Melanoma: Improving Clinical Outcomes Through Advances in Immunotherapeutics and Targeted Therapy

Metastatic melanoma has limited treatment options, but advances in the understanding of the oncogenic mutations that drive this cancer and how the immune system can be better modulated to fight melanoma provide a new generation of active approaches for patients. Targeting c-kit and BRAF-activating gene mutations with orally available specific inhibitors, like imatinib and PLX4032, has resulted in a high frequency of objective tumor responses. The use of antibodies that modulate costimulatory signaling, like anti-CTLA4 or anti-PD-1, result in reproducible durable tumor responses, although this occurs in only a small subset of patients. The response rates are higher when using adoptive cell transfer therapies. Therefore, novel advances arising from basic research knowledge are resulting in active treatments in the clinical setting and are reviewed here.

Most patients with advanced melanoma have poor outcomes with current therapeutic approaches. Neither of the only 2 agents approved by the US Food and Drug Administration (FDA) for the treatment of Stage IV melanoma, the chemotherapy drug dacarbazine and the immunotherapy cytokine interleukin-2 (IL-2), has demonstrated an impact on patient survival in a clinical trial compared with a placebo control group.¹ The past 20 years have provided plenty of examples of novel approaches and experimental drugs that have failed to improve on the low performance of standard-of-care therapies for melanoma in randomized clinical trials. Some examples include multiagent chemotherapy (ie, the Dartmouth regimen), combination of chemotherapy and immunotherapy in the so-called biochemotherapy regimens, vaccines (dendritic cells, vitespen [Oncophage]), the antisense bcl-2 oligonucleotide oblimersen (Genasense), the multitargeted tyrosine kinase inhibitor (TKI) sorafenib (Nexavar), the reactive-oxide species inhibitor elesclomol, among a long list of agents.^1-4 These numerous negative randomized clinical trials have given melanoma the reputation of being a graveyard for drug development.

Two main approaches are posed to change this grim picture; one based on immunotherapy with immune-activating antibodies or with adoptive cell transfer (ACT) therapy, and another based on small molecule—targeted therapies that specifically block pathways activated by oncogenes. Antigen-specific T cells reactive to infectious pathogens and tumor antigens generated in vitro and adoptively transferred to patients can provide clinical benefi t.^5-7 Response rates up to 70% have been achieved with an optimized combination of ex vivo, clonally expanded, tumor infiltrating lymphocytes (TILs), lymphodepletion, and IL-2 helper cytokine administration.^8-11 To make this approach more widely applicable and to avoid the long time required to expand antigen-specific cells ex vivo, T-cell receptor (TCR) engineering has been developed and tested in humans.¹² In addition, the improved knowledge of oncogenic events in melanoma has led to the development of specific oncogenic inhibitors and their very successful initial testing in clinical trials.^13-15

ADVANCES IN IMMUNOTHERAPY FOR

MELANOMA

The 2 major areas of progress in immunotherapy for melanoma are the clinical development of a series of immune-stimulating antibodies, which provide evidence that modulating the immune system can lead to objective and durable tumor responses, and the ex vivo generation of large quantities of tumor antigen—specific lymphocytes that have powerful antitumor activity when given in repeated infusions to patients with metastatic melanoma.

CTLA4-Blocking Antibodies

Costimulatory and coinhibitory molecules are key players in the activation step of the adaptive immune system and regulate the ability of antigen-specific T cells to expand and gain effector functions.¹⁶ CTLA4 has a pivotal role in this interaction, dampening immune responses to self-antigens.¹⁷ Antibodies that block CTLA4 have been in clinical development since 2001, providing evidence of benefit in some patients but without a definitive clinical trial leading to regulatory licensing.^18,19 The main reason for this lack of favorable clinical trial outcomes to date is the low objective response rate—in the range of 5% to 15%—in most series of patients with metastatic melanoma treated with the immunoglobulin (Ig) IgG1 anti-CTLA4 antibody ipilimumab (MDX-010, Medarex) and the IgG2 antibody tremelimumab (CP-675,206).^20-26 Most patients with objective tumor regression do have durable responses, but these are achieved at the cost of clinically significant inflammatory and autoimmune toxicities (grade 3 to 4) in 20% to 30% of patients.²⁷ The small percentage of patients with a response who are seemingly cured—relapses beyond 2 years are extremely rare—have not been enough to shift the overall survival curve compared with standard chemotherapy in the first reported, pivotal, randomized phase III clinical trial.²⁸ Overall, the proof of concept of antitumor activity with CTLA4 blockade has been achieved with a reproducible antitumor activity that is at least comparable to the FDA approved high-dose IL-2, but with markedly lower toxicities since IL-2 needs to be administered in an intensive care unit-like setting. The next steps should be guided to elucidate which subsets of patients are more likely to respond to this immune manipulation, as well as addressing the mechanisms leading to lack of tumor response.

Other-Immune Modulating Antibodies: Anti-PD-1, Anti-CD40, Anti-CD137, and Anti-OX40

Programmed-death 1 (PD-1) receptor

Immune-activating antibodies beyond antibodies that block CTLA4 are being clinically developed.²⁹ Similar to CTLA4, the PD-1 receptor is a negative immune-regulatory receptor expressed by activated lymphocytes that can be blocked with specific antibodies. PD-1 is crucial in modulating T-cell exhaustion, and its blockade may reactivate lymphocytes with antitumor activity.³⁰ A blocking antibody to PD-1 (MDX-1106/ONO-4538) and to its ligand are in clinical development, with early evidence of antitumor activity in some patients with melanoma.

CD40

CD40 is a key molecule required for the generation of fully functional CD8+ CTL, because it bypasses the need of CD4+ T-helper cells.^31-33 An activating antibody to CD40 (CP-870,893) is also in clinical development and has shown single-agent activity in patients with metastatic melanoma in phase I trials.³⁴

CD137 and OX40

The tumor-necrosis factor (TNF) super-family receptor CD137 (4-1BB) provides costimulatory signals to T cells.29,35 Administration of activating antibodies to CD137 resulted in regression of tumors in animal models,³⁵ which has opened the clinical testing of an antibody (BMS-663513) under clinical development for melanoma.29 OX40 is another member of the TNFR family (TNFR4), which is expressed on activated but not on resting CD4 cells. Its primarily role is to act as a late costimulatory receptor for CD4+ T cells. A fully murine antibody activating OX40 has been tested with limited activity, and humanized and fully human antibodies are advancing to the clinic.

Overall, these immune modulating antibodies have reproducible activity in patients with metastatic melanoma, to a level that is not too different from anti-CTLA4 antibodies. The potential of combining these antibodies to fully modulate a cancer-fighting immune system may lead to improved outcomes as demonstrated in preclinical models.³⁶

Adoptive Cell Transfer Immunotherapy in Melanoma

Most patients with metastatic melanoma do not mount efficient adaptive immune responses to cancer, either spontaneously or after vaccination. A main reason is the lack of nontolerized T cells that have been endogenously selected with TCRs specifically recognizing melanoma antigens and exerting antitumor cytotoxic activity. To overcome this limitation, ACT approaches have been designed with the common goal of increasing the pool of melanoma-targeted killer lymphocytes. Three main sources to generate large quantities of tumor antigen—specific T lymphocytes for ACT include: (1) autologous TILs expanded from tumor biopsies,¹⁰ (2) cellular cloning of antigen-specific lymphocytes expanded from peripheral blood mononuclear cells (PBMCs),^6,37 and (3) PBMCs genetically redirected to become tumor specifi c using viral vector-mediated transduction of TCR chains.^12,38

Tumor-infiltrating lymphocytes for adoptive cell transfer therapy

The basic approach includes the harvesting of patient tumors, which are minced and placed in ex vivo cell-culture systems with cytokines that allow the expansion of lymphocytes able to infiltrate tumors. These lymphocytes are cultured to large numbers and are then infused back into the patients from whom the tumors were harvested. Conditioning patients with chemotherapy or radiation therapy before infusing these lymphocytes back into the patient allows partially depleting the endogenous lymphocytes, providing an advantage for the ex vivo expanded TILs to repopulate the host. The TILs are further driven to expansion by the administration of high-dose IL-2. Response rates of up to 50% have been achieved using this approach, with an optimal combination of ex vivo clonally expanded TIL, lymphodepletion, and IL-2 helper cytokine administration,^8-10 and up to 70% when TIL are administered after a myelodepleting regimen.¹¹

Cellular cloning of antigen-specific T cells stimulated

from peripheral blood

Repetitive ex vivo stimulation of PBMCs with peptide antigens frequently allows the expansion of rare T cells specific for cancer. This requires a prolonged process of weekly antigen stimulation and cellular expansion of antigen-reactive cells, eventually providing large quantities of antigen- specific lymphocytes for ACT. Using this approach, occasional patients with metastatic melanoma have been shown to have objective tumor responses against melanosomal and cancer testis antigens.^6,37

T cell receptor—engineered adoptive cell transfer

using peripheral blood cells

Obtaining large numbers of tumor antigen—specific lymphocytes by expanding them from low- frequency cells in peripheral blood requires several weeks of ex vivo lymphocyte manipulation. When these cells are obtained from higher frequency tumor-specific lymphocytes from TILs, limitations include the need for tumor harvesting and for TILs to be present in that lesion, which allows their ex vivo expansion. What provides the fine antigen specificity of the tumor-specific lymphocytes used for ACT is just 2 genes—the alpha and beta chains of their TCRs. Since experimental data has demonstrated that the transfer of the 2 TCR genes is necessary and sufficient to endow recipient T cells with the specificity of donor cells,³⁹ then high-efficiency gene-transfer vectors can be used to genetically engineer lymphocytes with a new antigen specificity by transferring these 2 TCR genes.⁴⁰ The pioneering work by investigators at the Surgery Branch of the National Cancer Institute provided proof of principle that the ACT of TCR- engineered lymphocytes in humans is feasible and leads to objective tumor responses in patients with metastatic melanoma.^12,38 Using lymphocytes genetically modified to express 2 different TCRs, the response rate of ACT of TCR transgenic cells to patients with metastatic melanoma was in the range of 25%.³⁸ This is an approach that, when optimized with further clinical testing, has the potential to be more extensively used compared with TILs or peripheral-blood T-cell cloning, provided that the TCRs are matched with the major histocompatibility complex and peptide antigen presented by each cancer.

ADVANCES IN TARGETED THERAPY FOR

MELANOMA

Improved knowledge on the oncogenic events in melanoma has promoted the recognition that most of these lesions have mutually exclusive activating mutations in the mitogen-activated protein kinase (MAPK) pathway involving c-Kit, NRAS, or BRAF genes.⁴¹ These mutations render melanoma cells independent of the normal receptor tyrosine kinase (RTK)—mediated pathway regulation, and constitutively drive melanoma cells to proliferation and survival.

BRAF and Mek Inhibitors for Melanomas with BRAF^V600E Mutations

The most frequent mutation in the MAPK pathway is in the BRAF (v-RAF murine sarcoma viral oncogene homolog B1) gene, with 60% to 70% of malignant melanomas harboring a single nucleotide transversion from thymidine to adenosine, leading to a substitution of valine by glutamic acid at position 600 (termed BRAF^V600E).⁴² This mutation is in the activation loop of the kinase domain and leads to a 500-fold increased level of activity compared with the wild-type protein kinase.⁴¹ Several inhibitors have been tested with the aim of inhibiting this driver mutation. Early efforts using the multi targeted TKI sorafenib, which has some activity as a CRAF inhibitor but low activity as a BRAF^V600E inhibitor, were disappointing in the clinic.⁴³ Other not fully RAF-specific inhibitors in clinical development include RAF-265 and XL-281. In addition, BRAF^V600E mutant tumors have been reported to be exquisitely sensitive to inhibitors of Mek, which is the immediately downstream molecule from BRAF in the MAPK pathway.⁴⁴ However, current experiences with Mek inhibitors like CI-1040, PD0325901, or AZD6244 in the clinic have yet to fulfill this promise.⁴⁵

A specific RAF inhibitor is in full clinical development with very promising results, and others may follow soon. PLX4032 specifically inhibits the mutated BRAF^V600E kinase because it works in the activated-kinase conformation. The closely related tool, compound PLX4720, has been fully characterized, and it inhibits the mutated BRAF^V600E kinase at 13 nM, while the wild-type kinase requires more than 10 times higher concentration (160 nM) to inhibit its function in kinase assays,¹³ predicting its high specificity for BRAF^V600E mutant cell lines. In addition, since it binds to a RAF-selective pocket, it results in low activity against most other non- RAF kinases, which require concentrations 100 to 1000 times higher for kinase inhibition.¹³ In phase I testing, more than 80% of patients with BRAF^V600E mutations treated with PLX4032 had evidence of objective tumor response by RECIST [Response Evaluation Criteria in Solid Tumors] criteria.^14,46 This is a huge step forward in the treatment of metastatic melanoma, but acquired resistance develops, and side effects have included the development of low- grade squamous cell carcinomas of keratoachantoma subtype. Further understanding of this pathway, the effects of durable inhibition of oncogenic and wild-type BRAF, as well as the means by which tumor cells may escape this inhibition, will be areas of active investigation.

C-kit and NRAS Mutations in Melanoma

There are 2 other mutually exclusive activating mutations in smaller subsets of melanoma. Less than 5% of all melanomas, but up to 30% of acral, mucosal, and lentigo maligna melanoma, have activating mutations in c-kit gene.⁴⁷ These mutations are similar to those found in gastrointestinal stromal tumors and predispose to objective tumor responses to imatinib.^15,48 Approximately 15% of melanomas have activating mutations in NRAS genes, which also activate the MAPK pathway.⁴⁹ However, mutations in Ras have not been effectively targeted with drug therapy, and no specific targeted approach has provided benefit in the clinic in patients with this mutation.

CONCLUSIONS

Metastatic melanoma has been considered one of the most treatment-resistant cancer histologies. This notion is rapidly being replaced through the therapeutic advances derived from the wealth of knowledge about the oncogenic events that drive this cancer and its interaction with the immune system. The advanced knowledge arising from basic laboratory research is at the stage of efficient translation into the clinical setting, with demonstrable advances in treatment approaches for patients with metastatic melanoma. It is an exciting time for clinical translational research in this type of cancer.

Antoni Ribas, MD, is associate professor, Department of Medicine, Division of Hematology/Oncology, University of California, Los Angeles (UCLA), Los Angeles, California; associate professor, Department of Surgery, Division of Surgical Oncology, UCLA; and director, Tumor Immunotherapy Program Area, Jonsson Comprehensive Cancer Center, UCLA.

DISCLOSURES: Antoni Ribas has received honoraria from Roche and MannKind Corporation, and research support from Pfizer Inc.

REFERENCES

1. Tsao H, Atkins MB, Sober AJ. Management of cutaneous melanoma [Erratum published in N Engl J Med.2004;351(23):2461]. N Engl J Med. 2004;351(10):998-1012.

2. Chapman PB, Einhorn LH, Meyers ML, et al. Phase III multicenter randomized trial of the Dartmouth regimen versus dacarbazine in patients with metastatic melanoma. J Clin Oncol.1999;17(9):2745-2751.

3. Eigentler TK, Caroli UM, Radny P, Garbe C. Palliative therapy of disseminated malignant melanoma: a systematic review of 41 randomised clinical trials. Lancet Oncol. 2003;4(12):748-759.

4. Schadendorf D, Ugurel S, Schuler-Thurner B, et al. Dacarbazine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) in first-line treatment of patients with metastatic melanoma: a randomized phase III trial of the DC study group of the DeCOG. Ann Oncol.2006;17(4):563-570.

5. Walter EA, Greenberg PD, Gilbert MJ, et al. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N Engl J Med.1995;333(16):1038-1044.

6. Yee C, Thompson JA, Byrd D, et al. Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci U S A.2002;99(25):16168-16173.

7. Leen AM, Myers GD, Sili U, et al. Monoculture-derived T lymphocytes specifi c for multiple viruses expand and produce clinically relevant effects in immunocompromised individuals. Nat Med.2006;12(10):1160-1166.

8. Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science. 2002;298(5594):850-854.

9. Dudley ME, Wunderlich JR, Yang JC, et al. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J Clin Oncol.2005;23(10):2346-2357.

10. Rosenberg SA, Restifo NP, Yang JC, et al. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer.2008;8(4):299-308.

11. Dudley ME, Yang JC, Sherry R, et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol.2008;26(32):5233-5239.

12. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 2006;314(5796):126-129.

13. Tsai J, Lee JT, Wang W, et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci U S A.2008;105(8):3041-3046.

14. Flaherty K, Puzanov I, Sosman J, et al. Phase I study of PLX4032: Proof of concept for V600E BRAFmutation as a therapeutic target in human cancer [JCO abstract 9000]. J Clin Oncol. 2009;27(15s)(suppl).

15. Hodi FS, Friedlander P, Corless CL, et al. Major response to imatinib mesylate in KIT-mutated melanoma. J Clin Oncol.

2008;26(12):2046-2051.

16. Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol. 2004;4:336-347.

17. Chambers CA, Kuhns MS, Egen JG, Allison JP. CTLA- 4-mediated inhibition in regulation of T cell responses: mechanisms and manipulation in tumor immunotherapy. Annu Rev Immunol. 2001;9:565-594.

18. Ribas A. Update on immunotherapy for melanoma. J Natl

Compr Canc Netw. 2006;4(7):687-694.

19. Ribas A, Hanson DC, Noe DA, et al. Tremelimumab (CP-675,206), a cytotoxic T lymphocyte associated antigen 4 blocking monoclonal antibody in clinical development for patients with cancer. Oncologist.2007;12(7):873-883.

20. Ribas A, Camacho LH, Lopez-Berestein G, 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. 2005;23(35):8968-8977.

21. Downey SG, Klapper JA, Smith FO, et al. Prognostic factors related to clinical response in patients with metastatic melanoma treated by CTL-associated antigen-4 blockade. Clin Cancer Res. 2007;13(22 Pt 1):6681-6688.

22. Weber JS, O’Day S, Urba W, et al. Phase I/II study of ipilimumab for patients with metastatic melanoma. J Clin Oncol. 2008;26(36):5950-5956.

23. Camacho LH, Antonia S, Sosman J, et al. Phase I/II trial of tremelimumab in patients with metastatic melanoma. J Clin Oncol. 2009;27(7):1075-1081.

24. Wolchok JD, Neyns B, Linette G, et al. Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study. Lancet Oncol. 2010;11(2);155-164.

25. Kirkwood JM, Lorigan P, Hersey P, et al. Phase II trial of tremelimumab (CP-675,206) in patients with advanced refractory or relapsed melanoma. Clin Cancer Res. 2010;16(3):1042-1048.

26. Ku GY, Yuan J, Page DB, et al. Single-institution experience with ipilimumab in advanced melanoma patients in the compassionate use setting: lymphocyte count after 2 doses correlates with survival. Cancer. 2010;116(7):1767-1775.

27. Ribas A. Overcoming immunologic tolerance to melanoma: targeting CTLA-4 with tremelimumab (CP-675,206). Oncologist. 2008;13(suppl 4):10-15.

28. Ribas A, Hauschild A, Kefford R, et al. Phase III, Open- label, randomized, comparative study of tremelimumab (CP-675,206) and chemotherapy (temozolomide or dacarbazine) in patients with advanced melanoma [JCO abstract LBA9011]. J Clin Oncol. 2008;26(suppl):485s.

29. Melero I, Hervas-Stubbs S, Glennie M, et al. Immunostimulatory monoclonal antibodies for cancer therapy. Nat Rev Cancer.2007;7(2):95-106.

30. Barber DL, Wherry EJ, Masopust D, et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature. 2006;439(7077):682-687.

31. Schoenberger SP, Toes RE, van der Voort EI, et al. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature. 1998;393(6684):480-483.

32. Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature. 1998;393(6684): 474-478.

33. Bennett SR, Carbone FR, Karamalis F, et al. Help for

cytotoxic-T-cell responses is mediated by CD40 signalling.

Nature. 1998;393(6684):478-480.

34. Vonderheide RH, Flaherty KT, Khalil M, et al. Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. J Clin Oncol. 2007;25(7):876-883.

35. Melero I, Shuford WW, Newby SA, et al. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med. 1997;3(6):682-685.

36. Uno T, Takeda K, Kojima Y, et al. Eradication of established tumors in mice by a combination antibody-based therapy. Nat Med.2006;12(6):693-698.

37. Hunder NN, Wallen H, Cao J, et al. Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med.2008;358(25):2698-2703.

38. Johnson LA, Morgan RA, Dudley ME, et al. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood. 2009;114(3):535-546.

39. Dembic Z, Haas W, Zamoyska R, et al. Transfection of the CD8 gene enhances T-cell recognition. Nature.1987;326(6112):510-511.

40. Schumacher TN. T-cell-receptor gene therapy. Nat Rev Immunol. 2002;2(7):512-519.

41. Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted therapy. Nature.2007;445(7130):851-857.

42. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature.2002;417(6892):949-954.

43. Smalley KS, Flaherty KT. Integrating BRAF/MEK inhibitors into combination therapy for melanoma. Br J Cancer. 2009;100(3):431-435.

44. Solit DB, Garraway LA, Pratilas CA, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature. 2006;439(7074):358-362.

45. Banerji U, Camidge DR, Verheul HM, et al. The first-in- human study of the hydrogen sulfate (Hyd-sulfate) capsule of the MEK1/2 inhibitor AZD6244 (ARRY-142886): a phase I open-label multicenter trial in patients with advanced cancer. Clin Cancer Res.2010;16(5):1613-1623.

46. Chapman P, Puzanov I, Sosman J, et al. Completion of a phase I trial of the oncogenic BRAF-selective inhibitor PLX4032: early efficacy signal confi rmed in advanced melanoma. Proc. ECCO-ESMO, Berlin, Germany, 2009 (Abstract 6BA).

47. Curtin JA, Busam K, Pinkel D, Bastian BC. Somatic activation of KIT in distinct subtypes of melanoma. J Clin Oncol.2006;24(26):4340-4346.

48. Jiang X, Zhou J, Yuen NK, et al. Imatinib targeting of KIT-mutant oncoprotein in melanoma. Clin Cancer Res. 2008;14(23):7726-7732.

49. Curtin JA, Fridlyand J, Kageshita T, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353(20):2135-2147.