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European Biopharmaceutical Review

Promising Therapies

Karl-Hermann Schlingensiepen at Antisense Pharma GmbH summarises current approaches for the reversal of tumour-mediated immunosuppression and for cancer vaccination

Conventional cancer treatments with radiation and cytotoxic agents aim to destroy dividing cells in different stages of the cell cycle, depending on the cytotoxic agent. Since neoplastic cells divide rapidly, they can be destroyed, while the rapid multiplication of some normal cells can lead to the usual severe side effects. Nevertheless, in some malignancies, especially in testicular cancer and acute lymphoblastic leukaemia, refined cytotoxic schemes can cure a high percentage of patients. The downside is the occurrence of secondary malignancies, even with these indications. Surgery, as another cornerstone of current therapies, removes visible tumours.

Targeted therapies with kinase inhibitors and therapeutic antibodies have made considerable advances in many malignancies. The most recent successes in targeted therapies are two advanced immunotherapeutic approaches:

  • Tumour vaccination aims at stimulating the patientís immune system using tumour antigens. Vaccines designed to prevent cancer are beyond the scope of this review
  • Reversal of tumour induced immunosuppression overcomes tumour induced suppression of the anti-tumour immune response

Both approaches attack targets more precisely, inducing fewer side effects and markedly improving survival and the quality of the patientís life.

CANCER VACCINES: DELIVERING THE PROMISE

Pierre Van Der Bruggen et al identified the first tumour-specific antigen, MAGE, recognised by cytolytic T-cells in humans (1). Progress in the identification of tumour-associated antigens (TAA) and evolving vaccination techniques have enabled the development of cancer vaccines that are designed to induce the patientís adaptive immune response in an antigen-specific manner. However, the clinical benefits of cancer therapies might be limited by multiple mechanisms that tumours employ to interfere with effective antitumour response (2,3).

The full potential of immunotherapeutic strategies in oncology remains to be exploited. Currently, however, several immunomodulatory anti-cancer drugs have been developed which either have been approved or are in advanced clinical development. Examples of promising studies of both cancer vaccines and reversal of tumour-mediated immunosuppression suggest that immune-based interventions will provide therapeutic benefits in humans (see Table 1).

Table 1: Examples of immunomodulatory strategies that are either approved or in clinical development

           

 

Disease

 

 

Target

 

Principle

Clinical phase

Cancer vaccines

HRPC

Metastatic melanoma

Metastatic melanoma

PAP

Whole TAA spectrum

Defined TAA

Antigen-loaded APCs

Intradermal injection of whole tumour mRNA  

Intradermal injection of defined mRNAs

III/app (5,6)

 I/II (8)  

I/II (9)

Reversal of immuno-suppression

Metastatic melanoma

High-grade glioma

Metastatic melanoma, pancreatic and colorectal cancer

CTLA-4

TGF-β2

TGF-β2

Blocking antibody

Intratumoural administration of AS-ODN 

Systemic administration of AS-ODN

 III (13) 

III (IIb20)

I/II

App = approved; HRPC = hormone refractory prostate cancer; PAP = Prostatic acid phosphatase; APC = antigen presenting cells; CTLA-4 = cytotoxic T lymphocyte-associated antigen; TGF-β2 = transforming growth factor-β2; AS-ODN = antisense oligodeoxynucleotides

Antigen Loaded Cells

Therapeutic cancer vaccines are designed to treat cancer by stimulating the immune system to recognise and attack cancer cells. Several characteristics of prostate cancer provide a conclusive rationale for the application of cancer vaccines. As a slowly proliferating cancer type, prostate cancer is mostly resistant to cytotoxic drugs, whereas immunotherapy does not rely on high cell proliferation and because prostate cancer progresses slowly, multiple vaccinations are possible to induce effective antitumour response. Importantly, research has identified several TAA that represent potential targets for immunisation. Prostatic acid phosphatase (PAP), for example, is expressed in approximately 95 per cent of prostate cancers, and PAP expression is largely restricted to prostate tissue (4). Therefore, PAP has been considered a promising candidate for cancer vaccination (5).

An essential element in cancer vaccination is the activation of specific cytotoxic T-cells by antigen presenting cells (APCs). Sipuleucel-T (APC8015, Dendreon Corporation), the first cancer vaccine approved by regulatory authorities, consists of apheresisderived autologous APC loaded with recombinant human PAP. Patients undergo a series of three leukapheresis procedures to isolate APCs from peripheral blood mononuclear cells. APCs are then loaded ex vivo with a fusion protein containing PAP and the immune cell activator granulocytemacrophage colony-stimulating factor (GM-CSF) to enhance the immune response after vaccination (5). Approximately three days later, the protein-complexes are infused intravenously into patients. Several studies have shown that loading with the PAP/GM-CSF fusion protein activates APC, as demonstrated by increased expression of the immune cell activation marker CD54 on the surface of APC. A minimum of 50 x 106 autologous CD54+ cells are present in each dose of sipuleucel-T (6).

The safety and efficacy of sipuleucel-T in patients with metastatic hormone refractory prostate cancer (HRPC) were analysed in two randomised, double-blind, placebo-controlled trials (5,6). In the first study, 127 patients with asymptomatic metastatic HRPC received either sipuleucel-T (N=82) or placebo (N=45), and all patients were followed for survival for 36 months. After eight weeks of treatment, T-cell stimulation was eight times higher in sipuleucel-T-treated patients than in placebo-treated patients (P<0.001). The median time till disease progression was longer in sipuleucel-Ttreated patients (11.7 weeks) than in placebo treated patients (10 weeks), whereby this difference did not achieve statistical significance. However, median survival time was 25.9 months with sipuleucel-T and 21.4 months with placebo (P=0.01), suggesting a survival advantage of sipuleucel-T treated HRPC patients (7).

In the second study, 512 patients with metastatic HRPC were randomised to sipuleucel-T- (N=341) or control treatment (N=171). Patients had previously undergone radical prostatectomy (35 per cent), radiotherapy (54 per cent), combined androgen blockade (82 per cent) or chemotherapy (18 per cent). Disease progression had been observed in all patients either at metastatic sites or by serial measurement of prostate specific antigen (PSA). An intent-to-treat analysis of the primary end point and overall survival demonstrated that sipuleucel-T extended survival of sipuleucel-T treated patients by 4.1 months when compared to placebo (25.8 versus 21.7 months; P=0.032) (5). Sipuleucel-T provides important therapeutic options for prostate cancer and may also represent a milestone in the development of immunotherapeutic strategies.

Nucleic Acid Vaccination

Cancer vaccination might be mediated by direct injection of TAA or APC loaded with TAA. Additionally, nucleic acids encoding TAA are attractive candidate vectors for the development of vaccines. Injection of TAA encoding DNA has been shown to activate APC, which then present TAA to T-cells (8).

DNA vaccines are simple tools for clinically applicable in vivo transfection because large-scale DNA vaccines can be produced more easily than other vaccines including recombinant proteins. An alternative to DNA vaccines are mRNAbased vaccines. This type of vaccination provides important safety features because, in contrast to DNA, the half-life of mRNA is short, and mRNAs do not integrate into the genome or induce auto-antibodies.

Until recently, few clinical Phase I/II trials have used RNA as a vaccine (9,10). In a study on 15 patients with metastatic melanoma, the total RNA of one growing metastasis was extracted from each patient. After reverse transcription and cloning, cDNA libraries were transcribed into copy mRNA. Patients then received serial intradermal injections of autologous whole tumour mRNA in combination with GM-CSF as adjuvant. At the end of the trial, melanoma cell specific antibodies were detected in four of 15 patients. Five patients showed a favourable course of disease. The vaccine was proven to be safe because only mild and reversible side effects could be observed (9).

In their second RNA-vaccination study on 21 patients with metastatic melanoma, Weide et al used mRNA encoding six defined TAA (Melan-A, Tyrosinase, gp100, Mage-A1, Mage-A3, Survivin) instead of whole tumour mRNA (10). In contrast to the first study, patients received increasing doses of mRNA and a higher number of injections. Instead of using naked mRNA, nucleic acid preparations were protamine-stabilised. Again, GMCSF was applied as an adjuvant. In 10 patients, keyhole limpet haemocyanin (KLH) was added as helper antigen. Two patients showed a reproducible increase of vaccine-directed T-cells, whereas T-cell response was inconsistent between other patients. Clinical response was detected in one seventh of stage IV patients. Additionally, the number of immune suppressive cells was analysed; while the number of immune suppressive regulatory T-cells decreased in patients who received KLH as helper antigen, myeloid suppressor cells decreased in KLHuntreated patients. The authors concluded that both early Phase clinical trials encourage further investigation of mRNAvaccines. Recently, two Phase IIa studies on prostate cancer and non-small cell lung cancer have started.

REVERSAL OF TUMOUR-MEDIATED IMMUNOSUPPRESSION

The human immune system has evolved with effective mechanisms to attack tumours, but malignant cells can actively evade these mechanisms, resulting in tumour development. Even highly immunogenic cancer types manage to evade the immune system. A major mechanism is that malignant tumours actively suppress the anti-tumour responses of the immune system.

Currently, there are two approaches in advanced clinical development to reverse tumour-induced immunosuppression:

  • The antibody ipilimumab that is directed against the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) of regulatory T-cells (Treg)
  • The antisense-oligonucleotide trabedersen for specific silencing of transforming growth factor 2 (TGF-β2) expression

CTLA-4 Blocking Antibody

One approach has been to promote T-cell activity by blocking inhibitory signals from receptors such as the cytotoxic T lymphocyte-associated antigen (CTLA)-4 (11).

Effective T-cell activation requires the engagement of two separate signals. First, T-cell receptors bind to an antigen presented in the context of the major histocompatibility complex on APCs. The second signal requires the interaction of a costimulatory receptor on T-cells (for example CD28) and its ligand expressed on APCs, such as the peripheral membrane protein B7. This costimulatory signal results in T-cell proliferation, cytokine secretion and changes in gene expression. However, B7 binds either to activating CD28 or to inhibitory CTLA-4, a homologue of CD28 with higher affinity for B7. Interaction of CTLA-4 and B7 downregulates T-cell activation (12). The role of CTLA-4 as the main negative regulator of T-cell-mediated antitumour response was revealed by several in vitro and in vivo experiments; a blockade of CTLA-4 leads to enhanced T-cell functions such as cytokine production and proliferation. In a murine cancer model, anti-CTLA-4 monoclonal antibodies (mAb) inhibited the growth of both new and established tumours (13).

Ipilimumab, a human mAb to CTLA-4, inhibits the CTLA-4 mediated downregulation of antitumour response. In a recent Phase III trial, ipilimumab was administered with or without a glycoprotein peptide vaccine (gp100) in 676 patients with previously treated unresectable stage III or IV metastatic melanoma (14). Patients were randomly assigned to receive ipilimumab (N=137), gp100 (N=136) or ipilimumab and gp100 (N=403). Ipilimumab and gp100 were administered once every three weeks for four treatments. Analysis of the primary endpoint, overall survival, revealed that ipilimumab, either alone or with gp100, significantly improved overall survival when compared to gp100 alone (P=0.003; P<0.001). The median overall survival length with ipilimumab and ipilimumab plus gp100 was 10.1 months and 10.0 months, respectively. In comparison, overall survival time of patients who had received gp100 alone was 6.4 months. The data of this Phase III study are consistent with those of several Phase I/II trials, which demonstrated that CTLA-4 blockade is associated with objective responses in patients with metastatic melanoma (15).

TGF-β2 Silencing

Strategies that tumour cells employ to escape the immune system include, amongst others, the secretion of growth factors and cytokines. Soluble factors such as interleukin-10, and transforming growth factor-β (TGF-β) sustainably inhibit the activity of practically all types of immune cells, thereby forming an immunosuppressive environment around the tumour. Moreover, tumour-induced immunosuppression hampers the above-mentioned vaccination and immune activation strategies.

Aggressive tumours such as high-grade gliomas, advanced pancreatic carcinomas, malignant melanoma, and advanced colorectal cancers express huge amounts of TGF-β2. The resulting immunosuppression by TGF-β2 is one reason for the frequently observed failure of anti-cancer immunotherapies (16). Moreover, studies have demonstrated that TGF-β exerts numerous protumourigenic effects, such as proliferation, metastasis and angiogenesis, in addition to its immunosuppressive function. Therefore, inhibition of TGF-β tackles several oncogenic processes.

Table 2: Trabedersen treatment of patients with recurrent or refractive anaplastic astrocytoma in comparison to conventional chemotherapy

      Trabedersen (10μM) (N = 12) Chemotherapy (1) (N = 15)  Significance (p-value)  
Overall response rate (14 months) 42 per cent 0 per cent 0.034
Tumour control rate (14 months) 58 per cent 20 per cent 0.003
Tumour progression rate (14 months) 17 per cent 58 per cent 0.003
Survival rate (Two years) 83 per cent 42 per cent 0.100

Trabedersen is a TGF-β2-specific antisense oligodeoxynucleotides (ASODN). It has been developed to treat highgrade glioma and other solid tumours overexpressing TGF-β2 (17). Trabedersen represents a targeted therapeutic strategy and specifically binds TGF-β2-mRNA, thereby preventing TGF-β2 protein expression (18). In vitro experiments with different cancer cell lines demonstrated that both TGF-β2 secretion and tumour cell proliferation are markedly reduced by trabedersen when compared to untreated controls. In three clinical Phase I/II studies, very good safety and tolerability of trabedersen was demonstrated in 24 patients with recurrent high-grade glioma (19). Interestingly, median survival time after recurrence had exceeded the published data for chemotherapy (20). Recently, a randomised, active controlled Phase IIb clinical trial study was completed which evaluated efficacy and safety of two doses of trabedersen (10μM; 80μM) in comparison to currently approved gold standard chemotherapy (21). In this study, 134 out of 145 patients with either recurrent or refractory anaplastic astrocytoma (AA, N=39) or glioblastoma multiforme (GBM, N=95) received treatment. Trabedersen was administered locally using an intratumoural catheter and a portable pump allowing outpatient treatment. GBM-patients had a comparable overall survival rate among the three treatment groups (10μM trabedersen, 80μM trabedersen, chemotherapy). In the prespecified GBMsubgroup with the two prognostic factors age equal or less than 55 years and Karnofsky Performance Status more than 80 per cent (about 40 per cent of the GBM patients in this study), treatment with 10μM trabedersen resulted in long term survival rates at two and three years that were three times higher than those of patients who received standard chemotherapy.

In AA-patients, efficacy of 10μM trabedersen was even more pronounced. A significantly higher overall response rate of trabedersen treated patients was observed at 14 months when compared to patients who received standard chemotherapy (42 per cent versus 0 per cent; P=0.034). The duration of this response exceeded the treatment period. Similarly, the tumour control rate, the tumour progression rate, and the two-year survival rate were clearly improved under trabedersen (see Table 2). Importantly, these trabedersen effects were associated with a median overall survival benefit of 17.4 months (39.1 months versus 21.7 months, non-significant). In conclusion, these data demonstrate that TGF-β2 suppression by trabedersen is a promising immunotherapeutic treatment against malignant gliomas and other aggressive tumours. Based on these results, a pivotal Phase III study in recurrent or refractory AA-patients has started (SAPPHIRE). Furthermore, the intravenous application of trabedersen as second to forth line monotherapy is investigated in a Phase I/II dose-escalation study in patients with advanced pancreatic cancer, malignant melanoma, or colorectal cancer. Interim results show that trabedersen is safe and well tolerated. In addition, encouraging first efficacy results were also observed. Doseescalation has been completed and further 24 patients with pancreatic carcinoma or malignant melanoma are currently being enrolled for the treatment with a defined dose. A randomised, active-controlled study is in preparation.

CONCLUSION

Recently the knowledge of antitumour immune response as well as tumourmediated immunosuppression in man has significantly progressed. Both boosting the patientís immune system and reversing tumour-mediated immunosuppression are now clinically advanced highly promising therapies. Combination of both approaches may provide an even greater therapeutic benefit.

References

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  2. Finn OJ, Cancer vaccines: between the idea and the reality, Nat Rev Immunol 3(8): pp630-641, 2003
  3. Serafini P et al, Derangement of immune responses by myeloid suppressor cells, Cancer Immunol Immunother 53(2): pp64-72, 2004
  4. Jacobs EL and Haskell CM, Clinical use of tumor markers in oncology, Curr Probl Cancer 15(6): pp299-360, 1991
  5. Burch PA et al, Immunotherapy (APC8015, Provenge) targeting prostatic acid phosphatase can induce durable remission of metastatic androgen-independent prostate cancer: a Phase II trial, Prostate 60(3): pp197-204, 2004
  6. US Food and Drug Administration, FDA labelling information Ė Provenge, www.fda.gov/downloads/ BiologicsBloodVaccines/CellularGene Therapy Products/ApprovedProducts/ UCM210031.pdf, 2010
  7. Small EJ et al, Placebo-controlled Phase III trial of immunologic therapy with sipuleucel-T (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer, J Clin Oncol 24(19): pp3,089-3,094, 2006
  8. Pardoll DM and Beckerleg AM, Exposing the immunology of naked DNA vaccines, Immunity 3(2): pp165-169, 1995
  9. Weide B et al, Results of the first phase I/II clinical vaccination trial with direct injection of mRNA, J Immunother 31(2): pp180-188, 2008
  10. Weide B et al, Direct injection of protamine-protected mRNA: results of a Phase I/II vaccination trial in metastatic melanoma patients, J Immunother 32(5), pp 498-507, 2009
  11. Ribas A et al, Antitumor activity in melanoma and anti-self responses in a Phase I trial with the anti-cytotoxic T lymphocyte-associated antigen 4 monoclonal antibody CP-675,206, J Clin Oncol 23(35): pp8,968-8,977, 2005
  12. Egen JG, Kuhns MS and Allison JP, CTLA-4: new insights into its biological function and use in tumor immunotherapy, Nat Immunol 3 (7): pp611-618, 2002
  13. Leach DR, Krummel MF and Allison JP, Enhancement of antitumor immunity by CTLA-4 blockade, Science 271(5256): pp1,734-1,736, 1996
  14. Hodi FS et al, Improved Survival with Ipilimumab in Patients with Metastatic Melanoma, N Engl J Med [available: http://www.ncbi.nlm.nih.gov/ pubmed/20525992], 2010
  15. Robert C, Ghiringhelli F, What is the role of cytotoxic T lymphocyteassociated antigen 4 blockade in patients with metastatic melanoma?, Oncologist 14(8): pp848-861, 2009
  16. Kim S et al, Systemic blockade of transforming growth factor-beta signaling augments the efficacy of immunogene therapy, Cancer Res 68(24): pp10,247-10,256, 2008
  17. Vallieres L, Trabedersen, a TGFbeta2- specific antisense oligonucleotide for the treatment of malignant gliomas and other tumors overexpressing TGFbeta2, IDrugs 12(7): pp445-453, 2009
  18. Schlingensiepen KH, Schlingensiepen R, Steinbrecher A, Hau P, Bogdahn U, Fischer-Blass B and Jachimczak P, Targeted tumor therapy with the TGFbeta 2 antisense compound AP 12009, Cytokine Growth Factor Rev 17(1-2): pp129-139, 2006
  19. Hau P et al, Inhibition of TGF-beta2 with AP 12009 in recurrent malignant gliomas: from preclinical to Phase I/II studies, Oligonucleotides 17(2): pp201-212, 2007
  20. Schlingensiepen KH, Fischer-Blass B, Schmaus S and Ludwig S, Antisense therapeutics for tumor treatment: the TGF-beta2 inhibitor AP 12009 in clinical development against malignant tumors, Recent Results Cancer Res 177: pp137-150, 2008
  21. Bogdahn U et al, Targeted Therapy for High-Grade Glioma with the TGF-Ŗ2 Inhibitor Trabedersen: Results of a Randomized and Controlled Phase IIb Study, Neuro-Oncology [in press], 2010

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Karl-Hermann Schlingensiepen is Chief Executive Officer and cofounder of Antisense Pharma. Previously, he was Head of Business Development, Intellectual Property and Research & Development at Biognostik, market leader in the design and production of antisense molecules for biomedical research. Karl-Hermann studied medicine in Cambridge, UK, Harvard, US, and Goettingen, Germany. He completed his PhD thesis at the Max- Planck-Institute Goettingen (1988), where he then established a research group for molecular neuro- and tumour biology. As initiator and organiser of international antisense conferences in 1993 and 1995 he promoted scientific exchange and discussion in this field. He founded Antisense Pharma in 1998 to focus on the development of antisense drugs for oncological disorders and other unmet medical needs.
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