A mouse model for immunotherapy of osteosarcoma

Summary Corynebacterium parvum therapy of pulmonary micrometastases after amputation of the primary tumour has been studied in the Dunn osteosarcoma tumour of C3H/HeJ mice. Adjuvant antimetastatic C. parvum is efficacious in both intact and T cell-deficient mice. Repeated small doses of C. parvum have been shown to be as effective as or more effective than large single doses, and this finding was confirmed in two other metastasizing tumour models: the B16 melanoma and Lewis lung carcinoma. Commencing C. parvum preoperatively produces a greater reduction in weight of pulmonary metastases than postoperative therapy, although both are effective. C. parvum can reduce the enhanced growth of lung metastases produced by amputation of the primary tumour. The most effective route of administration was intravenous, followed by intraperitoneal therapy; and subcutaneous administration had no effect. Where measured, spleen weights correlated well with the therapeutic efficacy of the particular C. parvum regimen. As a result of these experiments showing effective use of C. parvum in doses that could be administered to man, certain implications for human immunotherapy are discussed.     

Immunotherapy of lung cancer with Corynebacterium parvum

Summary Thirty-one patients with inoperable carcinoma of the lung, excluding oat-cell carcinoma, were randomized to receive either chemotherapy alone, with methyl CCNU and vinblastine every 6–8 weeks (15 Pts) or such chemotherapy plus immunotherapy with IV infusions of Corynebacterium parvum (16 Pts). Prior duration of the disease was longer, and more patients had received previous therapy, in the immunotherapy group; these groups were otherwise very similar. In vitro lymphocyte response to phytohemagglutinin did not change significantly in either group, but the weaker response to Varidase declined in both groups after chemotherapy. An increased baseline level of circulating B lymphocytes was sharply reduced in the C. parvum group. There were no differences in -globulins or delayed skin test responses between immunotherapy and control patients at entry into this study or on follow-up. Median survival from entry was longer in the immunotherapy group (6 months) than in the control group (3 months), but this difference was not statistically significant and only two patients in each group lived for more than 11 months. It is conceivable that more benefit from C. parvum might have been recorded had more effective chemotherapy been available.     

Quantitative comparison of three C. parvum strains for immunotherapy of transplanted rat tumours

Summary Preparations of three commercially available killed bacterial suspensions designated Corynebacterium parvum have been compared for immunotherapy of transplanted rat tumours. The Wellcome CN6134 preparation was quantitatively superior to Institut Merieux IM1585 in suppressing local, subcutaneous, growth of tumour when injected in admixture with cells, for the control of thoracic malignant effusions when administered intrapleurally and for active specific immune stimulation where incorporated into vaccines of irradiated cells. The Wellcome CN5888 preparation was virtually ineffective. These observations, together with the recent re-classification of C. parvum into three distinct species of the genus Propionibacterium, i.e., P. acnes (Wellcome CN6134), P. granulosum (Wellcome CN5888), and P. avidum (Institut Merieux IMI1585) emphasize that the simple designation C. parvum is inadequate for the vaccines currently available for experimental or, more importantly, clinical immunotherapy.     

Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

Adjuvant immune stimulation with Corynebacterium parvum during maintenance chemotherapy of acute myeloid leukemia

Summary Of 93 consecutively treated patients with acute myeloid leukemia 36 (39%) achieved complete remission (CR). Thirty-five patients were randomized to receive either maintenance chemotherapy alone (C) or a combination of active nonspecific immunotherapy with Corynebacterium parvum and chemotherapy (C + I). Maintenance therapy was given monthly for 1 year or until relapse. The median survival time was 21 months for patients treated with chemotherapy alone, compared with 30 months for patients treated with chemotherapy and immunotherapy. The median remission duration was 15 months for patients treated with chemotherapy, compared with 18 months for chemotherapy and immunotherapy group. While no statistically significant difference in remission duration or survival time could be attributed to the use of immune stimulation, a plateau of 40% long-term time survivors was defined in the chemotherapy and immunotherapy group. Age and sex were found to be the major prognostic factors for achivement of CR. No difference was found in remission duration or survival between the two different induction schedules. Neither did the morphological subtype of AML (FAB classification) or the leukocyte count at diagnosis correlate with remission rate or survival.     

Adjuvant immune stimulation with Corynebacterium parvum during maintenance chemotherapy of acute myeloid leukemia

Of 93 consecutively treated patients with acute myeloid leukemia 36 (39%) achieved complete remission (CR). Thirty-five patients were randomized to receive either maintenance chemotherapy alone (C) or a combination of active nonspecific immunotherapy with Corynebacterium parvum and chemotherapy (C + I). Maintenance therapy was given monthly for 1 year or until relapse. The median survival time was 21 months for patients treated with chemotherapy alone, compared with 30 months for patients treated with chemotherapy and immunotherapy. The median remission duration was 15 months for patients treated with chemotherapy, compared with 18 months for chemotherapy and immunotherapy group. While no statistically significant difference in remission duration or survival time could be attributed to the use of immune stimulation, a plateau of 40% long-term time survivors was defined in the chemotherapy and immunotherapy group. Age and sex were found to be the major prognostic factors for achievement of CR. No difference was found in remission duration or survival between the two different induction schedules. Neither did the morphological subtype of AML (FAB classification) or the leukocyte count at diagnosis correlate with remission rate or survival.     

Intratumoral BCG and Corynebacterium parvum therapy of canine mammary tumours before radical mastectomy

Summary In two parallel studies, bitches with mammary tumour received single intralesional injections of BCG (1 mg: 10⁷ living bacteria) and Corybacterium parvum (10⁹ killed bacteria) (53 bitches) or C. parvum alone (129 bitches) at the same dosage. Control groups received injections, following the same protocol, of 1 ml BCG suspension medium diluted in saline in the first study (51 bitches) or no injections at all (120 bitches in the second study).A block dissection, including mammary tumours, adjacent mammary glands, and regional lymph nodes, was performed 2 weeks later in all animals. On the basis of histologically confirmed malignant tumours, 48 bitches (25 treated by immunotherapy and 23 controls) in the first study and 67 bitches (30 treated by immunotherapy and 37 controls) in the second study remained for postsurgical follow-up.The clinical tolerance of the treatment was generally good. No significant differences were found in cumulative survival rates between treated and control group in either studies.     

Analysis of the effect of periodate oxidation and phenol extraction on the antitumour properties of C. parvum

Summary The antitumour activity of C. parvum and its relationship to spleen weight increase have been analysed in a tumour model using syngeneic M₄ fibrosarcoma cells. It was found that treatment of C. parvum with low concentrations of periodate (2 mM) produced a minor decrease in the ability to abrogate tumour growth, following intratumour injection, although the capacity to increase spleen weight after IP injection was abolished. Higher concentrations of periodate (20 mM) produced complete inactivation of the bacteria. A phenol extract of C. parvum was weakly active in the intratumour test but amounts up to 1 mg injected IP were unable to increase the spleen weight. The data support the hypothesis that a surface carbohydrate is responsible for the increase in spleen weight. It is suggested that the antitumour activity may reside in a molecule that also reacts with periodate but at a slower rate, perhaps because of a more complex structure or a different location in the cell, for instance in the cytoplasm. The phenol extract was soluble in chloroform and at least two major components were glycolipids, so it is conceivable that a molecule of this type, which could be degraded by periodate, might be responsible for the antitumour activity of C. parvum. The precise biochemical nature of the active component is, as yet, undetermined.     

MANIPULATION OF THE TUMOUR-ASSOCIATED VASCULATURE TO IMPROVE TUMOUR THERAPY

ABSTRACT

Inhibition of tumour vascular growth, destruction of the tumour associated vasculature (TAV), and manipulation of the endothelial lining of the TAV provide powerful tools for anti-tumour therapy. We previously demonstrated that addition of TNF to chemotherapy improved tumour response. The major effect of TNF is an increased permeability of the tumour vascular bed resulting in augmented accumulation of co-administered drug in the tumour. As the TAV is recognised as a major candidate in tumour therapy it is becoming important to understand anti-vascular effects better. In our laboratory we examine the effect of immunotherapy on the TAV, and the effect of anti tumour-vascular therapy on tumours. This is studied in animal models, which exhibit similarities with the clinical setting, such as tumour perfusion treatment.     

PBT-3, a hepoxilin stable analog, causes long term inhibition of growth of K562 solid tumours in vivo

We demonstrate herein that daily administration of PBT-3 for 8 days to NU/NU mice bearing solid tumours derived from the s.c. administration of the leukemic cell line K562 results in inhibition of growth of the tumours in vivo, and this inhibition lasts for 60 days after stopping treatment with PBT-3 before recovery of tumour growth is re-established. Similar findings were observed when the mice were treated with Gleevec (STI-571). These results provide new evidence that PBT-3 is effective in controlling solid tumour growth in vivo and suggest that the PBT family may be useful in the development of new drugs in cancer therapy.     

Antitumour activity of endotoxin, concanavalin A and poly I: C and their ability to elicit tumour necrosis factor, cytostatic factors, and interferon in vivo

Summary Concanavalin A, endotoxin, poly I : C, and tumour necrosis serum (TNS) were compared for antitumour activity against Meth A sarcoma transplanted in syngeneic BALB/c mice and their capacity to induce tumour necrosis factor (TNF), heat-stable cytostatic factors, and heat-labile interferon in the blood of normal and Corynebacterium parvum-pretreated mice. All the agents induced hyperemia and inhibition of mitosis at 4 h, and by 24 h many tumours had a dark necrotic centre. Subsequent tumour growth was inhibited and in some of the treated mice tumours regressed completely. Poly A : U and normal mouse serum did not induce regression and their effects were less marked in all other respects, suggesting that these events may be linked. The necrotizing effects of concanavalin A and poly I : C are unlikely to be mediated by TNF, because neither agent could mimic endotoxin in eliciting RNase-resistant necrotizing and regressing activity in the serum of mice pretreated with C. parvum. Poly I : C did not induce strong cytostatic activity in the sera of C. parvum-treated mice, and for this and other reasons these factors are unlikely to be responsible for the observed effects. Concanavalin A, endotoxin, and poly I : C induced high levels of serum interferon but purified interferon had only weak antitumour activity in the Meth A system, suggesting that interferon may not be the mediator.From these and other data it is concluded that there is no clear relationship between the capacity of the agents to induce tumour necrosis and their capacity to elicit TNF, cytostatic factors, and interferon.     

Manipulation of the tumour-associated vasculature to improve tumour therapy

Inhibition of tumour vascular growth, destruction of the tumour associated vasculature (TAV), and manipulation of the endothelial lining of the TAV provide powerful tools for anti-tumour therapy. We previously demonstrated that addition of TNF to chemotherapy improved tumour response. The major effect of TNF is an increased permeability of the tumour vascular bed resulting in augmented accumulation of co-administered drug in the tumour. As the TAV is recognised as a major candidate in tumour therapy it is becoming important to understand anti-vascular effects better. In our laboratory we examine the effect of immunotherapy on the TAV, and the effect of anti tumour-vascular therapy on tumours. This is studied in animal models, which exhibit similarities with the clinical setting, such as tumour perfusion treatment.     

Immunotherapy of human colon cancer by antibody-targeted superantigens

T lymphocytes generally fail to recognize human colon carcinomas, suggesting that the tumour is beyond reach of immunotherapy. Bacterial superantigens are the most potent known activators of human T lymphocytes and induce T cell cytotoxicity and cytokine production. In order to develop a T-cell-based therapy for colon cancer, the superantigen staphylococcal enterotoxin A (SEA) was given tumour reactivity by genetic fusion with a Fab fragment of the monoclonal antibody C242 reacting with human colon carcinomas. The C242Fab-SEA fusion protein targeted SEA-reactive T cells against MHC-class-II-negative human colon carcinoma cells in vitro at nanomolar concentrations. Treatment of disseminated human colon carcinomas growing in humanized SCID mice resulted in marked inhibition of tumour growth and the apparent cure of the animals. Therapeutic efficiency was dependent on the tumour specificity of the fusion protein and human T cells. Immunohistochemistry demonstrated massive infiltration of human T cells in C242Fab-SEA-treated tumours. The results merit further evaluation of C242Fab-SEA fusion proteins as immunotherapy in patients suffering from colon carcinoma.     

Anti-metastatic effects of viral and non-viral mediated Nk4 delivery to tumours

The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 (also known as Interleukin (IL)32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metasasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate reduction in primary subcutaneous tumour growth. Overall, this study demonstrates the potential for Nk4 gene therapy of metastatic tumours, when delivered by AAV or non-viral methods.     

Oral Tolerance to Cancer Can Be Abrogated by T Regulatory Cell Inhibition

Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue – JBS fibrosarcoma (to induce oral tolerance to a cancer), or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p<0.05). Complete regression of tumours were only seen after Treg inactivation and occurred in all groups - this was not inhibited by tumour feeding. The cure rates for Treg inactivation were 60% during tolerisation, 75% during tumour growth and 100% during inactivation for both tolerisation and tumour growth. Depletion of Tregs gave rise to an increased number of Teff cells. Treg depletion post-tolerisation and post-tumour induction led to the complete regression of all tumours on tumour bearing mice. Oral administration of tumour tissue, confers a tumour growth advantage and is accompanied by an increase in systemic Treg levels. The administration of anti-CD25 Ab decreased Treg numbers and caused an increase in Teffs. Most notably Treg cell inhibition overcame established oral tolerance with consequent tumor regression, especially relevant to foregut cancers where oral tolerance is likely to be induced by the shedding of tumour tissue into the gut.     

Study of the mechanism of Corynebacterium parvum antitumour activity: Protective effect in mice submitted to sublethal doses of irradiation

Summary The antitumour activity of C. parvum against two different tumours, a lymphosarcoma grafted in XVII mice and a mammary carcinoma grafted in C₃H mice, was a radiosensitive phenomenon. A dose of X-rays as low as 100 rads was sufficient to abrogate the C. parvum-induced protection. The duration of this inhibition increased with augmentation of the X-ray dose. The stimulation of macrophage-phagocytic activity induced by C. parvum was not inhibited by a dose of 500 rads. A chronological parallelism has been demonstrated in the recovery of the C. parvum antitumour effect and the restoration of antibody responsiveness after the suppression of these two activities by 500 rads of X-rays in the case of the C₃H mice grafted with mammary carcinoma cells. No such concomitant recovery has been observed in XVII mice. In these mice, the recovery of C. parvum antitumour activity took place before the restoration of antibody responsiveness.     

Disseminated intravascular coagulation following intravenous Corynebacterium parvum injection in the rat

Summary Corynebacterium parvum (C. parvum) was administered by a single IV injection (14 mg/kg) to rats, and platelet counts, plasma fibrinogen concentrations and thrombin clotting times were monitored for up to 7 weeks. During this time histological and ultrastructural studies were also conducted. Thrombocytopoenia, hypofibrinogenaemia, and prolongation of the thrombin clotting time rapidly followed C. parvum injection and were accompanied by the appearance of platelet clumps and fibrin within blood vessels in a variety of tissues. This initial episode of disseminated intravascular coagulation (DIC) subsided 12–24 h after injection, but a more prolonged second episode of DIC occurred 1–3 days after injection. The results suggest that caution should be observed when systemic immunotherapy with C. parvum is proposed.     

Combination of vasostatin gene therapy with cyclophosphamide inhibits growth of B16(F10) melanoma tumours

Angiogenesis, i.e. formation of new blood vessels out of pre-existing capillaries, is essential to the development of tumour vasculature. The discovery of specific antiangiogenic inhibitors has important therapeutic implications for the development of novel cancer treatments. Vasostatin, the N-terminal domain of calreticulin, is a potent endogenous inhibitor of angiogenesis and tumour growth. In our study, using B16(F10) murine melanoma model and electroporation we attempted intramuscular transfer of human vasostatin gene. The gene therapy was combined with antiangiogenic drug dosing schedule of a known chemotherapeutic (cyclophosphamide). The combination of vasostatin gene therapy and cyclophosphamide administration improved therapeutic effects in melanoma tumours. We observed both significant inhibition of tumour growth and extended survival of treated mice. To our knowledge, this is one of the first reports showing antitumour efficacy of electroporation-mediated vasostatin gene therapy combined with antiangiogenic chemotherapy.     

Prise en charge du cancer du sein,avancées et perspectives

Breast cancer treatment has developed rapidly for the last 15 years, promoted by a better understanding of tumour growth biology. Targeted therapies have been rapidly expending: immunotherapy, targeted chemotherapy, endocrine therapy efficacy enhanced by mTOR inhibitors. Changes of molecular profiling tumours during the illness need to perform regularly biopsies and to adapt drugs. This article will focus on a high-level overview of main advances across systemic treatment.