Induction of Antitumor Immunity by Tumor Cells Treated with Abrin

[CANCER RESEARCH 42, 2872-2876, July 1982] 0008-5472/82/0042-OOOOS02.00 Induction of Antitumor Immunity by Tumor Cells Treated with Abrin

Hiroshi Shionoya,' Haruyoshi Arai, Nozomu Koyanagi, Shinzaburo Ohtake, Hiroshi Kobayashi, Takao Kodama, Hiroyuki Kato, Ta-Chen Tung, and Jung-Yaw Lin

Tsukuba Research Laboratories, Eisai Co., Ltd. 1-3, Tokaidai 5-chome, Toyosato-machi, Tsukuba-gun 300-26 [H. S., H. A., N. K., S. O.], and Laboratory of Pathology, Cancer Institute, Hokkaido University School of Medicine, Sapporo 060 [H. K., T. K., H. K.], Japan, and Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China [T. C. T., J. Y. L.]

ABSTRACT (1). This tumor grows s.c. with a LTD50 dose of 104 cells. The Meth-A tumor is maintained by serial passage in BALB/c mice. A Sarcoma 180 Abrin is known as a cytotoxic lectin. Immunization with Meth- ascites tumor is maintained in ICR mice. A tumor cells which were treated in vitro with abrin induced a Abrin. The abrin used in the present investigation was abrin C, one strong antitumor immunity in syngeneic BALB/c mice. The of the 4 isotoxins that are present in the seeds of A. precatorius. Abrin immunizing effect was stronger than that produced by an C has a weak affinity for binding to the galactose present in the 4 irradiated Meth-A tumor cell vaccine. Studies on the mecha isotoxins. The 50% lethal dose in mice, which was calculated 14 days nisms of the immunizing effect with the abrin-treated tumor after i.p. injection, was 15 fig/kg. The procedure used for the isolation of the abrin was as follows. Jequirity bean kernels (200 g) were allowed cells demonstrated that abrin acts as an immunoadjuvant. to swell in 1 liter of 5% acetic acid overnight at 4Â°and homogenized. Furthermore, the regression of a growing Meth-A tumor was The homogenate was then centrifuged at 10,000 x g for 20 min, and observed after abrin was injected into the tumor, while the the supernatant was fractionated with ammonium sulfate. The proteins induction of a strong antitumor immunity also occurred. It which precipitated between 35 and 95% saturation of ammonium appears, therefore, that the antitumor effects of abrin are sulfate were collected and dialyzed for 36 hr against 5 mM phosphate attributable to two kinds of activity: cytotoxicity and adjuvant buffer, pH 8.0, containing 1 mM EDTA. After dialysis, the extracts were activity. centrifuged, and the supernatants were applied to a Sepharose 4B affinity column (3 x 45 cm) preequilibrated with 5 mw phosphate buffer, pH 8.0, containing 0.2 M NaCI. The column was washed with the INTRODUCTION phosphate-buffered saline to elute those proteins which were nontoxic (the first peak) and those proteins which were toxic but weakly ad Abrin is known as a highly toxic, galactose-binding lectin sorbed on the column (the second peak). The proteins of the second (12, 18, 26). The antitumor activity of abrin in mice (2, 10, 14) peak were further purified by applying them to a DEAE-cellulose and in humans (3, 25) has been reported previously. column (2X15 cm) equilibrated with a 0.01 M Tris buffer, pH 8.6. Abrin is a glycoprotein with a molecular weight of 65,000 Two major peaks were eluted with a linear gradient elution of NaCI (0 isolated from the jequirity bean, the seeds of Abrus precatorius to 0.5 M) in 0.01 M Tris buffer, pH 8.6. The peak that was eluted at a L Abrin consists of 2 polypeptide chains, A and B, with higher NaCI concentration and that contained 35 mg of protein was the molecular weights of 30,000 and 35,000, respectively. The 2 preparation of abrin used in the present study. This preparation was polypeptide chains are linked to each other by an Sâ€”S bond. revealed in our experiments as a single protein band of sodium dodecyl Abrin binds to receptors containing galactose on the cell sur sulfate:polyacrylamide gel electrophoresis, with a consequent 95% purity of abrin. The concentration of the abrin solution was determined face through the B chain of the molecule. It penetrates into the by using the extinction coefficient (E^aonm)for abrin of 15.9 (19). cytoplasm and inhibits protein synthesis through the A chain Preparation of Tumor Cell Vaccine Treated with Abrin. The treat which inactivates the ribosomes of the cell, thus causing cell ment of the tumor cell in vitro with abrin abrogated tumorigenicity and, death (11, 13, 16). as a result, made it possible to use the tumor cells as a tumor vaccine. We found that tumor cells treated in vitro with abrin under The quantitative study on the decrease in the tumorigenicity of the suitable conditions lost their tumorigenicity and became avail Meth-A tumor cell after the incubation in vitro with abrin at 37Â°for 60 able for use as a potent tumor vaccine; we also found that the min was assessed by means of the estimation of the LTD50 of the tumor i.t.2 injection of abrin resulted in the regression of the tumor cell when the cell was transplanted i.p. into immunosuppressed BALB/ and induced strong antitumor immunity. c mice. The percentage ratios of the LTD5o of the untreated tumor cell to that of the abrin-treated one O.e., percentage of viability) were less The effect of abrin is compared with the effect of irradiation than 6, 0.2, and 0.06% at abrin concentrations of 0.1, 1, and 10 /ig/ and BCG as used for the same purpose, and the mechanism of ml, respectively. Accordingly, the procedure used for the preparation the antitumor adjuvant activity of abrin is discussed. of abrin-treated tumor cell vaccine was as follows. Ascites tumor cells obtained from mice which were inoculated with the tumor 7 days MATERIALS AND METHODS previously were washed 3 times with MEM (Nissui Pharmaceutical Co., Tokyo, Japan) and adjusted to a concentration of 1 x 108 cells/ml in Mice and Tumor Cells. Female BALB/c, BALB/c x DBA/2 F, MEM. To 1 ml of the suspension were added 1 ml of 6 /ig abrin per ml (hereafter called CD2F,), and ICR mice, 8 to 12 weeks old, were in MEM, 10 fil of 0.2 M glutamine, and 100 fil of 0.3 M N-2-hydroxy- supplied by Charles River Japan, Inc., Kanagawa, Japan. The Meth-A ethylpiperazine-A/'-ethanesulfonic acid; the mixture was incubated at ascites tumor is a methylcholanthrene-induced BALB/c fibrosarcoma 37Â°for 60 min while being shaken in a water bath. An aliquot of the cell suspension was diluted 50-fold by adding MEM. In this way, the 1To whom requests for reprints should be addressed. abrin-treated tumor cell vaccine containing 1 x 105 cells and 6 ng 2 The abbreviations used are: i.t., intratumoral; BCG, Bacillus Calmette-Guerin; abrin per 0.1 ml was prepared. LTDso, 50% lethal tumorigenic dose; Con A, concanavalin A; BSA, bovine serum albumin; MEM. Eagle's minimal essential medium; PBS, Dulbecco's phosphate- Inactivation of Tumor Cells. To inactivate the tumor cells, the cells were given a 9000-R irradiation from a cobalt source or were treated buffered saline; DTH. delayed-type hypersensitivity; i.d., Â¡ntradermal. Received October 12. 1981 ; accepted April 8, 1982. with glutaraldehyde according to the method reported by Sanderson

2872 CANCER RESEARCH VOL. 42

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1982 American Association for Cancer Research. Antitumor Adjuvant Activity of Abrin and Frost (23); i.e., the cells at a concentration of 5 x 107 cells/ml of RESULTS PBS containing 0.125% glutaraldehyde were incubated at 20Â°for 15 min and washed 3 times with PBS. Induction of Transplantation Resistance by Abrin-treated Con A-bound Tumor Cell Vaccine. A Con A (Sigma Chemical Co., Tumor Cell. The potency of antitumor transplantation resist St. Louis, Mo.)-bound Meth-A tumor cell vaccine was prepared accord ance induced by s.c. inoculation of an abrin-treated Meth-A ing to the methods reported by Martin era/. (15). To irradiated Meth-A tumor cells at a concentration of 2 x 107 cells/ml of PBS, an equal tumor cell was exhibited by means of the LTD50 of Meth-A volume of 50 Â¡igof Con A per ml in PBS was added; the mixture was tumor cells which were used to challenge mice. As shown in incubated at 37Â°for 30 min and washed 3 times with PBS. Table 1, the immunization with the abrin-treated Meth-A tumor Immunization with Tumor Vaccine and Tumor Challenge. Mice cell induced a strong antitumor transplantation resistance. were immunized s.c. with a tumor vaccine at an injection volume of 0.1 LTDso in immunized mice, compared with that of the nonim ml, once or 3 times at weekly intervals on the left side of the dorsum. mune group, increased 100 times after a single immunization Both immunized and normal mice were challenged s.c. with 1 x 103 to and 1000 times after repeated immunizations. 2X10' viable tumor cells on the right side of the dorsum, 2 weeks Effect of Doses of Abrin on Induction of Transplantation after the single immunization or 7 days after the 3 immunizations. The Resistance after Immunization with Abrin-treated Tumor mice were sacrificed 4 weeks after the tumor challenge, and both the Cells. After many trials concerning the induction of transplan tumor incidence and the tumor weight were recorded. tation resistance by abrin-treated tumor cells, it was noticed Winn Assay (27). Spleen cells from normal mice or mice immunized with abrin-treated Meth-A tumor cells were suspended in MEM con that the amount of abrin contained in the vaccine preparation taining 2% normal mouse serum. The spleen cells added to 2 x 106 was of critical importance for the induction of transplantation Meth-A tumor cells/ml were incubated at 37Â°for 30 min, and a 0.1-ml resistance. Therefore, the effect of different amounts of abrin volume of the incubation mixture was injected s.c. into the mice. Three injected into mice was investigated. To suspensions of Meth-A weeks later, the tumor incidence was determined. To deplete the glass- tumor cells of various cell concentrations, abrin was added to adherent cells, 1 x 108 spleen cells in 5 ml of MEM containing 2% the final concentrations of 1 to 2 jug/ml, and the suspensions normal mouse serum were plated in a 90-mm glass dish at 37Â°for 45 were incubated at 37Â°for 60 min. The number of cells in the min under a 5% CO2:95% air incubator. After the incubation, nonad- incubation mixtures were made to vary between 1 x 105 and herent cells were harvested. The process was repeated twice more. 5 X 107 cells/ml so as to fix abrin doses per mouse between Detection of Anti-Meth-A Antibody. For the complement-dependent cytotoxicity test, 0.1 ml containing 5 x 105 Meth-A tumor cells in MEM 0.2 and 200 ng, when the incubation mixtures were diluted to a cell concentration of 1 x 10" cells/0.1 ml. After the incuba was added to 0.1 ml of undiluted serum obtained from mice which were immunized with abrin-treated tumor cell vaccine, and the mixture was tion, the suspensions were diluted with MEM to a concentration incubated at 4Â°for 15 hr in a refrigerator. The cells were then washed of 1 x 10s cells/ml. Thus, abrin-treated tumor cell vaccines with MEM, added to 0.2 ml of 4-fold-diluted guinea pig serum, incu containing a constant cell number and various amounts of abrin bated at 37Â° while being agitated for 45 min, and finally given an were prepared. Mice were immunized s.c. once with a 0.1-ml addition of 0.2 ml of 0.25% trypan blue for viability counts. For indirect volume (1 x 10" cells) of the vaccines. As shown in Table 2, immunofluorescence staining, 5 x 105 Meth-A tumor cells in 0.1 ml of an antitumor transplantation resistance was induced at doses MEM were incubated with 0.1 ml of the undiluted serum being tested of 2 to 20 ng/mouse. On the other hand, high doses of 60 to for antibody. The cells were washed 3 times with MEM, and 0.1 ml of 10-fold-diluted fluorescein isothiocyanate-conjugated anti-mouse IgG 200 ng failed to induce the resistance. Comparison of Transplantation Resistance Induced by (Miles Laboratories, Elkhart, Ind.) was added to the sediments of the tumor cells; the mixed cells were then incubated at 4Â°for 30 min. The Abrin-treated Tumor Cells with That by Other Conventional cells were then washed 3 times with PBS, and the sediments were Tumor Cell Vaccine. The comparison of the immunogenicity resuspended in a small volume of 50% glycerin in PBS. The cells were between the abrin-treated Meth-A tumor cell and various other examined under a Tiyoda microscope with an incident UV illuminator. vaccines which were prepared by hitherto known treating meth In the controls, the test serum was replaced by normal mouse serum. ods was carried out by a determination of the potency of Humoral and Cellular Immune Response to BSA. Four groups of transplantation resistance. The immunogenicity of the abrin- CD2F, mice (6 mice/group) were given s.c. injections of 0.1 ml of 0.15 treated Meth-A tumor cell was, as shown in Table 3, signifi M NaCI containing 10 /ig of BSA (crystallized; Miles Laboratories) with cantly stronger than that of an irradiated tumor cell, the Con A- or without abrin on Day 0. On Day 14, the mice were given the bound tumor cell, or the glutaraldehyde-fixed tumor cell. secondary antigenic stimulation with the same dose of BSA and abrin Increase in Immunogenicity of Irradiated Tumor Cells by as at the primary immunization. Blood samples were removed from the retroorbital plexus on Day 21, and the serum antibody titer against Addition of Abrin. The dose of abrin that was mixed with an irradiated Meth-A tumor cell was adopted as a result of the BSA was then estimated by an agglutination reaction using tanned sheep RBC which were sensitized with BSA. DTH response to BSA findings shown in Table 2. Table 4 shows that the immunogen was assayed according to the procedures described by Robinson ef icity of the irradiated Meth-A tumor cell was significantly in al. (22), namely, mice were anesthetized with Nembutal and were given creased by merely adding as little as 6 ng of abrin to the tumor injections in the ear i.d. of 10 /il of 0.15 M NaCI containing 10 /Â¿gof cell vaccine to be injected. However, when the same amount BSA. Ear thickness before and after the challenge was measured, and of abrin and the tumor vaccine were injected separately into the increase in ear thickness (ear swelling) was calculated. the dorsum of mice, no increase in the immunogenicity of the Antiserum against Abrin. Rabbit anti-abrin antiserum was prepared tumor vaccine was observed. according to the method reported by Olsnes and Pihl (17). Statistical Analysis. The x2 and Fisher's exact probability tests were Specificity of Immunization with Abrin-treated Tumor Cells. To exclude the possibility of nonspecific stimulation of applied to evaluate statistically the tumor incidence between experi the immune system by the abrin-treated tumor cell, BALB/c mental and control groups. The mean difference between the experi mental and control groups was evaluated by Student's t test. LTD50 mice were immunized with either of 2 abrin-treated tumor cell was determined by Finney's probit method or by the method of Beh- lines, Sarcoma 180 and Meth-A, and challenged with each of rens-KÃ¤rber (5). the 2 tumors. The result (Table 5) revealed that the antitumor

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Table 1 Transplantation resistance induced by abrin-treated Meth-A tumor cells /totalNo.Immunization (s.c.) No. of mice with tumor when transplanted s.c.

of abrin- of im treated Meth-A muniza in tumorcells1 tions10"1 1 x 103 1 x X1051/1 X10629/64a X10712/12(100) LTD50(-fold)100 x 105 5a (6.7)* (45.3) 0/10a(0) 8/1 6a (50) 1 x 10s 3 IO710-Increase1000 NonimmuneNo. 0/13(0) 17/30(56.7)1 28/35 (80)1 131/131 (100)1 10/10(100)LTDÂ«,10" p < 0.01 by Fisher's exact probability test as compared with the respective nonimmune group. ' Numbers in parentheses, percentage.

Table 2 Table 6 Effect of various doses of abrin on immunogenicity of Meth-A tumor cells Tumor cell neutralization (Winn assay) with spleen cells obtained from mice immunized with abrin-treated Meth-A tumor cells Dose of abrin (ng/mouse) in abrin-treated tumor cell No. of mice with tumor takeSpleen Tumor vaccine (1 x 10* cells, 1 after 1 x 106s.c. immunization) challenge/total pa (80)"8/11 cells-de 0.20.62e2060200Nonimmune8/10 (77)4/9 cell:target cell pleted fromAbrin-treatedcells obtained (45)6/11 ratio250:1 spleencells0/6 spleencells2/6 (55)4/10 Meth-A immune (40)9/11 mice (1 x 105 cells, s.c., 2 100:1 0/6 0/66/6 (82)10/11 immunizations) 50:1 5/6 (91)19/19(100)NSCNS<0.01<0.01<0.01NSNS Abrin-injected mice 250:1 6/6 NonimmuneEffector 250:1Whole 5/5Adherent 7/7 a Compared with the nonimmune group by Fisher's exact probability test. 6 Numbers in parentheses, percentage. c NS, not significant. Table 7 Decrease in immunogenicity of abrin-treated Meth-A tumor cells by abrin removal Table 3 Transplantation resistance by abrin-treated Meth-A tumor cells in comparison No. of mice with tumor Immunizeds.c. (1 x 105cells, after 1 x 106s.c. chal with that by other conventional immunizations 1 immunization) with lenge Immunized s.c. with Meth-A tumor cells (1 x No. of mice with tumor Abrin-treatedMeth-A+ 105cells, 1 immuniza- after 1 x 106s.c. chal- washing0+ tion) lenge p" antiserum6+anti-abrin serum8Nonimmune9/14(62.5)"18/20(90.0)19/20(95.0)13/20(65.0)20/20(100)<0.01MS"NS<0.005normal rabbit AbrinIrradiation andCon (9000 R) (73.3)22/29(75.9)17/21 a Compared with nonimmune group by Fisher's exact probability test. AIrradiation 0 Numbers in parentheses, percentage. R)GlutaraldehydeNonimmune13/30(43.3)"22/30(9000 c Washed 3 times with 5-fold volumes of fresh MEM. (81.0)29/29(100)<0.05<0.05<0.02<0.000001 d NS, not significant. 8 One ml of abrin-treated Meth-A tumor (5 x 10' cells and 3 /ig of abrin per Compared with the group immunizedwith abrin-treated Meth-A tumor cell by ml, 37Â°,60 min) was added to 0.1 ml of normal or anti-abrin antiserum and X2test or by Fisher's exact probability test. incubated at 4Â°for 30 min. b Numbers in parentheses, percentage. Table 8 Table 4 Adjuvant activity of abrin on humoral and cellular immune response to BSA in Potentiation by abrin to immunogenicity of inactivated Meth-A tumor cells of mice with tu antibody re Immunized s.c. (1 x 10" cells, 1 mor/total after 1 x sponse, Iog2humoral ear swelling (x antibody0)1.5 titer (2" x 1 10~3cm)1 withIrradiatedimmunization) 10" s.c.challenge0/20 withAbrinImmunizeds.c. Â±1.0a" 2.8 Â±1.8Â° (O)6 (1 ng) + BSA cells mixed with abrin (6 13.3 Â±0.5Â° Abrin (10ng) + BSA 5.0 Â±1.266.6 ng) Â±0.4o 27.8 Â±4.4" Irradiated cells and abrin sepa 4/10(40)9/25 Abrin dOOng) + BSA rately BSA alone (control)Humoral 0DTH, 6.4 Â±1.5 Irradiated cells alone (36) Mean Â±S.E. Abrin alone 5/5(100)12/12(100)Pa<0.02<0.003<0.0004 " p < 0.01 by Student's f test as compared with the control. NonimmuneNo. <0.00001 0 p < 0.05 by Student's f test as compared with the control. 1Compared with the group immunized with irradiated cells mixed with abrin by Fisher's exact probability test. " Numbers in parentheses, percentage. resistance which was induced by abrin-treated tumor cells was specific. Table 5 Neutralizing Activity of Spleen Cells Immunized with Abrin- Specificity of immunity induced by abrin-treated tumor cells in BALB/c mice treated Tumor. Spleen cells from mice immunized with abrin- of mice treated Meth-A tumor cells were tested for their neutralizing Immunized s.c. (1 x 10' with tumor/ activity against Meth-A tumor cells by a Winn-type cytotoxicity withAbrin-treatedcells, 1 immunization) withMeth-A, s.c. total5/10 test. As shown in Table 6, the spleen cells from the immunized Meth-A 1 x 106 cells Abrin-treated Meth-A Sarcoma 180, 5 x 106cells 10/10 mice had a neutralizing activity, and the activity was not abol Abrin-treated Sarcoma 180 Meth-A, 1 x 10s cells 10/10 ished by any depletion of glass-adherent cells from the spleen Abrin-treated Sarcoma 180ChallengedSarcoma 180, 5 x 106 cellsNo. 1/10 cells used. Humoral antibody against Meth-A tumor cells in the

2874 CANCER RESEARCH VOL. 42

serum obtained from the immunized mice was not detected by immunoadjuvant, mitomycin C (Sankyo Co., Tokyo, Japan) and a fluorescent antibody technique or by a complement-depend viable BCG (Japan BCG Laboratory, Tokyo, Japan) were used, ent cytotoxic test. Accordingly, taking account of these results respectively. Two-mg/kg doses of mitomycin C (i.e., about 40 along with the specificity of the immunization mentioned above, /Â¿g/mouse),which was the optimal therapeutic dose of the drug it was confirmed that the antitumor resistance induced by in mice, were chosen (21). The dose of BCG was determined immunization with the abrin-treated Meth-A tumor cells was the as 2 x 107 bacteria as a result of the preliminary experiments result of a cell-mediated immunological phenomenon. which showed that 1 x 106 to 1 x 108 bacteria were necessary Lowering of Immunizing Effect by Washing or Adding Anti- to the enhanced induction of antitumor immunity by s.c. injec Abrin Antiserum to Abrin-treated Tumor Cell. Three states of tion of the mixture of inactivated Meth-A tumor cells and BCG. abrin in the abrin-treated tumor cell vaccine may be consid Mice were transplanted s.c. with 2 x 105 Meth-A tumor cells ered: (a) abrin that is irreversibly bound to the cell surface or and given i.t. injections of 15 ng of abrin on Days 1 and 8. The ingested into the cell; (Â£>)abrinthat is on the point of reversible cure rate was recorded on Day 30, and the cured mice were binding to the cell surface; and (c) an abrin molecule that is rechallenged with Meth-A to estimate the degree of acquired free in the vaccine preparation. In order to elucidate the effect antitumor immunity. The results are shown in Table 9. The cure of abrin of the latter 2 states (i.e., free or of reversible binding) rate after i.t. injection of abrin was superior to that of BCG (2 on the Â¡mmunogenicity of the abrin-treated tumor cell, the x 107 bacteria on Days 1 and 15), and the acquired immunity abrin-treated cells were washed or added to anti-abrin antise- in mice cured by abrin was stronger than that in those treated rum; the transplantation resistance in mice immunized with with mitomycin C (40 ng on Days 1 and 8). either of these vaccines was then compared with that of the abrin-treated tumor cell, the original vaccine. DISCUSSION The result (Table 7) revealed that the immunizing effect of Immunization with abrin-treated tumor cells induced a the abrin-treated tumor cell was abolished by washing or by stronger antitumor transplantation immunity than that produced adding anti-abrin antiserum; therefore, the result also indicated by irradiated tumor cells. The fact indicates not only that the that abrin molecules which were free or of reversible binding to tumor cells treated with abrin sufficiently maintained their im the tumor cell in the vaccine preparation were important for the munogenicity but also that abrin induced a higher specific enhanced immunogenicity of the abrin-treated tumor vaccine. antitumor immunity. The present study of the mechanism of the Accordingly, it was suggested that abrin acted as an immu- immunoaugmentative activity of abrin reveals that the molecule noadjuvant. bound to a tumor cell does not necessarily participate in the Immunopotentiative Activity of Abrin on Humoral and Cel augmentation of the immunizing effect by abrin, although the lular Immune Response to BSA. The effect of abrin on anti- molecules participate in the tumor cell killing; it also reveals BSA immune response in mice was investigated to confirm the that free abrin played an important role in the augmentative adjuvant activity of abrin. As shown in Table 8, abrin augmented effect. dose dependently the immune response to BSA in humoral With respect to the augmentative effect of a lectin treatment antibody formation as well as in DTH reaction. The immuno- of tumor cell vaccine, studies on Con A-bound tumor cell potentiative activity of abrin was reduced when it was injected vaccine have been reported (7, 8, 15), and, in these cases, s.c. separately from BSA (data not shown). contrary to the case of the abrin-treated tumor cell, Con A Treatment of Tumor by Intralesional Injection of Abrin and molecules bound to tumor cell were seen to be important for Subsequent Induction of Antitumor Immunity. Because abrin enhancing the Â¡mmunogencity of the vaccine (6). was found to be an immunoadjuvant and is also a toxin, an Data concerning adjuvant activity on immune response to experiment was designed to utilize both of the 2 activities of BSA may be important for 2 reasons, (a) BSA is a simple abrin, adjuvant activity and cytotoxicity, to treat Meth-A tumor protein and contains no carbohydrate residues in the molecule; in vivo. The cytotoxicity of abrin to Meth-A tumor cells in vitro no specific binding of abrin to BSA is therefore concerned in was dependent on a concentration of abrin as described under the adjuvant activity of abrin. Accordingly, it can be concluded "Materials and Methods." Therefore, a higher dose of abrin that the adjuvant activity of abrin depends solely on the inter was recommended for the treatment of the tumor through the action of abrin with the immune system of the host. Data in cytotoxic activity of abrin. However, the dose had to be in the Table 7 could not exclude completely the possibility that abrin range of 2 to 20 ng/mouse, which afforded the antitumor bound to a tumor cell might play some role in the adjuvant adjuvant activity of abrin, as already shown in Table 2. As activity of abrin. (b) If abrin is called an adjuvant, it will be positive controls of abrin as a cytotoxic agent and as an desirable to show evidence that abrin potentiates humoral as

Table 9 Injection of abrin, BCG, and mitomycin C i.t. on s.c.-growing Meth-A tumor and residual antitumor immunity in tumor- free mice of mice with tu x106-2mor/total after 1 oftumor-freemice/total43/57 re-challenge6/38(15.8)5/29(17.2)25/48(52.1)PÂ°NSC<0.01x 10' s.c. TreatedwithAbrinBCGMitomycin (75.4)"29/61 bacteria40,igNo. (47.5)58/58(100)Pa<0.01<0.01No. CDose/mouse15ng2X10' a Compared with abrin-treated group by Fisher s exact probability test or test. " Numbers in parentheses, percentage. 0 NS, not significant.

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well as cellular immune response to such a commonly used versuche. Arch. Exp. Pathol. Pharmakol., (62: 480-483, 1931. 6. Kataoka, T., Oh-hashi, F., and Sakurai, Y. Blastogenic potency of concan- antigen as BSA. avalin-A-bound L1210 leukemic vaccine associated with its immunogenic Features of the adjuvant activity of abrin are: (a) it is effective activity. Gann. 70: 155-164, 1979. in minute (ng) doses; (b) it is effective in a state of water 7. Kataoka, T., Oh-hashi. F.. Tsukagoshi. S., and Sakurai, Y. Induction of solution containing antigen, without making water-in-oil emul resistance to L1210 leukemia in BALB/c x DBA/2Cr F, mice with L1210 cells treated with glutaraldehyde and concanavalin A. Cancer Res., 37: sion; (c) abrin augments antitumor immunity in addition to 964-968, 1977. humoral immunity as well as inducing DTH reaction. Thus, 8. Kataoka, T., Oh-hashi, F., Tsukagoshi, S., and Sakurai, Y. Enhanced induc tion of immune resistance by concanavalin A-bound L1210 vaccine and an abrin, which is a toxic lectin, is also regarded as a lectin that immunopotentiator prepared from Coriolus versicolor. Cancer Res., 37: belongs to a new category of adjuvant active lectins. 4416-4419, 1977. Abrin should be able to bind to various Â¡mmunocytes by its 9. Klassen, L. W., Krakauer, R. S., and Steinberg, A. D. Selective loss of suppressor cell function in New Zealand mice induced by NTA. J. Immunol., specific galactose-binding activity. Its adjuvant activity may be I Â»9:830-837, 1977. related to selective binding of the molecule to certain kinds of 10. Lin, J. Y., Chang, Y. C., Hung, L. Y., and Tung, T. C. The cytotoxic effects of abrin and ricin on Ehrlich ascites tumor cells. Toxicon, )/: 379-381, immunocytes. For instance, it has been reported that suppres 1973. sor cells from the embryonic liver of mice have a high affinity 11. Lin, J. Y., Kao, W. Y., Chen, C. C., and Tung, T. C. Effect of crystalline abrin to peanut agglutinin, a galactose-binding lectin (20); that the on the biosynthesis of protein, RNA, and DNA in experimental tumors. Cancer Res., 30: 2431-2438, 1972. natural thymocytotoxic autoantibody which develops in NZB 12. Lin, J. Y., Lee, T. C., and Tung, T. C. Isolation of antitumor proteins abrin-a mice also specifically exerts its cytotoxicity upon suppressor and abrin-b from Abrus precatorius. Int. J. Peptide Protein Res., 12: T-cells (9, 24); and that the antibody specifically recognizes 311-317, 1978. 13. Lin, J. Y., Pao, C. C., Ju, S. T., and Tung, T. C. Polyribosome disaggregation terminal galactose residues on the cell surface (4). in rat liver following administration of the phytotoxic proteins, abrin and ricin. The most interesting facts observed with regard to the adju Cancer Res., 32. 943-947, 1972. 14. Lin, J. Y.. Tserung, K. Y., Chen, C. C.. Lin, L. T., and Tung, T. C. 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2876 CANCER RESEARCH VOL. 42

Hiroshi Shionoya, Haruyoshi Arai, Nozomu Koyanagi, et al.

Cancer Res 1982;42:2872-2876.

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