Gamma-globulin inhibits tumor spread in mice

Yehuda Shoenfeld and Pnina Fishman1

Research Unit of Autoimmune Diseases, Department of Medicine `B', Sheba Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Tel-Hashomer 52621, Israel
1 Laboratory of Clinical and Tumor Immunology, The Felsenstein Medical Research Center, Rabin Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Petach-Tikva 49100, Israel

Correspondence to: Y. Shoenfeld


    Abstract
 Top
 Abstract
 Introduction
 Methods
 References
 
Intravenous (i.v.) Ig is the human serum Ig fraction that is mainly composed of IgG prepared from plasma pools of over 15,000 healthy blood donors and is suitable for i.v. use. High-dose i.v. Ig is currently used to treat patients with diverse autoimmune conditions. Autoimmunity and malignancy co-exist frequently, and share etiological and pathological mechanisms. Since the two diseases are similarly treated, we studied the efficacy of i.v. Ig as a treatment for malignant conditions. The administration of i.v. Ig to mice inoculated i.v. with melanoma or sarcoma cells induced a statistically significant inhibition of metastatic lung foci and prolongation of survival time. Similar results were seen with SCID mice inoculated with SK-28 human melanoma cells. In a different model, melanoma was induced in the foot pad, followed by leg amputation, after the development of the tumor lesion. A lower number of melanoma recurrences and prolongation of survival time were demonstrated in the i.v. Ig-treated groups. In vitro studies revealed that i.v. Ig was found to stimulate the production of IL-12, an anti-tumor and anti-angiogenic cytokine. Moreover, it enhanced NK cell activity, thus explaining its beneficial effect in SCID mice (which lack B and T but possess NK cells). The results indicate that i.v. Ig acts as an anti-tumor agent. Since it has only minor side effects and is used extensively for other clinical conditions, i.v. Ig may be considered as a potential therapy for the prevention of tumor spread in humans.

Keywords: IL-12, i.v. Ig, melanoma, NK cells, sarcoma


    Introduction
 Top
 Abstract
 Introduction
 Methods
 References
 
High dose {gamma}-globulins were first employed in treating patients with immunodeficiencies (1). Following the observation of an increased number of platelets in a child with Wiscott–Aldrich syndrome treated with i.v. {gamma}-globulin, the compound was used successfully in patients with autoimmune thrombocytopenia [idiopathic thrombocytopenic purpura (ITP)] (2). The positive results in ITP in children and adults (9) prompted the introduction of this therapy in diverse autoimmune states including systemic lupus erythematosus (SLE), dermatopolymyositis, Guillain–Barre' syndrome, multiple sclerosis, rheumatoid arthritis and others (38).

Recently we showed the efficacy of human i.v. Ig as a treatment for the murine experimental model of anti-phospholipid syndrome (APS) and SLE (9,10): i.v. Ig prevented fetal loss in APS and abrogated the clinical manifestations of SLE.

Autoimmune conditions and malignancy co-exist frequently (11,12). Cancer may develop in patients with autoimmune diseases, while autoimmune conditions may follow malignancy. An interesting association is represented by myasthenia gravis with thymoma. Thymectomy in such patients may result in a complete cure of the myasthenia gravis, while a few patients may develop a therapy-resistant SLE (13). Another interesting relationship was reported by us, in which melanoma in mice was treated successfully with autoantibodies derived from patients with vitiligo (14,15).

The cancer–autoimmunity similarities and co-existence led to the implementation of therapies used in cancer for the treatment of autoimmune diseases and vice versa (cytotoxic agents, non-steroidal anti-inflammatory drugs, cytokines etc.).

These similarities led us to challenge the hypothesis that i.v. Ig may help in the treatment of malignant diseases. Indeed, in this study, we demonstrated that i.v. Ig effectively prevented the metastatic spread of melanoma and sarcoma.


    Methods
 Top
 Abstract
 Introduction
 Methods
 References
 
Tumor cells
Tumor cell lines of murine (B-16-F10 melanoma and MCA-105 sarcoma) or human (SK-28 melanoma) origin were used. The SK-28 cell line was purchased from ATCC (Rockville, MD), and the B-16 and MCA-105 cells were kindly donated by Dr E. Keidar (Hebrew University, Jerusalem, Israel). Cells were routinely maintained in RPMI medium containing 10% FCS. The cells were transferred to a freshly prepared medium twice weekly.

Gamma-globulin preparations
Human {gamma}-globulin suitable for i.v. (i.v. Ig) was obtained from each of the following manufacturers: Miles (Biological Products Division, West Heaven, CT), Baxter (Gammagard, New Jersey, NJ), Isiven (Lucca, Italy) and Sandoz (Novartis, Basel, Switzerland).

Experimental animal models
C57BL/6J mice or SCID mice (2–3 months old) were used during the study and were purchased from Harlan Laboratories (Bicester, UK).

To examine the efficacy of i.v. Ig in vivo, several types of tumor models were used.

Inoculation of melanoma or sarcoma cells to naive mice by the i.v. mode. B-16 F10 melanoma or MCA-105 sarcoma cells were i.v. inoculated to 30 C57BL/6J mice. Three weeks later, the mice were sacrificed and lungs removed. In the B-16 model, black metastatic foci were counted using a dissecting microscope. In addition, lung weight was evaluated in some experiments. In the MCA-105 model, lung weight and survival time were measured.

Inoculation of melanoma cells to the footpad. B-16 F10 melanoma cells (2.5x105) were injected to 30 mice to the foot pad where the cancerous cells developed locally. Two weeks later (or when tumor mass reached 1 cm3), the leg was amputated. Tumor recurrence at site of amputation, as well as the survival time, was monitored.

Inoculation of human tumor cells to SCID mice. Thirty SCID mice were inoculated i.v. with 1x106 SK-28 human melanoma cells. Three weeks later the mice were sacrificed and black metastatic foci were counted.

Treatment modalities
Several treatment modalities were applied.

Intravenous administration. Mice were treated by i.v. injection of high dose i.v. Ig (25 mg/mouse, parallel to 400 mg/kg body wt in humans).

In the lung metastatic model, mice were treated on day 0, 7 and 14.

In the leg amputation model, the i.v. Ig was i.v. administered 1 day before, and on day 7 and 14 after amputation.

Subcutaneous (s.c.) administration. Mice were treated with a low dose (25 µg/mouse) of Ig twice weekly, until sacrificed.

Effect of i.v. Ig on IL-12 production
To explore the mechanism by which i.v. Ig prevents tumor metastases, we examined its effect on the production of IL-12, a cytokine which possesses anti-tumor and anti-angiogenic activity. Mononuclear cells were fractionated from heparinized blood of 10 healthy volunteers using a Ficoll-Hypaque gradient. Samples of 5x106 mononuclear cells/ml were preincubated with 250 µg/ml i.v. Ig for 4 h. Then, RPMI containing 10% FCS with and without 0.0075% of the mitogen Staphylococcus aureus Crown (Calbiochem Pansorbin, New Jersey, NJ) was added for an additional 18 h. At the end of the incubation period the supernatant was collected, centrifuged and filtered through a 0.22 µm sterile filter and kept at 70°C until assayed. The level of IL-12 in the supernatants was analyzed using a commercial kit from R & D (Minneapolis, MN).

Effect of i.v. Ig on NK cell activity
The effect of i.v. Ig on the activity of human peripheral blood NK cells was assayed by a standard 4 h 51Cr-release assay using K562 leukemia cells as targets. Peripheral blood mononuclear cells were separated from 10 healthy volunteers using a Ficoll-Hypaque gradient. Cells (5x105) were cultured in 96-well plates and preincubated with 250 µg/ml i.v. Ig for 4 h. Intravenous Ig was then washed out and cells were resuspended in RPMI containing 5% FCS. K562 cells were used as targets and labeled with 100 µCi of Na2[51Cr]O4 at 37°C for 1 h. Cells (1x104) were resuspended and mixed with the effector cells at an E:T ratio of 1:50 in a volume of 200 µl. After 4 h of incubation at 37°C in 5% CO2, plates were centrifuged and supernatants were counted in a {gamma} counter (LKB, New Jersey, NJ). NK cytotoxicity was calculated using the following equation: % lysis = [(c.p.m. experiment – c.p.m. spontaneous)/(c.p.m. maximal – c.p.m. spontaneous)]x100, where c.p.m. spontaneous and maximal were determined by measuring c.p.m. of the supernatants of target cells alone or in the presence of 1% SDS. The spontaneous release was <8% of the maximal release throughout the experiment.

Statistical analysis
Values in the figures and text are expressed as mean ± SEM of at least four observations. Statistical analysis of data was carried out using Student's t-test. P < 0.05 was considered statistically significant.

Results
Since there were no significant differences between the various types of i.v. Ig used, our results are representative of the i.v. Ig preparations.

Effect of i.v. Ig on the development of lung metastases in mice inoculated i.v. with melanoma or sarcoma cells
B16-F10 melanoma. Intravenous treatment with i.v. Ig resulted in a statistically significant inhibition of lung melanoma foci (control: 51.8 ± 9; i.v. Ig: 16.4 ± 3.6, P < 0.001).

Subcutaneous treatment with low-dose i.v. Ig (250 µg/mouse) given twice weekly till the mice were sacrificed resulted in a statistically significant inhibition of 58.6 ± 11% (P < 0.001) of the lung metastatic foci.

MCA-105 sarcoma. Intravenous Ig decreased lung weight in the treated mice (control: 382.4 ± 41.7; i.v. Ig: 214.8 ± 19.7 mg, P < 0.001).

In a different group (30 mice) where survival time was monitored, in the i.v. Ig group mice started to die on day 50, while on day 110, 27% were still alive. In the control group they all died by day 40 (Fig. 1aGo).



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Fig. 1. Survival time of mice inoculated with MCA-105 sarcoma (A) and melanoma (B) following treatment with i.v. Ig.

 
Effect of i.v. Ig on melanoma recurrence and survival time in amputated mice
Both high- and low-dose i.v. Ig treatments were effective and significantly reduced tumor recurrences (P < 0.01, Table 1Go). In the experiments where survival time was determined, the untreated mice died by day 28, while in the i.v. Ig-treated group, they started to die on day 32 and 50% were still alive after 118 days (Fig. 1bGo).


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Table 1. Tumor recurrence following amputation
 
Effect of i.v. Ig on the development of SK-28 human melanoma in SCID mice
SCID mice developed metastatic melanoma foci in the lung following i.v. inoculation of SK-28 human melanoma cells. The number of metastatic foci as well as lung weight were significantly lower in comparison to those of the control group (P < 0.001 and P < 0.01 respectively) (Fig. 2Go).



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Fig. 2. Lung metastatic foci and lung weight of SCID mice, i.v. inoculated with SK-28 human melanoma and treated with i.v. Ig.

 
Effect of i.v. Ig on the production of IL-12
IL-12 production was tested in the presence and absence of mitogen in the culture system. A marked increase in the production of IL-12 was observed in both cases (P < 0.001) (Fig. 3Go).



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Fig. 3. IL -12 production by mononuclear cells incubated with S. aureus Crown mitogen and subjected to i.v. Ig.

 
Effect of i.v. Ig on NK cell activity
A significant increase in the activity of NK cells following preincubation with i.v. Ig was observed (56 ± 9 versus 89% ± 12, P < 0.01).

Discussion
The efficacy of i.v. Ig treatment in murine melanoma and sarcoma was demonstrated in this study. Its effect (at either high or low doses) was evidenced by the reduction in the number of lung metastases and prolonged survival time of mice.

The rationale of using i.v. Ig, an effective therapy for autoimmune conditions, in the treatment of cancer stems from the bidirectional relationship between autoimmunity and cancer.

Intravenous Ig was found to affect autoimmune conditions through multifactorial mechanisms (16). These are divided into humoral mechanisms which include Fc blockade by the i.v. Ig, effects on autoantibody binding and production, via the idiotypic anti-idiotypic network, prevention of immune complex formation, and neutralization of microbial toxins (17). Intravenous Ig also exerts its effects via cellular mechanisms entailing immune modulation of T and B cell number and function, as well as inhibition of anti-inflammatory cytokine production (18,19).

The mechanism through which i.v. Ig prevents metastatic spread may also be multifactorial (Table 2Go). Practically, i.v. Ig can affect each step in the process of metastatic spread, from angiogenesis to direct killing (lysis) of the malignant cell.


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Table 2. Proposed mechanisms for anti-metastatic effects of i.v. Ig.
 
In this study, i.v. Ig was shown to stimulate production of IL-12, a cytokine known to induce an anti-cancer effect by activating NK cells and by inhibiting angiogenesis (2022). Indeed, enhanced NK activity of peripheral blood cells was observed in this work following incubation with i.v. Ig. Recent clinical studies have indicated the effective role of IL-12 in cancer treatment (23), although toxicity of the cytokine has been reported. (24). Cavalo et al. (23) compared the effects of local and systemic rIL-12 administration in mice harboring and invasive 7-day-old moderately differentiated and spontaneously metastasizing mammary adenocarcinoma (TSA). Whereas the immune events elicited via the two routes of rIL-12 administration seem to be the same, systemic rIL-12 was markedly more effective; tumor destruction was dependent on a prompt antitumor response resulting from the cooperation of several subsets of reactive cells. The reactions that seem to play a key role are: (i) indirect inhibition of angiogenesis by secondary cytokines (mainly IFN-{gamma}); (ii) systemic activation of leukocyte subsets capable of producing proinflammatory cytokines, cytotoxic T lymphocytes and antitumor antibodies; and (iii) destruction of tumor vessels by polymorphonuclear cells. The markedly higher efficacy of systemic rIL-12 seems to rest on its ability to recruit these systemic reactions more quickly and efficiently than local rIL-12.

Thus, the use of i.v. Ig, which has minimal adverse effects, may be preferable. The stimulation of NK cells by i.v. Ig may explain its potency also in SCID mice, lacking a normal immune system while still having potent NK cells (25). Masci et al. (26) demonstrated an increase of peripheral blood lymphocytes with the NK surface phenotype in patients treated with i.v. Ig for genital herpes simplex virus infection.

There might be other possibilities for the efficacy of i.v. Ig as an inhibitor of tumor growth: Vassilev et al. (27) showed that i.v. Ig contains antibodies to the Arg–Gly–Asp (RGD) sequence which defines the binding site of a variety of adhesive proteins. Thus, such antibodies may neutralize some adhesion molecules which are essential for the metastatic process.

It is conceivable that part of the effect of i.v. Ig on metastatic spread may involve the Fc rather than the F(ab)2. It is well known that primary tumors harboring the Fc receptor are of a low capability to metastasize (28).

Previous studies were carried out where i.v. Ig was given to patients to prevent infection rather than treat the tumor. In these cases, the efficacy of i.v. Ig as an anti-cancer agent was not evaluated.

The first to raise the possibility of employing normal Ig to treat malignant conditions was Moore (29). He did not use the pooled IgG recognized today, but rather employed Ig purified on tumors, thus alluding to the idea that the normal repertoire of Ig contains anti-tumor specific antibodies. In two other studies, Cafiero et al. (30,31) infused i.v. Ig with antibiotics to patients with colon carcinoma to prevent infections following the resection of the tumor. Indeed, these studies were shown to be beneficial to the patients on a short follow-up basis. However, no analysis was carried out to evaluate the effect of i.v. Ig in the long run and especially on the incidence of tumor recurrences. Moreover, in chronic lymphocytic leukemia (32,33), mortality in the group treated with i.v. Ig was lower than in the controls. In another study with chronic lymphocytic leukemia patients, a lower number of lymphocytes was recorded in the treated group, thus pointing to the anti-leukemic effect of i.v. Ig.

Our results with the amputation model support the administration of i.v. Ig prior to surgery. The multifactorial activity of i.v. Ig may affect tumor spread occurring upon surgical intervention. The spread may be due to tumor bleeding, as well as to the effect of the anesthesia, as has been shown in sham operations (34). Similar observations were noted in a study of Carmeli et al. (35) where regression in Kaposi's sarcoma (KS) was found in an HIV patient treated with i.v. Ig. This group pointed to several mechanisms including targeting NK cells by i.v. Ig to produce antibody-dependent cellular cytotoxic activity against KS, immune regulation, clearance or suppression of growth factors and cytokines as well as clearance and suppression of viruses involved in the pathogenesis of KS.

In this study, i.v. Ig was effective at both high and low doses. As i.v. Ig is prepared from the plasma of 10,000–20,000 healthy subjects, it is an expensive product. Therefore, our results showing that a low dose (x100 less) is as effective as the regular high one (of 400 mg/kg body wt) have practical implications.


    Acknowledgments
 
This work was supported by ARP BIOMED Ltd, Israel.


    Abbreviations
 
APSanti-phospholipid syndrome
ITPidiopathic thrombocytopenic purpura
KSKaposi's sarcoma
SLEsystemic lupus erythematosus

    Notes
 
Transmitting editor: I. Pecht

Received 18 February 1999, accepted 20 April 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 References
 

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