Abstract:
There are provided anti-tumor vaccines which contain as active ingredient tumor cells which have been pressure treated so as to augment their antigenic properties, tumor cells treated with cholesteryl hemisuccinate (CHS) and subsequently pressure treated, or plasma membranes from either of, or membrane proteins shed from either of these cells, or a combination of any of these. According to another embodiment, tumor cells are treated with cholesteryl hemisuccinate or by the application and release of pressure, and subsequently with a cross-linking agent. Such cells, plasma membranes obtained from these and proteins shed from the surface of these are effective active ingredients in anti-tumor vaccines.

Description:
This application is a continuation of application Ser. No. 803,556, filed Dec. 2, 1985, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to anti-tumor vaccines for use in human and veterinary medicine. The active ingredient of such vaccines are tumor cells the antigenicity of which has been increased by certain treatments, plasma membranes obtained from such treated cells, or proteins shed from the surface of such treated cells. The invention further relates to the production of such vaccines and to methods of treatments based on the use of such vaccines. 
     Experiments have demonstrated that EL-4 T-leukemia cells treated in vitro as set out above, plasma membranes obtained from such treated cells, or proteins shed form the surface of such cells or membranes can be used as active ingredient in vaccines which effectively immunize laboratory animals against the subsequent challenge by such tumor cells. 
     BACKGROUND OF THE INVENTION 
     Cancer vaccines made from autologous cell preparations are based on the well established fact that tumor cells bear specific neo-antigens. The immunogenicity of cancer cells is too weak to elicit a pronounced immune reaction capable of coping with tumor development in cancer patients. Also artificial coupling of such tumor cells with strong antigens failed to give adequate results. 
     Since 1976 the &#34;vertical displacement&#34; approach, Borochov et Shinitzky, Proc. Nat. Acad. Sci., U.S.A. 73, 4526-30 (1976) has been used to enhance the immunogenicity of tumor cells. Increase of membrane lipid microvisocosity results in an exposure of latent antigens which render the system more immunogenic. It has been shown that rigidification of the membrane by incorporation of cholesteryl hemisuccinate (CHS) enhances the exposure of the effective antigens and that such cells can be used with a greater degree of success for immunization of mice against certain tumors, Shinitzky et al., Proc. Natl. Acad. Sci. U.S.A. 76 5313-5316 (1979). This provides a potential anti-tumor vaccine. 
     The clinical potential of these findings was examined by delayed type hypersensitivity reaction (skin-test) in patients with solid tumors against their autologous irradiated tumor cells. Only upon enrichment with CHS a strong response could be detected, Skornick et al. Cancer Immunolo. Immunother. 11, 93-96 (1981). 
     The vertical displacement of proteins was used to shed off blood group antigens of human erythrocytes, Muller and Shinitzky, Exp. Cell Res. 136, 53-62 (1981). This illustrates that by the combination of CHS and hydrostatic pressure it is possible to isolate surface proteins without the use of detergents which are undesired as they tend to denature such proteins. 
     SUMMARY OF THE INVENTION 
     There are provided effective vaccines for use in human and veterinary medicine. The anti-tumor vaccines contain as active ingredient certain specific types of tumor cells which have been treated so as to augment their antigenicity, or plasma membranes obtained from such treated cells, or tumor antigens shed from the surface of such treated cells (proteins shed from such surfaces of treated cells). There is provided a process for augmenting the antigenicity of tumor cells by the application and release of relatively high pressures. There is further provided a process for chemically treating such cells (as for example by means of cholesteryl hemisuccinate and the subsequent pressure treatment); there is also provided a process for augmenting the antigenicity of such pressure treated or cholesteryl hemisuccinate treated cells, by means of a suitable cross-linking agent (formaldehyde or suitable bifunctional agent). There is further provided a process for isolating certain surface constituents from such treated cells: these may be plasma membranes or tumor antigen associated proteins. Any of these can be used as active ingredient in a suitable carrier to provide effective anti-tumor vaccines. 
     The methods of the present invention essentially maintain the biological activity of the cells or of their constituents used for such vaccines. The immunogenicity is essentially maintained and thus the resulting vaccines can be used for the immunization and also for the treatment of patients afflicted with certain types of tumor. 
     The cross-linking is advantageously effected by treating in vitro suitable tumor cells with a chemical cross-linking agent in a suitable medium, such as PBS buffer. Advantageous results were obtained by treating cells, or cells pretreated with cholesteryl hemisuccinate, in a buffer solution (PBS) containing about 2 per cent formaldehyde or 2 per cent glutaraldehyde for about 1 h at room temperature. Under such conditions chemical cross-linking results in a certain fixing of the antigenic display on the cell surface, augmenting the antigenicity of such surface layer. Cells thus treated, plasma membranes obtained from such cells, or proteins shed from such cells have advantageous antigenic properties when used in vaccine form. 
     It has been established that tumor cells of certain laboratory animals, and especially mice, when treated as set out above, have a substantially augmented antigenicity against such tumor cells. Such cells, and the plasma membranes and surface proteins shed from the surface of such cells (treated as set out above), are effective ingredients in vaccines. The invention is of special applicability to solid tumors. Experiments have shown that when administered prior to the challenge with viable tumor cells or shortly after the application of such cells, as those used for vaccine preparation, the incidence of tumors is greatly reduced, and survival after challenge is prolonged. The rate of tumor growth and of metastatic dissemination are reduced. 
     Preliminary experiments with tumor cells and of immunogens shed from human solid tumors treated with cross-linking agents demonstrate that they have an effectivity in anti-cancer vaccination and that they have an effectivity in allogeneic treatment according to the type and stage of the tumor. 
     There have been carried out tests with treated cells and with proteins shed from tumor cell plasma membranes either by a combination of CHS treatment and hydrostatic pressure or by hydrostatic pressure alone followed by cross-linking. Experimental tumors tested were the T-leukemia EL-4 in C57B1 mice and chemically induced carcinoma LSP-1 in hamsters. 
     The cells and immunogens shed from these two types of tumor cells were found to be immunogenic as evident by a substantial delay in tumor development when immunization with such proteins was given prior to tumor implantation. 
     Experiments were carried out with human patients with tumor cells treated with a cross-linking agent and with immunogens shed from their CHS-pressure or pressure alone treated tumor cells and with plasma membranes of these. Skin reaction tests were made with 9 patients who displayed only a weak reaction with CHS treated cells, and the results indicate that precipitated membrane fragments or protein aggregates or soluble membrane proteins have an immunogenic potential stronger than that of CHS treated cells. The results are summarized in Table 2. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Either hydrostatic pressure alone or a combination of treatment of the cells with CHS and application of hydrostatic pressure were used to obtain cells with augmented antigenicity or membrane proteins or plasma membranes which were used as active ingredient in the vaccines of the invention. 
     Most of the proteins thus obtained stay in solution as soluble proteins, presumably through support of bound phospholipids which are shed off simultaneously. The shedding off seems to result from vertical or lateral displacement of the freely diffusible protein upon increase of the lipid microviscosity. In practice, this is brought about by pretreatment with CHS, to moderately rigidify the plasma membrane lipid layer, followed by hyperrigidification with hydrostatic pressure, followed by further treatment with a cross-linking agent. 
     The procedure used is outlined in the following. For CHS incorporation a sterile medium (&#34;PVP medium&#34;) consisting of 3.5% polyvinyl pyrrolidone, PVP (40.000 MW), 1% bovine serum albumin and 0.5% glucose in phosphate buffered saline, PBS, is used. An ethanolic solution of 5 mg/ml CHS is diluted 1:50 or 1:100 into the PVP medium to form homogenous dispersion of 50 or 100 μg/ml CHS. Tumor cells are incubated in the PVP-CHS dispersion (10 6  cells per ml) at room temperature for up to 90 min. The incorporation of CHS, and therefore the increase in the plasma membrane microviscosity, is approximately linear with incubation time up to 90 min. Attention is given to the possibility of shedding of tumor immunogens into the medium during the longer incubation periods. 
     Pressure (French Press) is applied as previously described, Muller C. P. and Shinitzky M. Exp. Cell Res. 136, 53-62(1981). The CHS enriched cells or cells without any pretreatment are washed and dispersed in sterile PBS as a dense dispersion (˜10 8  cells per ml) in a capped Eppendorff plastic tube (˜1.5 ml) filled to the top. A short needle stuck through the cap serves as a vent for pressure equalization. Both the tube and the needle are filled with PBS without any air bubbles to avoid cell rupture. The tubes (up to 4) are then placed in a 40 ml pressure bomb (Aminco) filled with PBS and sealed. Pressure is applied gradually to reach after 15 min the maximum (up to 1500 atm.) and after 30-60 min it is slowly released (˜15 min). Under such conditions the viability of most cells is preserved (˜80%), though the morphology of the cells can be markedly distorted, presumably due to loss of membrane material. The pressurized cells are centrifuged and both the supernatant and the cells are collected. The supernatant is then ultracentrifuged for 1 hr at 110.000 g to separate between shed membrane fragments or protein aggregates (&#34;shed fragments&#34;) which constitute the precipitate and soluble monomeric or oligomeric proteins (&#34;shed soluble proteins&#34;) which remain in the supernatant. When required, further fractionation of the shed soluble proteins is carried out by gel filtration. The pressure treated cells and plasma membranes of these are also effective in vaccines. 
     The above described method for producing tumor cells of increased antigenicity and for isolation of tumor immunogens (as well as other membrane proteins like receptors and enzymes) as well as plasma membranes has important advantages over the conventional methods for isolation of membrane proteins which are based on membrane disintegration with detergents. In the latter, the detergent has an adverse denaturation effect on the isolated proteins and its subsequent separation is difficult and not always complete. In the novel method the cells and the isolated proteins retain their natural structure and activity presumably because of the strong association with a small amount of membrane phospholipids which prevent their aggregation. The shed immunogens can be obtained in a relatively large quantity within only a short period of time (hours). The pressurized cells treated with cross-linking agents, with or without irradiation, contain activated antigens and can be used for vaccination. A further improved antigenicity is obtained by chemical cross-linking. 
     HUMAN STUDIES 
     Solid tumors, mostly gastric carcinoma, bowel carcinoma and malignant melanoma, are used. Tumor cells are isolated from the viable portion of the growth immediately after surgery by mechanical means only. Cells are treated with CHS and pressure as described above or by pressure alone. The immunogens are prepared at concentrations required for skin tests (10 6  cells, or the material shed from them, in 0.2 ml sterile PBS) or for treatment [(2-5) 10 7  cell, or the material shed from them in 1 ml sterile PBS]. Decision on which immunogen to use and the timing of the skin test or treatment are made according to the availability of material and the clinical condition of the patient. 
     EXAMPLE 
     Immunization Against the T-Leukemia EL-4 
     EL-4 cells (10 8  in 1 ml phosphate buffered saline, PBS) were subjected to hydrostatic pressure of 1500 atmospheres for 90 min at room temperature. Cells were separated by centrifugation and the supernatant was divided to fractions of 0.1 ml each used as a unit vaccine per mouse. Pressure treated cells were chemically cross-linked with 2% glutaraldehyde. Groups of ten C57B1 mice were treated as follows: 
     Group I--each animal was injected twice (7 days apart) with irradiated 10 7  EL-4 cells in 0.1 ml PBS. (Control). 
     Group II--each animal was injected twice (7 days apart) with a unit of soluble vaccine described above. 
     Group III--each animal was injected twice (7 days apart) with 10 7  cells after pressurization at 1200 atm. for 15 minutes and irradiation. 
     Group IV--isolated plasma membranes after chemical cross-linking of 10 7  EL-4, treatment: 1200 atm. for 15 min (2% glutaraldehyde). 
     Group V--cells as in Group IV chemically cross-linked and pressure of 1500 atm. for 90 minutes. 
     Group VI--control: each animal injected twice (7 days apart) with 0.1 ml of PBS. 
     Seven days after the second injection the treated mice were challenged with 10 5  viable EL-4 cells injected intraperitoneally and survival time after this challange was determined. The mean survival time of each group (of 10 animals) was determined and this is presented in Table 1. 
     
                       TABLE 1______________________________________                  Survival time (days)Vaccine                after challenge______________________________________Group I irradiated         30   cellsGroup II   soluble            45   antigensGroup III   pressurized irradiated cells                      90Group IV   membranes from cross-linking                      90   agent treated cellsGroup V Surface proteins shed from                      70   Group IV cellsGroup VI   Control            21______________________________________ 
    
     These results clearly demonstrate the immunogenic capacity of the proteins shed from EL-4 cells by hydrostatic pressure, as well as that of the pressurized cells and of their isolated plasma membranes. Each experimental group consisted of 10 animals. 
     
                                           TABLE 2__________________________________________________________________________SKIN REACTIONS 36 HOURS AFTER INTRADERMAL INJECTIONOF AUTOLOGOUS TUMOR MATERIAL(*)Subject                    Skin reaction(**)(sex, age)   Tumor              1  2 3 4   5   PPD test__________________________________________________________________________  B. M. Metastatic malignant                induration                      ±                         ±                           ±                             -   -   ±  male, 73   melanoma     erythema                      ++ + + ++  +  A. E. Primary breast carcinoma                induration                      -  ±                           - -   -   +  female, 56         erythema                      +  + ±                             +   -  A. S. Metastatic breast                induration                      -  + ±                             +   ±                                     +  female, 62   carcinoma    erythema                      ±                         + ±                             ++  ++  A. G. Primary carcinoma of the                induration                         ±                           ±                             +   ±                                     +  male, 61   sigma        erythema + + ++  ++  H. I. Local recurrence of breast                induration ±                             ++  +++ +  female, 30   carcinoma    erythema   ±                             +++ +++  L. B. Carcinoma of the                induration                      +  ±                           + +   +   +  male, 72   stomach      erythema                      +  ±                           + +   ++  R. A. Local recurrence of breast                induration                      -  ±                           - ++  +   +  female, 74   carcinoma    erythema                      ±                         - ±                             ++  +++  G. Y. Primary colon carcinoma                induration                      ±                         + - ++  -   +  female, 62         erythema                      ±                         + ±                             ++  +  B. Y. Hairy cell leukemia                induration                      -  - - -   -   -  male, 68           erythema                      -  - - -   -__________________________________________________________________________ (*) patients with weak skin reaction to CHS treated cells. (**) 1. 10.sup.6 untreated tumor cells in 0.2 ml phosphate buffered saline (PBS). 2. 10.sup.6 CHS enriched cells in 0.2 ml PBS. 3. 10.sup.6 CHS enriched cells in 0.2 ml PBS after application of hydrostatic pressure (900 atm. 15 min at 4°). 4. Material precipitated after 20 min centrifugation at 100.000 g of the supernatant from 3 (suspended in PBS). 5. The supernatant of 4. + = &lt;2 cm erythema diameter, &lt;0.5 cm induration; ++ = 2 cm diameter; 0.5-1 cm induration; +++ = &gt;2 cm diameter; &gt;1 cm induration.