Mocetinostat + checkpoint inhibitorsSpectrum-Selective HDAc inHibitor

the rationale and therapeutic promise of combining HDAc inhibitors with immune

checkpoint inhibitors

February 2016

Mocetinostat

the rationale and therapeutic promise of combining HDAc inhibitors with immune checkpoint inhibitors

the Hypothesis for combining HDAc inhibitors with immune checkpoint inhibitors Major breakthroughs in the treatment of patients with multiple types of cancer have been achieved by drugs targeting the immune checkpoint pathway. Treatment with agents targeting the programmed death receptor-1 (PD-1) and ligand (PD-L1) have resulted in robust tumor responses and survival benefit in patients with a number of tumor types, including melanoma, renal, lung, head and neck, and bladder cancers.23,24 Despite the promise of PD-1/PDL-1 inhibitors as single-agent therapies, the majority of patients either do not respond or eventually progress due to innate or acquired resistance. Possible mechanisms of resistance to immune checkpoint inhibitors include (i) downregulation of PD-L1; (ii) a decrease in the number of tumor-infiltrating lymphocytes; (iii) the inability of T-effector lymphocytes to recognize tumor cells; and (iv) the presence of negative (immune suppressive) immune regulatory cells in the tumor microenvironment including regulatory T cells [Tregs] and myeloid-derived suppressor cells [MDSCs].16 Research is now focused on the co-administration of PD-L1 inhibitors with other therapeutic agents to treat or prevent resistance potentially resulting in enhanced activity of PD-1/PD-L1 pathway inhibitors. Based on scientific rationale, one key class of molecules for immune checkpoint inhibitor-based combination therapy are spectrum-selective histone de-acetylase (HDAC) inhibitors.

There is growing evidence that the combined anti-tumor and immunomodulatory effects of Class I HDAC inhibitors may increase the efficacy of immune checkpoint inhibitors.17,22,31

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There is growing evidence that the combined anti-tumor and immunomodulatory effects of Class I HDAC inhibitors may increase the efficacy of immune checkpoint inhibitors.17,22,31

clASS i HDAc inHibitorS

PD-1 serves as an “on/off” switch on cytotoxic T cells, while its ligand, PD-L1, is overexpressed in a variety of tumors as well as tumor immune-infiltrating cells. Binding of PD-1 to PD-L1 deactivates CD4+ and CD8+ T cells and inhibits cytokine production, allowing tumors to escape and survive.5 Antibodies that block the PD-1 receptor have been shown to restore T-cell function and result in profound clinical responses in some patients with various solid tumors, including non–small cell lung cancer (NSCLC), bladder, and head and neck carcinomas.24 Similarly, antibody-mediated blockade of PD-L1 has induced durable tumor regression (objective response rate of 6%–32%) and prolonged stabilization of disease (rates of 12%–41% at 24 weeks) in patients with several advanced cancers, including NSCLC, melanoma and renal-cell cancer.5,23,28

The rationale for combining these two therapeutic classes is based on the ability of Class I HDAC inhibitors to:

• ”Prime” the adaptive immune system to program and activate naïve T cells to target tumor antigens2,19

• “Prime” the innate immune system to stimulate natural killer [nk] cell activity against tumor cells14,26

• Increase the number of cancer-killing CD8+ and CD4+ tumor-infiltrating lymphocytes2,14

• Deplete immunosuppressive cells, such as Tregs and MDSCs17,25

• Make tumors more recognizable to the immune system by upregulating PD-L1 and the major histocompatibility (MHC) Class I and II molecules that present tumor antigens13,14,17

In addition, combination therapy with checkpoint blockade immunotherapy has the added advantage of inducing a memory response unattainable with single-agent cytotoxic and targeted therapies.13

the complementary mechanisms of Action of HDAc inhibitors and checkpoint inhibitors In addition to the immunostimulatory effects noted above, several studies show that certain HDAC inhibitors are relatively nontoxic to normal cells or tissues, but are selectively cytotoxic against a wide range of cancer cells. These characteristics make them an attractive partner for combination therapy with treatments such as PD-L1 inhibitors that reverse the defective immune checkpoint regulation associated with cancer cells.1 Supporting this concept, recent data demonstrate that Mirati’s Class I/IV HDAC inhibitor, mocetinostat, generated robust and durable upregulation of PD-L1 and PD-L2 in melanoma cell lines, as well as patient tumor samples,31 and also addressed other potential immune checkpoint inhibitor resistance mechanisms by increasing tumor cell immunogenicity and enhancing the T cell–mediated adaptive immune response.17

The immunomodulatory properties of mocetinostat and other Class I HDAC inhibitors are mediated through several distinct routes that complement checkpoint inhibition, as summarized in Table 1.

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tHe role of HDAc inHibitorS in cancer

HDACs are comprised of a large family of proteins that are divided into four groups, including Class I (HDAC 1, 2, 3, 8), Class II (HDAC 4, 5, 6, 7, 9, 10), Class III (Sirtuins), and Class IV (HDAC 11). HDACs and their counterparts, histone acetyl transferases (HATs), work together to regulate gene activity by respectively deleting or adding acetyl groups to histone and non-histone lysine molecules.3 The dynamic and tightly controlled process of histone acetylation is important in the regulation of gene expression. In human cancers, histone lysine acetylation can become dysregulated through the upregulation of HDACs and/or inactivation of HATs, resulting in the activation or silencing of genes that contribute to a cell’s transformation into cancer. Dysregulation of histone acetylation can lead to aberrant gene expression which, in turn, can activate oncogenes, inactivate tumor suppressor genes and lead to uncontrolled cell growth and avoidance of immune system detection – ultimately resulting in tumor progression.17

HDAC inhibitors have the potential to restore normal gene expression and also display potent anti-tumor effects in vitro.1,26 As single agents, certain HDAC inhibitors have demonstrated clinical efficacy in cutaneous T-cell lymphoma and are approved for that indication. HDAC inhibitors have also shown single-agent clinical activity in diffuse large B-cell lymphoma (DLBCL),15 malignant melanoma,30 and a variety of other hematological malignancies and solid cancers.1,17

Mocetinostat

enhancement of tumor antigenicity by:

• upregulating antigens necessary for co-stimulation in multiple tumor types19,20,26

• elevating proteins associated with the processing and presentation of tumor peptides.9,15 This increases the expression of certain glycoproteins on tumor cells (but not normal cells) making them sensitive to destruction by the innate immune system4,32

• Increasing expression of other phagocytosis (“eat-me”) signals9

Induction of immunogenic cell death:

• Vorinostat (pan-inhibitor of Class I and Class II HDACs) triggered apoptosis and stimulated the release of important mediators of immunogenic cell death17,27

• Christiansen et al. showed that MC38 colon carcinoma cells treated with a pan-HDAC inhibitor, were efficiently taken up by dendritic cells10

enhance T-effector cell function:

• HDAC inhibitors enhance cytotoxic CD8+ T-cell activity and have no (or minimal) effects on CD4+ T cells2,17

Decreased Treg cell function:

• Class I HDAC inhibitors reduced Treg numbers and Treg activity in multiple tumor models6

• In addition to effects on Tregs, treatment with Class I HDAC inhibitors transformed tumor-induced myeloid-derived suppressor cells into cancer-eliminating cells in vitro33

• Class I HDAC inhibitors can restore antigen expression on tumor-associated macrophages, reversing immune suppression and delaying tumor growth7,12

HDAC inhibitors:

• upregulate expression of signals on cancer cells, making it easier for T-effector cells to recognize and eliminate tumors9

• enhance dendritic cells’ recognition of the tumor as “non-self,” which primes T cells and other cancer-killing immune cells against the tumor11

• Drive the elimination of tumor cells and reduce tumor-induced immune suppression. eliminating cancer cells on the periphery of the tumor makes the core tumor mass more accessible to cytotoxic immune cells17

• Are distinguished from other cancer therapies by not only inducing apoptotic tumor cell death, but also by producing and presenting tumor-specific antigens17

• Increase ligands expressed by tumor cells that activate the innate immune system’s natural killer cells17

• Activate tumor-destroying CD8+ cytotoxic T cells and upregulate CD4+ and CD8+ memory T cells that recognize tumor antigens and lock them into memory for future identification and defense29

• Reverse the immune-suppressive tumor microenvironment by reducing tumor-driven recruitment of Tregs and myeloid-derived suppressor cells17

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table 1. immune functions Associated With class i HDAc inhibitors

clASS i HDAc inHibitor functionSignificAnce of HDAc inHibitorS

in cancer treatMent

note: CLASS II HDACS (HDACS 4–7/HDACS 8–10) AnD PAn-HDAC InHIBIToRS CAn HAVe IMMuno-SuPPReSSIVe eFFeCTS THAT CouLD CounTeRACT THe IMMunoSTIMuLAToRy ACTIonS oF CLASS I HDAC InHIBITIon.17

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mocetinostat, a Spectrum-Selective class i/iv HDAc inhibitor, may be better Suited than pan-HDAc inhibitors for combination treatment with pD-1/pD-l1 inhibitorsHDAC inhibitors are categorized by the types of HDACs they inhibit (see InSeT, Page 3: The Role of HDAC Inhibitors in Cancer).

Mocetinostat is a spectrum-selective Class I/IV HDAC inhibitor specifically targeting HDACs 1, 2, 3, and 11. Class I HDACs are of particular interest from an immunostimulatory and immune-priming perspective because of their effects on a broad range of immune system functions. For example, treatment with Class I HDAC inhibitors was shown to enhance nk cell and CD8+ T cell functions and reduce Treg numbers and function in multiple tumor models,17

resulting in an enhanced tumor immune response. In contrast, Class II HDAC inhibitors directly enhance the immunosuppressive function of murine Treg cells.17 In addition, Class II HDAC inhibitors (HDACs 4–7/HDACs 8–10) and pan-HDAC inhibitors can have immunosuppressive effects that could counteract the immunostimulatory actions of Class I HDAC inhibition.17 Class I and II inhibitors have different effects on MDSCs that may be implicated in resistance to immune checkpoint inhibitors. This is highlighted by a study showing that inhibition of Class I HDACs caused highly immunosuppressive, tumor-induced MDSCs in the bone marrow to differentiate into macrophages and dendritic cells capable of stimulating an immune system attack on the tumor cells. This effect is distinct from the effects of pan-HDAC inhibition, which causes myeloid cells to differentiate into immunosuppressive MDSCs that limit immune system responsiveness.17,33 The immunosuppressive activity of pan-HDAC inhibitors is highlighted by the fact that they have been used to limit cytokine production and immune responses following allogenic transplant in autoimmune diseases, including rheumatoid arthritis.8,30 Therefore, data suggest that the use of mocetinostat, a Class I/IV HDAC inhibitor, would be more appropriate in combination with a checkpoint inhibitor than a Class II or pan-HDAC inhibitor, such as vorinostat (which could result in immunosuppression and a decrease in the activity of the checkpoint inhibitor).17,18,33

Further, while mocetinostat generated robust and durable upregulation of PD-L1 and PD-L2 in melanoma cell lines and patient tumor samples, these effects were not observed with HDAC 6 or Class IIa–specific inhibitors.31 This observation is consistent with a related study by Woods et al. showing that a treatment with a Class I HDAC inhibitor and PD-1 blocking antibodies in murine models of metastatic melanoma resulted in

delayed tumor growth and increased survival compared to either single agent.31 ongoing studies confirm the induction of PD-L1 expression following mocetinostat treatment of non-small cell lung cancer (nSCLC), head and neck squamous cell carcinoma (HnSCC), and urothelial cancer cell lines on mouse models (Mirati, data on file). In addition, mocetinostat induced the expression of molecules that mediate the presentation of tumor antigens including HLA-DR, HLA-A, and HLA-B or markers facilitating an innate nk cell–directed immune response, including MICA and MICB. Studies in syngeneic mouse models indicated that mocetinostat increased CD4/CD8+ T-effector lymphocytes and decreased tumor T regs in vivo (Mirati, data on file). These preclinical studies indicate that mocetinostat exhibits immunomodulatory properties consistent with Class I HDAC inhibitors.

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… data suggest that the use of mocetinostat, a Class I/IV HDAC inhibitor, would be more

appropriate in combination with a checkpoint inhibitor than a Class II or pan-HDAC,

such as vorinostat (which could result in immunosuppression and a decrease in the

activity of the checkpoint inhibitor).

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figure 1. mocetinoStAt: Key Anti-cAncer ActivitieS complementing pD-1/pD-l1 inHibition

Mocetinostat, a Class I/IV HDAC inhibitor, “primes” the immune system to attack a cancer cell by inducing immunogenic cell death and upregulating MHC Class I and II molecules, making the cancer cell more recognizable. Mocetinostat also upregulates

PD-L1, the target of PD-L1 antibodies. In addition, spectrum-selective HDAC inhibitors, such as mocetinostat, increase the number of CD8+ tumor-infiltrating lymphocytes

while depleting immunosuppressive cell types, such as T regs and MDSCs.

Moce�nostat

Tumor Immunity

Tumor Cell Markers

Tumor Cell

Tumor cell markers and immune cell types associatedwith increased tumor cell immunogenicity

HLA I

HLA II

MICA

MICB

CD86 (B7-2)

PD-L1

Immune Cell Types

Immune Cell

T-Regs

MDSC

T-Effector (CD8)

conclusion: immunogenicity and priming of the tumor microenvironmentThere is increasing evidence that indicates Class I HDAC inhibitors may enhance the efficacy of immune checkpoint inhibitors such as PD-L1 antagonists, especially spectrum-selective Class I HDAC inhibitors, like mocetinostat, that selectively target specific HDAC isoforms.

This evidence suggests that combining a spectrum-selective HDAC inhibitor with an immuno-oncology therapy may provide an important therapeutic option in treating a wide range of cancers, and provides a strong scientific rationale for further research in this area.

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Mocetinostat

FeBRuARy 2016 • MoCeTInoSTAT + CHeCkPoInT InHIBIToRS

1. Acharya MR, et al. Rational development of histone deacetylase inhibitors as anticancer agents: A review. Mol Pharmacol. 68.4 (2005):917-932.2. Agarwal P, et al. Gene regulation and chromatin remodeling by IL-12 and type

I IFn in programming for CD8 T cell effector function and memory. J Immunol. 183.3 (2009):1695-1704.

3. Akimova T, et al. Histone-protein deacetylases and T-cell immune responses. Blood. 119.11 (2012):2443-2451.

4. Armeanu S, et al. natural killer cell-mediated lysis of hepatoma cells via specific induction of nkG2D ligands by the histone deacetylase inhibitor sodium valproate. Cancer Res. 65 (2005):6321-6329.

5. Bramer JR, et al. Safety and activity of anti–PD-L1 antibody in patients with advanced cancer. n engl J Med. 366.26 (2012):2455-2465.

6. Bridle BW, et al. HDAC inhibition suppresses primary immune responses, enhances secondary immune responses, and abrogates autoimmunity during tumor immunotherapy. Mol Ther. 21.4 (2013):887-894.

7. Chang yC, et al. epigenetic control of MHC class II expression in tumor-associated macrophages by decoy receptor. Blood. 111.10 (2008):5054-5063.

8. Choi SW, et al. Histone deacetylase inhibition regulates inflammation and enhances Tregs after allogeneic hematopoietic cell transplantation in humans. Blood. 125.5 (2015):815-819.

9. Chou SD, et al. Histone acetylation regulates the cell type specific CIITA promoters, MHC class II expression and antigen presentation in tumor cells. Int Immunol. 17.11 (2005):1483-1494.

10. Christiansen AJ, et al. eradication of solid tumors using histone deacetylase inhibitors combined with immune-stimulating antibodies. Proc natl Acad Sci uSA. 108.10 (2011):4141-4146.

11. Dudek AM, et al. Immature, semi-mature, and fully mature dendritic cells: toward a DC-cancer cells interface that augments anticancer immunity. Front Immunol. 4 (2013):438.

12. Hagemann T, et al. ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. J Immunol. 176.8 (2006):5023-5032.

13. Harshman LC, et al. PD-1 blockade in renal cell carcinoma: to equilibrium and beyond. Cancer Immunol Res. 2 (2014):1132.

14. Jazirehi AR, et al. Histone deacetylase inhibitor sensitizes apoptosis-resistant melanomas to cytotoxic human T lymphocytes through regulation of TRAIL/DR5 pathway. J Immunol. 192.8 (2014):3981-3989.

15. khan An, et al. Histone deacetylase inhibitors induce TAP, LMP, tapasin genes and MHC class I antigen presentation by melanoma cells. Cancer Immunol Immunother. 57.5 (2008):647-654.

16. kim JW & eder JP. Prospects for targeting PD-1 and PD-L1 in various tumor types. oncology. 28.3 (2014):15-28.

17. kroesen M. HDAC inhibitors and immunotherapy; a double edged sword? oncotarget. 5.16 (2014):6558-6572.

18. Licciardi PV & karagiannis TC. Regulation of immune responses by histone deacetylase inhibitors. ISRn Hematol. 2012 (2012):690901.

19. Maeda T, et al. upregulation of costimulatory/adhesion molecules by histone deacetylase inhibitors in acute myeloid leukemia cells. Blood. 96.12 (2000):3847-3856.

20. Magner WJ, et al. Activation of MHC class I, II, and CD40 gene expression by histone deacetylase inhibitors. J Immunol. 165.12 (2000):7017-7024.

21. northrop Jk, et al. epigenetic remodeling of the IL-2 and IFn-gamma loci in memory CD8 T cells is influenced by CD4 T cells. J Immunol. 177.2 (2006):1062-1069.

22. oki y, et al. Immune regulatory effects of panobinostat in patients with Hodgkin lymphoma through modulation of serum cytokine levels and T-cell PD1 expression. Blood Cancer J. 4 (2014):e236.

23. Robert C, Schachter J, Long GV, et al. Pembrolizumab versus Ipilimumab in Advanced Melanoma. n engl J Med. 2015;372(26):2521.

24. Romano e & Romero P. The therapeutic promise of disrupting the PD-1/PD-L1 immune checkpoint in cancer: unleashing the CD8 T cell mediated anti-tumor activity results in significant, unprecedented clinical efficacy in various solid tumors. J Immunother Cancer. 3 (2015):15.

25. Shen L, et al. Class I histone deacetylase inhibitor entinostat suppresses regulatory T cells and enhances immunotherapies in renal and prostate cancer models. PLoS one. 7.1 (2012):e30815.

26. Skov S, et al. Cancer cells become susceptible to natural killer cell killing after exposure to histone deacetylase inhibitors due to glycogen synthase kinase-3-dependent expression of MHC class I-related chain A and B. Cancer Res. 65.23 (2005):11136-11145.

27. Sonnemann J, et al. The histone deacetylase inhibitor vorinostat induces calreticulin exposure in childhood brain tumour cells in vitro. Cancer Chemother Pharmacol. 66.3 (2010):611-616.

28. Topalian SL, et al. Safety, activity, and immune correlates of anti–PD-1 antibody in cancer. n engl J Med. 366.26 (2012):2443-2454.

29. Töpfer k, et al. Tumor evasion from T cell surveillance. J Biomed Biotechnol. 2011 (2011):918471.

30. Vojinovic J & Damjanov n. HDAC inhibition in rheumatoid arthritis and juvenile idiopathic arthritis. Mol Med. 17.5-6 (2011):397-403.

31. Woods DV, et al. Class I HDAC inhibition upregulates PD-1 ligands in melanoma and increase the efficacy of PD-1 Blockade. Abstract presented at: AACR 2015. Accessed April 19, 2015.

32. yamanegi k, et al. Sodium valproate, a histone deacetylase inhibitor, augments the expression of cell-surface nkG2D ligands, MICA/B, without increasing their soluble forms to enhance susceptibility of human osteosarcoma cells to nk cell-mediated cytotoxicity. oncol Rep. 24.6 (2010):1621-1627.

33. youn JI, et al. epigenetic silencing of retinoblastoma gene regulates pathologic differentiation of myeloid cells in cancer. nat Immunol. 14.3 (2013):211-220.

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endnotes

forward looking StatementsCertain statements contained in this Backgrounder, other than statements of fact that are independently verifiable at the date hereof, contain “forward-looking” statements, within the meaning of the Private Securities Litigation Reform Act of 1995, that involve significant risks and uncertainties. For more detailed disclosures and discussions regarding such forward looking statements, please refer to Mirati’s filings with the u.S. Securities and exchange Commission (“SeC”), including without limitation Mirati’s filings on Forms 10-k, 10-Q, and 8-k. Forward looking statements are based on the current expectations of management and upon what management believes to be reasonable assumptions based on information currently available to it. Such statements can usually be identified by the use of words such as “may,” “would,” “believe,” “intend,” “plan,” “anticipate,” “estimate,” “expect,” and other similar terminology, or by statements that certain actions, events or results “may” or “would” be taken, occur or be achieved. Such statements include, but are not limited to, statements regarding Mirati’s development plans and timelines, potential regulatory actions, expected use of cash resources, the timing and results of clinical trials, and the potential benefits of and markets for Mirati’s product candidates. Forward looking statements involve significant risks and uncertainties and are neither a prediction nor a guarantee that future events or circumstances will occur. Such risks include, but are not limited to, potential delays in development timelines or negative clinical trial results, reliance on third parties for development efforts, changes in the competitive landscape, changes in the standard of care, as well as other risks described in Mirati’s filings with the SeC. We are including this cautionary note to make applicable, and to take advantage of, the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 for forward-looking statements. The information in this Backgrounder is given as of the date below and Mirati expressly disclaims any obligation to update or revise any forward-looking statements, whether as a result of new information, future events or otherwise, unless required by law.

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About mirati therapeutics

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Mirati Therapeutics develops molecularly targeted, single agent and immuno-oncology combination therapies intended to treat cancer. Mirati’s approach combines the three most important factors in oncology drug development, 1) researching and developing drug candidates that target genetic and epigenetic drivers of cancer, 2) designing creative and agile clinical development strategies that select for patients whose tumors are dependent on specific driver alterations, and 3) leveraging a highly accomplished targeted oncology leadership team. The Mirati team uses a blueprint – proven by their prior work – for developing potential breakthrough cancer therapies, with accelerated development paths, in order to improve outcomes for patients. Mirati is advancing three drug candidates through clinical development for multiple oncology indications.