Non–small Cell Lung Cancer: Novel Immunotherapy Strategies

Cancer cells derive from normal cells by genetic mutations. The alterations and rearrangements in these mutations should be viewed by the immune system as “non-self.” The presence of cancer cells should therefore result in the activation of the immune system. The cytolytic power of the immune system can be harnessed to destroy cells that express “non-self” antigens on their surfaces; this effect is observed in the rejection of foreign tissue following organ transplantation. The goal of immunotherapy in non–small cell lung cancer (NSCLC) is to manipulate the immune system to recognize the “non-self” antigens that are expressed in tumor cells. In order to achieve this, the immunotherapeutic agent must overcome the defenses of the tumor cells, such as surface proteins that downregulate the host T cells, preventing the immune system from being activated by the tumor.

There are 2 approaches to manipulating the immune system in patients with NSCLC as well as other malignancies. One is the use of molecules that directly modulate the immune system. Such molecules include talactoferrin, BMS-936558 (an anti-PD1 IgG4 monoclonal antibody), and ipilimumab (Yervoy, Bristol-Myers Squibb), which has been approved for the treatment of melanoma. The second approach is to employ tumor-specific vaccines such as melanoma-associated antigen-A3 (MAGE-A3) vaccine, the granulocyte-macrophage colony-stimulating factor (GMCSF) vaccine (GVAX, Biosante Pharmaceuticals), belagenpumatucel-L (Lucanix, NovaRx) and the transforming growth factor β2 (TGFβ2) antisense vaccine combined with human recombinant GMCSF (TAG). There is also a new derivation of the latter, with activity against a wider spectrum of TGFβ proteins, a bi-shRNA-furin and GMCSF augmented autologous tumor cell vaccine (FANG, Gradalis, Inc).

Immunomodulation

There are a variety of immunomodulators that show potential to be used for immunotherapy in NSCLC. For example, the 80 kilodalton human recombinant lactoferrin protein talactoferrin is produced in Aspergillus niger. Lactoferrin is expressed in immune cells and throughout the body. This protein is found in high concentrations in milk and colostrum, and has a central role in establishing the immune system, including gut-associated lymphoid tissue (GALT), in infants. Oral talactoferrin is transported by M cells into Peyer’s patches, structures in the GALT that act as antigenic sampling sites. They detect luminal antigens, induce an immunological response, and prevent antigen translocation across the mucosal epithelium. When talactoferrin is introduced into Peyer’s patches, it induces migration and maturation of dendritic cells.¹

BMS-936558 (MDX-1106) is a human IgG4 monoclonal antibody directed against PD1. The programmed death receptor PD1 has a central role in T-cell regulation. BMS-936558 binds with high affinity to PD1, blocking its interaction with the ligands PDL-1 and PDL-2, thereby influencing T-cell regulation.² BMS-936558 has no antibody-dependent or cell-dependent cytotoxicity (ADCC/CDCC).

Ipilimumab is a monoclonal antibody directed against cytotoxic T lymphocyte-associated antigen 4 (CTLA4).³ CTLA4 blocks T-cell activation. Therefore, by blocking CTLA4, ipilimumab promotes T-cell activation and augments the immune response to the endogenous tumor cell population.

Vaccine Therapy

Several conditions must be satisfied in order for a vaccine to be successful. First of all, the vaccine must take advantage of a tumor-associated antigen that is expressed almost, if not completely, exclusively in the tumor cell population rather than in healthy cells. Mutations in the cancer cells should confer this exclusivity of novel antigen expression. A successful vaccine should also induce an effective cellular or humoral immune response. Many modern vaccines use adjuvants, which enhance the response in several ways: they promote the recruitment of antigen-presenting cells; they modulate cytokines to augment the immune response; and they help to sustain antigen presentation so that memory can be developed, which results in a more vigorous immune response to the tumor vaccine.

Many of these vaccines are now engineered to interfere with immune tolerance to tumor antigens, as in the use of “knockdown” TGFβ2. TGFβ2 inhibits processes such as activation of antigen-presenting cells, natural killer (NK) cell function, and T-cell and B-cell functions. Thus, knocking down expression of TGFβ2 would augment the activation of these components.⁴Another example of this approach is the inhibition of CTLA4, which is the mechanism of action for ipilimumab.

A lot of research is dedicated to the identification of antigen sources that could be used for immunotherapy in NSCLC in order to present these antigens to the dendritic cell population in the human body. These include naked DNA, mRNA, viral-like particles, viral vectors, bacterial vectors, recombinant proteins, novel peptides and proteins, and tumor cells (Figure).⁵ This article will focus on recombinant proteins, peptides, and tumor cells.

Figure. Antigen Sources for Immunotherapy in NSCLC

A lot of research is dedicated to the identification of antigen sources that could be used for immunotherapy in NSCLC.
Source: Romero P. Clin Lung Cancer. 2008;9(suppl 1):S28-S36.

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Protein-based vaccines

The MAGE-A3 vaccine is a protein vaccine that is currently in phase 3 trials. MAGE-A3 is an almost exclusively tumor-specific antigen. MAGE-A3 has increased expression with increasing stage of disease and is associated with a poor prognosis. In lung cancer, approximately 30% of tumors express the MAGE-A3 antigen. The MAGE-A3 vaccine is composed of purified recombinant MAGE-A3 protein fused to protein D. The vaccine incorporates a second-generation adjuvant system.^6-9

Peptide-based vaccines

The BLP25 liposome vaccine L-BLP25 (Stimuvax, Oncothyreon) simulates the mucin MUC1.¹⁰ The normal form of MUC1 is expressed on a number of epithelial surfaces throughout the human body, particularly the oral and gastrointestinal mucosa. In most epithelial cancers, an aberrant form of MUC1 is highly expressed, rendering it an attractive therapeutic target. Stimuvax mimics an exposed core peptide that forms a part of the oncogenic protein. MUC1 is involved in cell-cell adhesion. The aberrant form of MUC1 functions as an oncogene, promoting invasion and metastasis of tumor cells and conferring resistance to genotoxic agents. These characteristics contribute to the poor prognosis associated with the aberrant form of MUC1. MUC1 can affect antitumor immune response, as it endogenously suppresses T-cell function, impairs dendritic cell function, and prevents NK cell binding.¹¹ One of the key characteristics of MUC1 that enable its role in promoting tumor growth is its strongly glycosylated extracellular region, which is known to bind growth factors. By accumulating growth factors near the tumor cell surface, MUC1 promotes cancer cell growth. Furthermore, the hydrophilic nature of the glycoslyated region of MUC1 acts as a barrier to hydrophobic anticancer drugs.

MUC1 is observed in a variety of epithelial malignancies, expressed in approximately 90% of breast cancer; almost all NSCLC and nasopharyngeal cancer; about 80% of renal cell, colorectal, ovarian, and head and neck cancer; and in 70% to 80% of mesothelioma, gastric, prostate, and pancreatic cancers (Table). MUC1 also occurs in more than one-half of multiple myeloma and in approximately one-third of esophageal cancers.

Table. Overview of MUC1 Expression in Human Tumors

Key: H&N SCC — head and neck squamous cell carcinoma; NSCLC — non–small cell lung cancer; RCC — renal cell carcinoma
Sources: 1. Rakha EA, et al. Mod Pathol. 2005;18:1295-1304;
2. Data on file, Merck Serono; 3. Langner C, et al. Mod Pathol. 2004;17:180-188; 4. Baldus SE, et al. Histopathology. 2002;40:440-449; 5. Chauhan SC, et al. Mod Pathol. 2006;19:1386-1394; 6. Croce MV, et al; Pathol Oncol Res. 2001;7:284-291; 7. Zhong XY, et al. Zentralbl Pathol. 1993;139:281-286. 8. Utsunomiya T, et al. Clin Cancer Res. 1998;4:2605-2614; 9. DeNardo SJ, et al. Clin Cancer Res. 2005;11:7187S-7194S; 10. Qu CF, et al. Br J Cancer. 2004;91:2086-2093; 11. Saad RS, et al. Diagn Cytopathol. 2005;32:156-159; 12. Pileri S, et al. Eur J Haematol Suppl. 1989;51:52- 59; 13. Cloosen S, et al. Br J Haematol. 2006;135:513-516; 14. Kijima H, et al. Anticancer Res. 2001;21:1285-1289.

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Stimuvax contains the BLP25 lipopeptide as well as the adjuvant monophosphoryl lipid A to enhance the immune response. Liposomal components include cholesterol, dimyristoyl phosphatidylglycerol (DMPG), and dipalmitoyl phosphatidylcholine (DPPC), which have a direct effect on the immune system and also enhance targeted uptake by dendritic cells. The proposed mechanism is that Stimuvax is taken up by antigen-presenting cells, then processed and presented to both major histocompatibility complex (MHC) class I and class II cells.¹⁰ The MHC class I cells are cytotoxic T cells that are then activated and will directly target cancer cells that express the MUC1 antigen. The class II cells are helper T cells, which in turn stimulate the B-cell system and the production of endogenous antibodies. These antibodies result in antigen-dependent, cell-mediated cytotoxicity against the tumor cell population. Both of these mechanisms result in cancer cell death.

Tumor cell-based vaccines

There are a variety of tumor cell-based vaccines in development. Lucanix was derived from 4 human NSCLC cell lines, which were transfected with pCHEK/human TGFβ2 antisense (pCHEK/HBA-2), a vector containing the TGFβ2 antisense transgene, and then irradiated.¹²

The tumor cell-based TGFβ2 Antisense + rhGMCSF (TAG) vaccine was developed using a more sophisticated approach. The patient’s autologous tumor cells are transfected with TGFβ2 antisense and recombinant human GMCSF, irradiated, and then used in the vaccination process.¹³

Much of the immune inhibitory activity of the TGFβ proteins is known to be associated with the isoform TGFβ1, which is unaffected by TGFβ2 antisense. By combining a furin RNA inhibitor with the immunostimulation provided by GMCSF, researchers were able to produce the FANG vaccine, with broad TGFβ-blocking activity as well as the ability to activate T cells.¹⁴

In summary, there are reasons for optimism in the future of NSCLC therapy. There has been recognition and characterization of tumor-associated antigens that have a relatively high level of specificity. Antigen processing and presentation is now better understood, particularly the role of antigen-presenting cells such as dendritic cells. The role of immunomodulatory cytokines, including TGFβ2, interleukin (IL)-2, and IL-10, that are capable of augmenting the immune response has been clarified. Furthermore, the role of homeostatic controls such as suppressor T cells, CD4⁺ T cells, and CD25⁺ T cells, has been recognized. Tools have been developed during the past decade that allow the manipulation and monitoring of the immune response with greater specificity. Finally, the recent positive results of clinical trials of immunotherapeutic agents in prostate cancer and melanoma lead to an expectation of positive results in other highly refractory malignancies such as NSCLC.

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