is induced by mixing antigen with Tween and Pluronic;

FIG. 9 is a graph showing data on CTL induction by antigen mixed with squalane and Pluronic;

FIG. 10 is a graph showing data on CTL induction by antigen mixed with squalane and Pluronic;

FIG. 11 shows the effect of ovalbumin and different antigenic agents on CTL responses;

FIG. 12 is a graph of anti-gp 120IIIb antibody induction in monkeys using different antigenic preparations;

FIG. 13 shows the antitumor activity against HOPE2 cells after 10 days after one immunization with soluble E7 protein in adjuvant;

FIG. 14 shows the antitumor activity against HOPE2 cells on days 10 and 19 after 2 immunizations with soluble E7 protein in adjuvant.

Antigen formulations

The antigen preparation used in this invention has been described above. One of ordinary skill in the art will recognize that the same agents can be readily prepared and expected to have equivalent properties in inducing a CTL response, and that these agents can be readily tested for their properties using the same techniques as those described in the examples below.

The antigen preparation (AF) used in the following examples of the invention comprises approximately 2.5% squalane, 5% oxirane and Tween 80, which are formulated with phosphate buffer. In particular, AF emulsifiers include: each 1mL of water having a pH of 7.4 contained 15mg of squalane, 37.5mg of Poloxamer 401 (Pluronic L121), 6mg of polysorbate 80 (Tween 80), 0.184mg of potassium chloride, 0.552mg of potassium monophosphate, 7.36mg of sodium chloride, and 3.3mg of sodium dibasic phosphate (dehydrate). Microfluidization (microfluidics 110F) was performed using conventional techniques with a back pressure of 11-14,000psi, gradually reduced to atmospheric pressure, cooled in an ice bath, and encapsulated.

In other examples, the antigen is mixed with a mixture of microfluidized squalane (S), pluronic (P) and Tween (T) to final concentrations of Tween 800.2%, pluronic 1.25% and squalane 5%, respectively. To determine the necessity of the subcomponent in the induction of an antigen-specific immune response, squalane-tween 80, pluronic-tween 80 or squalane-pluronic was formulated at the same concentration. Pluronic, squalane or tween 80 was prepared separately to examine the effect of the monomeric components in CTL responses. Tween 80 was replaced by tween 20, tween 40 or amphoteric detergent in the ovalbumin system to test the effect of different tween derivatives in CTL induction. Similarly, an Eicocore or Triacontone was used in place of squalane in the three-component formulation, and polyethylene glycol 1000, Pluronic L62LF and Tetronics 1501 and 150R were used in place of the complex ethylene oxide-propylene oxide polymer (Pluronic). Analogs that differed as two component preparations were variously combined to determine their induction of ova-specific CTLs. They are cholesterol-tween 80, squalane-tween 80 or olive oil-tween 80. For stability studies, the squalane-tween 80 fluidized mixture was mixed with dextrose to a final concentration of 5%. The excipients were mixed throughout the process in the microfluidics instrument to form a stable emulsion. In some experiments, the two-component formulation and different concentrations of MDP mixed induced CTL and humoral responses. Table 1 is a comprehensive summary of the various formulations used in this study.

TABLE 1

Effect of various substitutes in three-or two-component systemsAlternatives in three component formulations Kill rate at E: T100: 18TP 84 tween 40(T) 66 tween 20(T) 48T1501(P) 0T150R1(P) 0 pluronic L62LF (P) 47 eicosane (S) polyethylene glycol triacontane (S) amphoteric detergent (T)Alternatives in two-component formulationsST 76PT 45SP 26 cholesterol + Tween 800 squalane + Tween 29(T) 65 squalane + Tween 8042 olive oil + Tween 8069One-component preparationPluronic L1210 squalane 0 Tween 800 squalane + Tween 80+ 5% dextrose 86

^*Repeat CTL assays

Syntex adjuvant formulation (microfluidization; SAFm) as adjuvant control, it included two parts. The first part comprises phosphate buffered saline containing squalane at a final concentration of 5%, Pluronic at 1.25%, and Tween 80 (excipient or 1-SAF) at 0.2%. The second part comprises N-2-acylmuramyl-L-threonine-D-isoleucine amine, a cell wall component of mycobacteria. The antigen was mixed with microfluidized excipient (part I) to obtain a homogeneous emulsifier for immunization. The addition of MDP resulted in the formulation of SAFm and a simple vortex stirring. The concentration of MDP in the mixture will be determined as the case may be if there is an optimal concentration for inducing CTLs. As an adjuvant control, mice were also immunized with soluble antigen mixed with alum according to the manufacturer's protocol (Pierce Chemical, Rockford, Ill.) or with Complete Freund's Adjuvant (CFA).

This antigen preparation is used to induce a cytotoxic T lymphocyte response in mice. One of ordinary skill in the art will recognize that such mouse models may indicate that cytotoxic T lymphocyte responses can be induced in humans, domestic animals or agricultural animals using equivalent experiments or treatments. The amount of antigen preparation and the amount of antigen to be administered in order to induce the desired cellular response may be determined empirically without prior experimentation by routine methods well known to those of ordinary skill in the art. Thus, if such a mixture is desired for treatment with minimal side effects, the skilled artisan will determine the minimum level of induction of a cytotoxic T lymphocyte response when applied to a human, domestic animal or agricultural animal, and thus immunity to the desired antigen. In general, the mixture can be administered by injection by any conventional method, but it is particularly preferred to administer the mixture intramuscularly topically, so that the emulsion remains stable for several days or weeksMethod

The following materials and methods were used in the examples listed below, unless otherwise noted:

mouse

C57BL/6(H-2^b) And BALB/c (H-2d) female mice, purchased from HarlenSpragne (San Diego, California).

Antigens

Ovalbumin (ova, grade VII, Sigma (Sigma) chemicals, st. Beta-galactosidase (beta-gal, grade VIII; BRL) was used in its native state after 2 minutes of alkaline digestion in 1M NaOH. Recombinant gp120 was purchased from bioengineering, USA.

Tumor cells and transfectants

The tumor cell line used is Ia^-Is EL₄(C57BL/6，H-2^bThymoma) and P₈₁₅(DBA/2，H-2^dMast cell tumor). Moore previously equaledCells54, 777: 1988 describes ova-expressing EL₄Derivative EG of transfectant₇-ova. Beta-gal expression transfectant P_13.1Is 10⁷ P₈₁₅Cells and 10mgPst ILinearized PCH 110 (pharmaciLKB bioengineering technology, Piscataway, NJ) and 1mgPvu ILinearized PSV₂neo (Southern et al,J.Mol.Appl.Genes.1: 327, 1982) in 1ml of Phosphate Buffered Saline (PBS), followed by 400. mu.g/ml of antibiotic G₄₁₈The product of the screening in (1). The C3-4 transfectant was induced from BALB/C hybridoma IgM662 by transformation of a plasmid encoding the β -gal gene fused to exons 3, 4 of the IgM heavy chain. (Rammensee et al,immunogenetics (immunogenetics)30, 296: 1989). The gp160 IIIb-expressing 3T3 fibroblasts 15-12 were supplied by NIH dr. K^bTransfected L cell lines were provided by dr. D^dAnd L^dTransfected L cell lines were provided by dr.ted Hensen, st.louis washington university.

Immunization

Such as Moore, etcSame as aboveAnd carbon et al (Journal for experimental medicine169: 603, 1989), after cytoplasmic loading with 200. mu.l of 25X 10⁶The mice were immunized intravenously with the splenic cell suspension. When the ova-antigen preparation or the beta-gal antigen preparation is used for immunization, 30 mu g of each protein antigen is injected subcutaneously into the foot pad or the tail root of each mouse. Each injection included 67. mu.l of microfluidised antigen preparation (prepared according to conventional procedures) and 30. mu.g of protein antigen in a final volume of 200. mu.l.The final volume was formulated with HBSS, see Whittaker handbook (Welkerstille, Md.). The concentration of MDP is 0-300. mu.g. It is noted that mice were immunized with antigen soluble in CFA or alum in a final volume of 200. mu.l.

In vitro stimulation of effectors

Splenocytes obtained from normal mice or immunized mice that have been exposed to antigen at least 14 days ago (30X 10)⁶) And 1.5X 10⁶EG7-ova (with 2000rad, radiation) or 1.5X 10⁶C3-4 cells (irradiated with 2000rad radiation) in 24-well plates, 37 ℃, 7% CO₂Incubate under air conditions for ova response or β -gal response. All tissue cultures were performed in complete medium with IMDM medium, supplemented with 10% Fetal Calf Serum (FCS), 2mM glutamine, gentamicin and 2X 10, according to the Whittaker Manual (Welkersville, Md.)^-5M2-mercaptoethanol. In vitro depletion experiments, splenocytes stimulated or stimulated in vivo with monoclonal antibodies (mAbs) RL.172 (anti-CD)₄) Or mAbs 3.168 (anti-CD)₈) Processing to remove CD₄ ⁺Or CD₈ ⁺Cells (a cell of Sarmiena et al,journal of immunology125: 2665, 1980 and Ceroding et al,nature of nature34: 98, 1985), mAb rl.172 and mAb 3.168 were given by dr.Cytotoxicity assays

Target cell (1X 10)⁶) Using 100 μ Ci [ alpha ], [⁵¹Cr]Chromate mark 60 minutes. Labeling target cells to allow the peptide to bind to the target cells⁵¹For Cr, 50. mu.l of 1mg/ml peptide dissolved in HBSS was added. After washing, 10⁴Labeled target cells and serially diluted effector cells at 200. mu. lRP₁₀Culturing at 37 deg.C for 4 h. 100 μ l of supernatant was aspirated and specific killing was calculated according to the following formula: the percentage of specific killing was 100 × { (CTL release-natural release)/(maximum release-natural release) }. In all experiments, the natural release caused by detergent in the absence of Cytotoxic T Lymphocytes (CTL) was < 25% of the maximum release.

Detection of antibody responses in mice and monkeys

Each well of the 96-well plate was covered with 50. mu.l of ova or gp 120150 ng in HBSS on the bottom of a U-shaped plate (Costar, Cambridge, MA) and incubated overnight. To detect the response of anti-gp 120 and anti-ova antibodies in mice, plates were blocked with 1% BSA for 1 h. Mu.l of serially diluted serum was added to each well and incubated for 2 hrs. Plates were washed and 50. mu.l of HRPO-conjugated goat anti-mouse IgG diluted 1: 1000 in 1% BSA was added to each well. After 1h incubation, plates were washed, 100. mu.l of substrate was added to each well, and OD was measured after 10 to 15 minutes₄₅₀The value is obtained. For testing monkey responses against gp120 antibody, all procedures were identical except for 5% plain goat serum plate and diluted serum in Hank's balanced salt solution.

Synthesis of peptides

The peptides synthesized according to the amino acid sequences 253-276(SEQ ID No. 1: EQLESIINFEKLTEWTSSNVMEER; one standard letter used herein represents each amino acid) of ovalbumin (ova 253-276) and the peptides synthesized according to the amino acid sequences 308-322(18IIIb sequence) of gp120IIIb were assembled by solid phase peptide synthesis using a practical biosystems 430A synthesizer. The hydroxybenzotriazole esters are formed by linking a preformed symmetrical anhydride with additional asparagine, glutamine and arginine. Method for coupling efficiency reference Kaister et al (Biochemical journal34: 595, 1970) are controlled by the ninhydrin reaction. With Tam et al inJournal of the American chemical Association21: 6442, "Low-high" procedure described in 1983, peptides were released from the support with HF and the peptides were extracted from the resin with 10% ethyl acetate. After lyophilization, the peptides were desalted on a Sephadex G-25 column, and then the sample peptides were subjected to a Vydame preparative C-18 column and HPLC purification by reverse phase chromatography. The purified peptide (98%) was dissolved in HBSS to a final concentration of 1mg/ml and diluted to the desired concentration in complete medium.

Cyanogen bromide disinfectantTransforming

A protein sample (e.g.. beta. -galactosidase) was treated with 100-fold molar excess of cyanogen bromide in 100mM trifluoroacetic acid. The reaction can be carried out for more than 18 hours at room temperature (about 20 ℃) with stirring. After the completion of the reaction, the peptide fragment was separated from the reaction product by using SEP-PAK C-18 instrument (Waters), eluted with 95% acetonitrile, and lyophilized.

Alkaline digestion

Protein samples (e.g.,. beta. -galactosidase) were treated with 1N NaOH, boiled for 2 minutes, and the resulting peptide fragments were separated using a C-18 CEP-PAK instrument (Wasters), eluted with 95% acetonitrile and freeze-dried.Example 1: priming of CTL restricted by MHC class I molecules

Moone et al, UCLA Symp. mol.cell.biol.113, 1989 and Carbone and BevamJournal for experimental medicine171: 377, 1990. It was indicated that immunization of mice with splenocytes harboring soluble ova in the cytoplasm induced ova-specific class I molecule-restricted CTL responses. With El expressing ova₄Transfectant EG₇Ova stimulates splenic lymphocytes already in vivo contacted with antigen in vitro and serves as target for ova-specific CTL mediated killing. This study also demonstrated that EG₇Ova transfectants or ova-loaded splenocytes induced CD₈ ⁺Effector recognition genetic background is H-2K^bPeptide ova-258-276 cleavage of EG₇Ova and killing EL with ova-258-276₄A cell. Therefore, to determine whether soluble antigens can induce endogenous class I molecule-restricted CD₈ ⁺T cell pathway, using the above system to detect whether an antigen preparation can bring soluble antigen into a class I molecule-restricted pathway.

a)ova

C57BL/C mice were immunized once subcutaneously or by tail root injection with different amounts of ova (30. mu.g-1 mg per mouse) with or without antigen preparations. After at least 2 weeks of immunization, splenocytes from the immunized mice are taken and usedEG₇In vitro stimulation of ova transfectants. ova was effective at a low concentration of 30. mu.g as well as 1 mg. Therefore, 30. mu.g of ova immunized mouse spleen cells, together with EG, were commonly used in CTL studies₇EG lysis by energy after 5 days of in vitro culture of ova₇-presence of ova-specific effectors of ova to assess their inducibility.

Mice were injected with up to 1mg of soluble antigen in HBSS and no CTL priming occurred. (FIG. 1A). However, immunization of mice with 30 μ g ova dissolved in the above antigen preparation (indicated as AF in the figure) resulted in a significant transfectant-specific CTL response (FIG. 1C). Also, Eg was generated by ova-AF-immunized splenocytes₇The extent of killing of ova was compared to that of splenocytes from mice immunized with ova-loaded splenocytes (FIG. 1B).

When using EG₇Spleen lymphocytes from mice immunized with β -galactosidase did not show a second CTL response upon in vitro stimulation, indicating that the specificity of CTL initiation in vivo was Ag-specific. No discovery was madeovaOva-specific CTLs were induced.

b) Beta-galactosidase enzyme

Similar results were obtained with the other soluble antigen, β -gal. To determine the response of β -gal specific CTL, the β -gal expressing C3-4 transfectant from BALB/C was used as target. Immunization of BALB/c with soluble β -gal produced a slight CTL response. Therefore, to detect specific CTL responses, spleen lymphocytes were collected at least up to 8 weeks and cultured for 5 days in the presence of irradiated C3-4 transfectants.

FIG. 2B shows that 30. mu.g β -galactosidase in AF can induce a strong specific CTL response against transfectants. At a 3: 1 effective target ratio (E: T), β -gal-AF immunized mice showed about 80% specific C3-4 killing. However, effectors isolated from β -gal immunized mice solubilized in HBSS killed only 20% of the same target with the same effective target ratio. Because of EL₄And P₈₁₅None express MHC class II gene products and cleavage is homology-limiting, so the specific effects of ova and beta-galThe substance is restricted by MHC class I molecules.

To examine the utility of antigen preparations, mice were immunized with soluble antigens embedded in type 2 liposomes, one of which was a pH sensitive liposome. One week later, splenocytes were stimulated in vitro as described above and treated with⁵¹EG labeled with Cr₇-ova or EL₄And (6) detecting. Representative results presented in figure 3 indicate that ova in liposomes are unable to initiate substantial CTL induction in mice. Similar results were obtained when ova were immunised in alum.Example 2: recognition of epitopes by CTL

The carbon and Bevan materials are,same as aboveConfirmation by EG₇Immunization of C57BL/6 mice with ova transfectants and cytoplasmic ova-loaded spleen cells induced CTL recognition of EL4 cells harboring ova 258-276 epitopes. To examine whether soluble ovalbumin in AF also induced the same CTL response, splenocytes were prepared from immunized mice and EG was used in vitro₇Ova stimulation Effector vs EL coated with ova 258-276 peptide or with control peptide isolated from myelin basic protein (MBP84-102)₄The effect of the cells was examined. The results demonstrate that ova-AF-initiated CTLs have similar effects to those initiated by transfectants or cytoplasmic-borne ova. (FIGS. 1A, 1B and 1C). Eg efficient lysis of ova-AF-initiated effector cells₇Ova and each 10⁸Untransfected EL cells with 50. mu. gova peptide₄Cells, but not lysed, coated with 10. mu.g of MBP peptide/10⁸EL of cells₄A cell.

Carbone and Beran indicated above that CTLs induced by transfectants expressing β -gal in the β -galactosidase system and splenocytes with cytoplasmic soluble β -gal could lyse the transfectants expressing β -gal and P815 cells that were untransfected with β -gal digested with alkali. CTL induction when soluble β -gal was immunized in AF was equally specific. (FIG. 2).Example 3: the CTL effector is CD ₈ ⁺ T cells

The following shows that soluble antigen in AF induces CD₈ ⁺Effector T cells. Spleen obtained from immunized miceThe cells were cultured in vitro with the irradiated transfectants for 5 days. Thereafter, anti-CD for cells was collected₄Or against CD₈And complement of (3) to deplete CD₄ ⁺Or CD₈ ⁺T cells. The depleted population is then paired in the ova system⁵¹Cr-EG₇Detection of ova or in beta-gal systems⁵¹Cr-P_13.1And (6) detecting. The results in FIG. 4 show that the entire effector cell population demonstrates CD in the ova system₈ ⁺Depletion of T cells terminates cytolytic activity, however, CD₄ ⁺Depletion of T cells against EG₇Cleavage of ova had no effect.

Also in the beta-gal system, CD₈ ⁺Depletion of T cells terminates the cytolytic activity of β -gal-antigen preparations in immune spleen cells.Example 4: soluble ova-initiated CD in AF ₈ ⁺ T cells

To demonstrate in vivo CD initiation by ova-AF₈ ⁺T cell population and restricted in secondary response in vitro, CD₄ ⁺Or CD₈ ⁺Cell populations were depleted from splenocytes from ova-AF immunized and normal mice. Subsequently, the thus treated population was isolated in vitro with EG alone₇CD of-ova or mice immunized with ova-AF₄ ⁺And CD₈ ⁺CD of T cells together or with ova-AF immunized mice₄ ⁺Or CD₈ ⁺CD of T cell and normal mouse₄ ⁺Or CD₈ ⁺Different combinations of cells are activated together. FIG. 5 shows a boot CD₈ ⁺Cells are necessary to demonstrate the 2 nd CTL response in vitro. These data also show that CD is required for an effective 2 nd CTL response in vitro₄ ⁺T cells, priming does not require CD₄ ⁺T cells. Similarly, CD is required for the beta-gal specific second CTL response in vitro₈ ⁺T cells.

The above examples demonstrate the effect of an antigenic preparation on the induction of a class I molecule-restricted CTL response by a soluble protein antigen. AntigensThe agent mediates soluble antigen-induced CTL priming and has similar activity as that induced by transfectants and cytoplasmic-laden soluble ova or β -gal splenocytes. In the ovalbumin System, EG₇Ova, splenocytes cytosolic with ova and ova-AF including: (a) class I molecule restricted CD₈ ⁺CTL; (b) CTL identification of targets sensitized by ova 253-276 synthetic peptide; (c) after only one immunization, there were long-lived CTLs. In the beta-galactosidase system, beta-gal-AF-induced CTL recognized the beta-gal expressed transfectant C3-4 and also untransfected P sensitized by alkaline digestion of beta-gal₈₁₅A cell. This is similar to CTL induced by immunization with splenocytes loaded with β -galactosidase in the cytoplasm. The AF-induced ova-specific CTLs are unique in that neither ova encapsulated in pH sensitive liposomes nor in alum can induce CTL priming in vivo.

These examples show that AF and its equivalents used above are useful in human therapy and in the development of CTL vaccines for the induction of various cancers, viral diseases.Example 5：

This is a specific example showing that AF described above induces class I-molecule-restricted CTL initiation in HIV soluble gp 120.

The gp160 IIIB-expressing cell line (15-12) was generated from the Balb/c fibroblast differentiated 3T3 cell line. It was obtained from doctor Ron germanin and Jay Berzofsky, Bethesda, m.d. national institutes of health. The gp160 IIIB-expressing cell line is used to activate splenic lymphocytes that have been contacted with antigen in vivo in vitro and to serve as a target in the induction of gp 160-specific CTLs. Balb/c mice were immunized 1601 times with 10 μ g gp with or without AF and injected plantar and caudal root. Splenocytes from immunized mice were taken 2 weeks after immunization, stimulated in vitro with irradiated gp160 transfectants, and after 5 days of culture, the priming effect was assessed by the presence of a heteroeffector that had the ability to lyse gp160 transfectants without lysing untransfected cell lines. Figure 7 lists the results, with AF and gp120 promoting CTL responses.

The following examples demonstrate antigen formulations using only one or two parts of the inventionThe use of (1). These examples demonstrate that CTL responses can be induced with only two of the above three components.Example 6: determination of the Key Components necessary for the Induction of CTL

In order to examine whether all the above-mentioned components are necessary for the induction of antigen-specific CTL, mice were immunized with ovalbumin of microfluidized preparations of different 2-component combinations among the above-mentioned AF three-components. The 2-component combination used was as follows: the squalane/tween is dissolved in PBS, the squalane/pluronic is dissolved in PBS or the pluronic/tween is dissolved in PBS. Another group included ova immunized mice formulated in a partial system, that is, PBS with squalane, pluronic or tween alone. The three-component antigen preparation was adjusted, one component at a time being excluded and replaced with PBS.

The antigen preparation described above comprises: 0.300g Tween 80(Aldrich, Wis.), 1.875g Pluronic L₁₂₁(BASF, NJ) and 7.5g squalane (Aldrich, Wis.) in 50ml PBS.

The preparation with 2 components is: squalane/tween: 50ml of 0.300g of Tween 80 and 7.5g of squalane in PBS were prepared.

Pluronic/tween: 1.875g Pluronic L₁₂₁And 0.300g Tween 80, in 50ml PBS.

Pluronic/squalane: 1.875g Pluronic L₁₂₁And 7.5g of squalane, in 50ml of PBS.

Then by means of a microfluidics instrument: the samples were processed and stored in aliquots at 4 ℃ for use, model 110T, microfluidics, Inc.

Ovalbumin (Sigma, MO) was weighed out and made up into a 0.3mg/ml solution in PBSS. The stock solution was combined with a 2-component formulation in the following amounts: 5 parts of a 0.3mg/ml ovalbumin solution, 3.3 parts of a 2-component preparation and 1.7 parts of PBSS.

The formulation was stirred on ice until injection and all solutions were combined immediately prior to injection.

Each mouse was injected bilaterally posteriorly into the plantar area with 200. mu.l of the ova 30. mu.l formulation, and the remaining solution was injected subcutaneously into the tail root. Splenocytes were collected 2-4 weeks later.

Splenocytes were prepared 2 weeks after immunization and stimulated in vitro with irradiated EG 7-ova. After 5 days of culture, the content of the culture medium is measured within 4h⁵¹Cr-EG7-ova or⁵¹Cr-EL₄To detect the presence of ova-specific CTLs. The data presented in FIGS. 8-10 demonstrate that ovalbumin preparations in microfluidised 2-component systems can initiate ova-specific CTL in vivo.

We further evaluated the relative role of each component in their ability to induce CTLs when used in combination with protein antigens. In the immune process, soluble antigen is mixed with microfluidized excipient to obtain stable homogeneous emulsion with the particle size of 250-300 μm. To further determine the role played by the squalane-tween 80-pluronic (STP) formulation components in inducing CTLs, we immunized mice with ova in squalane-tween 80(ST) mixture, pluronic-tween 80(PT) mixture or squalane-pluronic (SP) mixture, with ova in squalane (S), tween 80(T) or pluronic (P) as a control (naive mice). Also, ova-SAFm (containing 70. mu.g MDP) or ova-alum was used as adjuvant in control naive mice. As a positive control, mice were immunized with splenocytes cytosolically loaded with soluble ova. Other combinations and substitutions are also possible and the results are shown in Table 1.

To ascertain the CTL initiation process, mice were immunized 1 time. After 2 weeks of immunization, splenocytes were mixed with EG7-ova (OVA-expressing EL4 cells) irradiated with radiation for 5 days, and the pairs were examined⁵¹Cr-EG7-ova or⁵¹Cr EL4 cells. The results (FIG. 11) show that 30 μ g of ova used in combination with STP or ST initiated a class I-restricted CTL response in mice. Ova-specific CTLs initiated by ova in STP or ova in ST induced better than splenocytes harboring soluble ova in the cytoplasm. Ova in PT or SP can induce ova-specific CTL responses in mice but are weak and not persistent, unlike SAFm, the addition of MDP to ST preparations did not disrupt ova-specific CTL induction in mice (table 2). Mice were immunized when ova were mixed with individual components S, P or TOr does not induce ova-specific CTL when mice are immunized with ova-SAFm or ova-alum. Immunization of mice with up to 1mg ova solubilized in (a) HBSS, (b) SAFm or (c) alum-absorbed ova did not initiate ova-specific CTL.

TABLE 2

Non-blocking of ST + MDP induces ova-specific CTL responses

Percent cytotoxicity of immunized mice ^*

ova-ST-MDP ova-ST-MDPIrritant substance Target material ^** E-T ova-ST ova-ST 300 ug mouse 72 μ g miceEG7-ova EG7-ova 100∶1 0 100 82 76

33∶1 0 86 67 62

11∶1 0 33 39 25

3∶1 0 6 13 3

1∶1 0 0 0 0

3∶1 0 0 0 0

^*Mice were immunized with ova in 30 μ g of different formulations

^**Percent cytotoxicity was calculated by subtracting the kill rate on antigen-non-expressing cell lines.Example 7: essential Components for the production of ova-specific antibodies

Mice were immunized 3 times with 30 μ g ova in HBSS, STP, ST, PT or SP every 2 weeks. Mice were also immunized with ova-SAFm as a positive control, since SAFm is known to induce a strong antibody response. 7 days after the 2 nd and 3 rd immunizations, the mice were sacrificed by exsanguination and sera were tested for ova-specific antibody responses. The results are shown in Table 3 and indicate that the same anti-ova response was shown after 2 immunizations of mice with ova dissolved in STP, ST or SAFm.

TABLE 3 Induction of anti-ova antibody response30 μ gova formulation/animal Number of mice reacted/number of mice injected 1/serum dilutionHBSS 0/3 ＜1/20，＜1/20，＜1/20STP 3/3 ＜1/4860，＞1/4860，＜1/4860ST 3/3 ＞1/4860，＞1/4860，＞1/4860PT NA NA NA NASP NA NA NA NASAF-M 3/3 1/4860， 1/4860， 1/4860

^*NA is not obtainedExample 8: induction of HIV gp 120-specific CTL

HIV gp120IIIB was used as a second antigen system to test its CTL-inducing ability in STP, ST or MP-T. Mice were immunized with 1 μ g gp120IIIb in HBSS, STP, PT or ST. With 1. mu.g of SAFm or CFA: (Complete freund's adjuvant) or gp120IIIb immunized mice in RIBI adjuvant system containing MPL (monophosphoryl lipid a) and TDM (trehalose dimycolate) served as controls. Three weeks later, splenocytes were prepared and stimulated in vitro with mitomycin-treated transfectant cells 15-12 or with 18IIIb peptide. After 5 days of culture, the obtained effector cells were tested for the target vaccinia: gp160 IIIB or P of parenteral vaccinia infection₈₁₅The function of the cell. The results showed that the gp 120-squalane-tween 80 formulation induced a specific CTL response in mice, but not the gp 120-squalane-tween 80-pluronic or gp120-HBSS formulation.

TABLE 4 Induction of gp 120-specific CTL responses in mice

Percentage of cytotoxic activity in immunized mice (%) ^* Irritant substance Target material ^** E-T gp120-HBSS gp120-ST gp120-STP18IIIb/IL2 vac∶gp120 100∶1 23 42 NA^***

33∶1 23 38 NA

11∶1 0 0 NA

3∶1 0 35 NA18IIIb/IL2 15-12 100∶1 0 50 0

33∶1 0 35 0

11∶1 0 27 0

3∶1 0 18 018IIIb/IL2 3T3+18IIIb 100∶1 0 59 13

33∶1 0 59 2

11∶1 0 57 0

3∶1 0 29 015-12 vac∶gp120 100∶1 35 84 NA

33∶1 19 65 NA

11∶1 12 37 NA

3∶1 0 22 NA

1∶1 0 0 NA

^*Mice were immunized with 1 μ g gp120III in different formulations

^**Percent cytotoxicity (%)

^***NA; is not obtained.Example 9: induction of gp 120-specific humoral responses in mice

To induce a gp 120-specific humoral response, mice were immunized 3 times with 1 μ g gp120IIIb every 2 week interval. Mice were sacrificed by exsanguination and gp120IIIb was detected by solid phase ELISA to verify the presence of IgG antibodies. The results indicate that gp120-ST is more antigenic than gp120-HBSS, gp120 SAFm (Table 5) or gp 120-STP.

TABLE 5 Induction of anti-gp 120 antibody responseAnimal containing 1 μ g gp120 preparation Number of mice reacted/number of mice injected 1/serum dilutionHBSS 0/3 ＜1/20，＜1/20，＜1/20STP 1/3 ＜1/20，＞1/4860，＜1/20ST 3/3 ＞1/4860，＞1/4860，＞1/4860PT 3/3 ＞1/4860，＞1/4860，＞1/4860SP 2/3 ＜1/20，1/540，1/540Saf-M 2/3 1/180，＞1/4860，1/540Example 10: induction of gp 120-specific antibody response in monkeys

Monkeys were immunized with gp120-SAFm, gp120-STP, gp120-ST or gp120-HBSS (2 per group). A group of monkeys were immunized with gp160 IIIb-containing recombinant vaccinia as a control, immunized at 2-week intervals, and bled 2 and 3 weeks after the 2 nd immunization. Serial dilutions of the stock and immune sera obtained from each monkey were assayed for anti-gp 120 activity by ELISA as described in materials and methods. The data (FIG. 12) show that immunization of mice with gp120-STP or gp120-SAFm induces similar responses. The 1 monkey in the gp120-ST immunization group induced a response similar to that induced by the gp120-SAFm or gp 120-immunization group. Nor did 1 monkey immunized with gp120-ST induce a strong anti-gp 120 response after 2 weeks of immunization.Example 11: activity of AF in combination with HPV16E7 in vivo

1. Production of recombinant HPV16E7 protein for immunization

a) Cloning and PCR of E7 Gene

The HPV16E7 gene was cloned in a plasmid supplied by dr. karen vosden (Ladwing research institute), which encodes the E7 gene from the cancer cell line Caski. The coding region was amplified by PCR using primers encoding the 5 'and 3' ends of the genes flanked by BamHI and Sal I cloning sites. Binding of PCR product of E7 to PGEX-4T₁The expression vector (Pharmacia bioengineering) thus formed the PGEX · E7 expression plasmid. Coli line XL transfected with the plasmid₁Blue (Stratagene). The E7 sequence obtained from the cloning plasmid was identical to the E7 sequence obtained from Caski cells.

b) Production and purification of bacterially expressed E7

PGEX. E7A bacterial expression plasmid encodes a glutathione-S-transferase (GST) fusion protein containing GST at the amino terminus and an E7 protein and a fibrinolytic site at the carboxy terminus. Purified E7 protein was prepared according to the manufacturer's instructions for PGEX-4T-1 vector (Pharmacia Biotech). isopropyl-b-D-thiogalactosidase is added into a culture medium to induce bacteria containing PGEX & E7 expression plasmids to express the fusion protein. The cells were lysed with gentle sonication and harvested, the lysate was passed through a glutathione Sepharose 4B column (Pharmacia bioengineering), after binding of the fusion protein to the matrix, the resin was washed to remove non-specifically bound protein, and the bound fusion protein was digested with thrombin to release the E7 protein from the GST fusion moiety.

The E7 protein preparation was analyzed by SDS-PAGE and the concentration of E7 protein was determined by Bradford method (BioRad) to obtain 9mg of soluble E7 protein per 1L of bacterial culture.

Formation of X21E 7 transfectants

Insertion of the coding sequence of the HPV16E7 protein into the IDEL suitable eukaryotic expression plasmid INPEP₄In (1). Expression of E7 in this vector is controlled by the cytomegalovirus promoter/enhancer transcription element. In addition, the first 3 nucleotides of the coding sequence for E7 were removed and replaced by an immunoglobulin light chain leader sequenceInstead, the monomer G was detected by Northern blotting after transfection of the mouse cell line X21, placed directly upstream, in frame with the E7 coding region₄₁₈Resistant clones were used for the E7 messenger product. Each clone showed a detectable E7 message. The generation of E7 protein was then confirmed by Nestern blot assays of cell lysates of clones 4E7 and 1C 72.

In vivo Activity of E7/AF soluble antigen

C3H female mice (H2) were used in this study^k/kHarlan Spnague Dawley). Animals were kept in a book according to "Instructions for Care and application for laboratory animals" (DHHS Press No. NIH 86-23, Bethesda, MD: NIH, 1985), with food and water ad libitum. The E7 transfectant cell line HOPE was used in this study₂(H2^k/k). Tumor cell lines were cultured by serial passage in vitro.

Western blot detection and subsequent in vitro passaging repeat showed that the cell line maintained the expression of the cytoplasmic antigen of E7. Homologous C3H mice were injected subcutaneously with 15000 in vitro passaged cells to initiate tumors.

Tumors were measured in two perpendicular directions over 2 weeks. Tumor volume can be calculated according to the following formula:

V(mm³)＝(L×W²)/2

l is the longest axis, measured in millimeters

W is vertical axis (mm)

The data in table 6 represent tumor mice (number of tumor bearing mice/number of all mice receiving injections). The data in FIGS. 13 and 14 represent mean tumor size (mm) for each treatment or control group³). Each treatment group was compared to an untreated control group. HOPE injection₂After 10 days of cell culture, when most tumors are accessible (about 50-75 mm)³) And (6) carrying out treatment. Treatment was initiated by immunizing mice with soluble E7 protein in AF or alum adjuvant (subcutaneous, total volume 0.2 ml). Immediately before immunization, AF and E7 proteins were in Hank's planeThe balanced salt solution (HBSS) was mixed for 60 seconds, so that each mouse received 0.2ml of E7 protein 30. mu.g or 90. mu.g. This was mixed with E7 protein according to manufacturer's instructions A1(Pieme Chemicals Co.) so that each mouse received 0.2ml of 90. mu.g of E7 protein. Animals in the 2 nd treatment group received the 2 nd immunization 9 days later (19 days after tumor inoculation). As previously described, booster immunizations were prepared immediately prior to vaccination.

In this example (Table 6: Xp #233), mice (30. mu.g or 90. mu.g each) that received 71 injections of soluble E dissolved in AF had only 4/8 and 5/8 measurable tumors 41 days after tumor cell inoculation. In contrast, all mice immunized with alum soluble E7 protein (8/8) developed motility. Progressive tumors. In addition, as shown in fig. 13, significant tumor growth inhibition was observed only in the treatment group immunized with E7 protein dissolved in AF, compared to the control group (untreated) or alum-treated group. No inhibition of tumor growth (figure 13) or an increase in tumor inhibition rate (table 6) was found in mice receiving 1 injection of E7 in alum.

Although some impaired tumor growth was observed in mice receiving 2 injections of E7 in alum, similar results were observed in treatment groups receiving 2 immunizations on days 10 and 19 post-tumor challenge (FIG. 14 and Table 6).

The results show that, as calculated from the reduction of the number of tumor-bearing mice and the inhibition of tumors, with the immunization of soluble E7 in AF, a significant antitumor activity was observed. In contrast, all animals immunized 1 or 2 times with alum soluble E7 protein had progressive tumor growth. In summary, immunization of mice with soluble E7 protein dissolved in AF significantly inhibited tumor cell growth, whereas immunization with soluble E7 dissolved in alum did not.

Claims (9)

1. A composition comprising a papillomavirus antigen mixed with a microfluidised antigen preparation comprising:

(a) a stabilizing detergent for a detergent composition,

(b) a micelle-forming agent, and

(c) a biodegradable and compatible oil that is capable of forming,

said antigen preparation is formulated as a stable oil-in-water emulsion, substantially free of immunostimulatory peptides and said composition is capable of inducing a specific cytotoxic T lymphocyte response to an antigen contained in the composition when administered to an animal selected from the group consisting of a human, a domestic animal and an agricultural animal.

2. The composition of claim 1, wherein the papillomavirus antigen is a human papillomavirus antigen.

3. Use of the composition of claim 2 in the manufacture of a medicament for the treatment of cervical cancer.

4. Use of a composition according to claim 2 for the preparation of a medicament for the treatment of condyloma acuminatum.

5. The composition of claim 1, wherein said antigen is selected from the group consisting of an HPV16E 6 antigen, an HPV16E7 antigen, an HPV 18E 6 antigen, an HPV 18E 7 antigen, an HPV 6E 4 antigen, an HPV 11E 4 antigen, and an HPV 11L 1 antigen.

6. The composition of claim 1, wherein the detergent is selected from polysorbate 80, tween 20, tween 40, tween 60, amphoteric detergent 3-12, tebol HB7, and Span 85.

7. The composition of claim 1 wherein said micelle-forming agent is selected from the group consisting of poloxamer 401, pluronic L62LF, pluronic L101, pluronic L64, polyethylene glycol 1000, quaternary ketone 1501, quaternary ketone 150R1, quaternary ketone 701, quaternary ketone 901, quaternary ketone 1301, and quaternary ketone 130R 1.

8. The composition of claim 1, wherein the oil is selected from the group consisting of squalene, squalane, eicosane, tetratetracontane, squalane, glycerol, and vegetable oils.

9. The composition of claim 1, comprising squalane, polysorbate 80, polaxamer 401.