WO2014194293A1

WO2014194293A1 - Improved methods for the selection of patients for pd-1 or b7-h4 targeted therapies, and combination therapies thereof

Info

Publication number: WO2014194293A1
Application number: PCT/US2014/040388
Authority: WO
Inventors: Solomon Langermann; Rena May; Shannon Marshall
Original assignee: Amplimmune, Inc.
Priority date: 2013-05-30
Filing date: 2014-05-30
Publication date: 2014-12-04

Abstract

The present disclosure relates to improved methods for selecting patients who would be amenable for PD-1 and B7-H4 pathway targeted therapies and combination therapies, and for treating such patients. In particular, the disclosure pertains to improved PD-1 targeted therapies and combination therapies for treating patients who have failed treatment with BRAF/MEK inhibitors or other inhibitors of the RAS-RAF-MEK-ERK pathway. The disclosure further pertains to improved PD-1 targeted therapies and combination therapies to overcome resistance caused by "tumor dormancy" and to prevent the selection/outgrowth of rapidly, progressing, resistant tumors in the presence of various small molecule inhibitors. The present disclosure additionally provides a PD-1 targeted therapy which involves the administration of an immunomodulatory molecule such as a PD-1-binding fusion protein/antibody (e.g., an anti-PD-1 antibody, a B7-DC-Ig, a B7-H1-Ig, etc.) with a BRAF inhibitor ("BRAFi") or other small molecule as an initial treatment regimen in such selected patients.

Description

IMPROVED METHODS FOR THE SELECTION OF PATIENTS FOR PD-1 OR B7-H4 TARGETED THERAPIES, AND COMBINATION THERAPIES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to United States Patent Application Nos. 61/828,952 filed May 30, 2013 and 61/903,432 filed November 13, 2013 which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention generally relates to improved methods for selecting patients who would be amenable for PD-1 and B7-H4 pathway targeted therapies and combination therapies.

BACKGROUND OF THE INVENTION

Cell Mediated Immune Responses

The immune system of humans and other mammals is responsible for providing protection against infection and disease. Such protection is provided both by a humoral immune response and by a cell-mediated immune response. The humoral response results in the production of antibodies and other biomolecules that are capable of recognizing and neutralizing foreign targets (antigens). In contrast, the cell-mediated immune response involves the activation of macrophages, natural killer cells (NK), and antigen- specific cytotoxic T-lymphocytes ("CTLs"), and the release of various cytokines in response to the recognition of an antigen (Dong, C. et al. (2003) "Immune Regulation by Novel Costimulatory Molecules,"

Immunolog. Res. 28(l):39-48).

The ability of T cells to optimally mediate an immune response against an antigen requires two distinct signaling interactions (Viglietta, V. et al. (2007) "Modulating Co-Stimulation," Neurotherapeutics 4:666-675; Korman, A.J. et al. (2007) "Checkpoint Blockade in Cancer Immunotherapy," Adv.

Immunol. 90:297-339). First, antigen that has been arrayed on the surface of antigen-presenting cells (APC) must be presented to an antigen-specific naive CD4⁺ T cell. Such presentation delivers a signal via the T cell receptor (TCR) that directs the T cell to initiate an immune response that will be specific to the presented antigen. Second, a series of co- stimulatory and co- inhibitory signals, mediated through interactions between the APC and distinct T cell surface molecules, triggers first the activation and proliferation of the T cells and ultimately their inhibition. Thus, the first signal confers specificity to the immune response whereas the second signal serves to determine the nature, magnitude and duration of the response.

The immune system is tightly controlled by co- stimulatory and co- inhibitory ligands and receptors. These molecules provide the second signal for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses against infection while limiting immunity to self (Wang, L. et al. (March 7, 2011) "VISTA, A Novel Mouse Ig Superfamily Ligand That Negatively Regulates T Cell Responses," J. Exp. Med. 10.1084/jem.20100619: l-16; Lepenies, B. et al. (2008) "The Role Of Negative Co stimulators During Parasitic Infections," Endocrine, Metabolic & Immune Disorders - Drug Targets 8:279-288). Of particular importance is binding between the B7.1 (CD80) and B7.2 (CD86) ligands of the Antigen Presenting Cell and the CD28 and CLTA-4 receptors of the CD4⁺ T- lymphocyte (Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2: 116-126; Dong, C. et al. (2003) "Immune Regulation by Novel Costimulatory Molecules," Immunolog. Res. 28(l):39-48; Lindley, P.S. et al. (2009) "The Clinical Utility Of Inhibiting CD28-Mediated

Costimulation," Immunol. Rev. 229:307-321). Binding of B7.1 or of B7.2 to CD28 stimulates T cell activation; binding of B7.1 or B7.2 to CTLA4 inhibits such activation (Dong, C. et al. (2003) "Immune Regulation by Novel Costimulatory Molecules," Immunolog. Res. 28(l):39-48; Lindley, P.S. et al. (2009) "The Clinical Utility Of Inhibiting CD28-Mediated Costimulation," Immunol. Rev. 229:307-321 ; Greenwald, R.J. et al. (2005) "The B7 Family Revisited," Ann. Rev. Immunol. 23:515-548). CD28 is constitutively expressed on the surface of T cells (Gross, J., et al. (1992) "Identification And Distribution Of The Costimulatory Receptor CD28 In The Mouse," J. Immunol. 149:380-388), whereas CTLA4 expression is rapidly up-regulated receptor (Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2: 116-126), binding first initiates T cell proliferation (via CD28) and then inhibits it (via nascent expression of CTLA4), thereby dampening the effect when proliferation is no longer needed.

Further investigations into the ligands of the CD28 receptor have led to the identification and characterization of a set of related B7 molecules (the "B7 Superfamily") (Coyle, A.J. et al. (2001) "The Expanding B7

Superfamily: Increasing Complexity In Costimulatory Signals Regulating T Cell Function^ Nature Immunol. 2(3):203-209; Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2: 116-126; Greenwald, R.J. et al. (2005) "The B7 Family Revisited;' Ann. Rev. Immunol. 23:515- 548; Collins, M. et al. (2005) "The B7 Family Of Immune -Regulatory Ligands," Genome Biol. 6:223.1-223.7; Loke, P. et al. (2004) "Emerging Mechanisms Of Immune Regulation: The Extended B7 Family And

Regulatory T Cells." Arthritis Res. Ther. 6:208-214; Korman, A.J. et al.

(2007) "Checkpoint Blockade in Cancer Immunotherapy," Adv. Immunol. 90:297-339; Flies, D.B. et al. (2007) "The New B7s: Playing a Pivotal Role in Tumor Immunity " J. Immunother. 30(3):251-260; Agarwal, A. et al.

(2008) "The Role Of Positive Costimulatory Molecules In Transplantation And Tolerance," Curr. Opin. Organ Transplant. 13:366-372; Lenschow, D.J. et al. (1996) "CD28/B7 System of T Cell Co stimulation," Ann. Rev.

Immunol. 14:233-258; Wang, S. et al. (2004) "Co-Signaling Molecules Of The B7-CD28 Family In Positive And Negative Regulation Of T Lymphocyte Responses," Microbes Infect. 6:759-766). There are various known members of the B7 Superfamily: B7.1 (CD80), B7.2 (CD86), the inducible co-stimulator ligand (ICOS-L; B7-H2), the programmed death- 1 ligand 1 (PD-Ll ; B7-H1), the programmed death- 1 ligand 2 (PD-L2; B7-DC), B7-H3, B7-H4 and B7-H6 (Collins, M. et al. (2005) "The B7 Family Of Immune- Regulatory Ligands," Genome Biol. 6:223.1-223.7; Tseng, S.Y. et al. (2001) "B7-DC, A New Dendritic Cell Molecule With Potent Costimulatory

Properties For T Cells," J. Exp. Med. 193(7):839-846). B7-H1 /PD-1 Interactions

B7-H1

B7-H1 (PD-L1, CD274) is a particularly significant member of the B7 Superfamily as it is pivotally involved in shaping the immune response to tumors (Flies, D.B. et al. (2007) "The New B7s: Playing a Pivotal Role in Tumor Immunity " J. Immunother. 30(3):251-260; United States Patents Nos. 6,803,192; 7,794,710; United States Patent Application Publication Nos. 2005/0059051 ; 2009/0055944; 2009/0274666; 2009/0313687; PCT

Publication No. WO 01/39722; WO 02/086083). B7-H1 is a 55kDa type 1 transmembrane protein. It has been speculated to play a major role in suppressing the immune system during particular events such as pregnancy, tissue allografts, autoimmune disease and other disease states such as hepatitis. Dormant tumor cells are believed to over-express B7-H1, which may explain how such cells are able to evade immune surveillance and persist for years or decades (Quesnel, B. (2013) "Tumor Dormancy: Long- Term Survival in a Hostile Environment," In: SYSTEMS BIOLOGY OF TUMOR DORMANCY, ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 734, H. Enderling et al. (eds.) Springer Science+Business Media, NY; Chapter 9, pp. 181-200; Lee, J.-Y. et al. (2012) "Remembering To Be Tolerant," Science 335:667-668).

B7-H1 is broadly expressed in different human and mouse tissues, such as heart, placenta, muscle, fetal liver, spleen, lymph nodes, and thymus for both species, as well as liver, lung, and kidney in mouse only (Martin- Orozco, N. et al. (2007) "Inhibitory Co stimulation And Anti- x \or

Immunity," Semin. Cancer Biol. 17(4):288-298). In humans, B7-H1 protein expression has been found in human endothelial cells (Chen, Y. et al. (2005) "Expression ofB7-Hl in Inflammatory Renal Tubular Epithelial Cells," Nephron. Exp. Nephrol. 102:e81-e92; de Haij, S. et al. (2005) "Renal Tubular Epithelial Cells Modulate T-Cell Responses Via ICOS-L And B7- HI" Kidney Int. 68:2091-2102; Mazanet, M.M. et al. (2002) "B7-H1 Is Expressed By Human Endothelial Cells And Suppresses T Cell Cytokine 170: 1257-1266), syncyciotrophoblasts (Petroff, M.G. et al. (2002) "B7 Family Molecules: Novel Immunomodulators At The Maternal-Fetal Interface," Placenta 23:S95-S101), resident macrophages of some tissues, or in macrophages that have been activated with interferon (IFN)-y or tumor necrosis factor (TNF)-a (Latchman, Y. et al. (2001) "PD-L2 Is A Second Ligand For PD-1 And Inhibits T Cell Activation," Nat. Immunol 2:261-268), and in tumors (Dong, H. (2003) "B7-H1 Pathway And Its Role In The Evasion Of Tumor Immunity ," J. Mol. Med. 81 :281-287). In the mouse, B7- Hl protein expression is found in heart endothelium, islets cells of the pancreas, small intestines, and placenta (Martin-Orozco, N. et al. (2007) "Inhibitory Co stimulation And Anti-lumor Immunity ," Semin. Cancer Biol. 17(4):288-298).

Additionally, studies indicate that human and rodent cancer cells, and stromal cells and immune cells in the cancer microenvironment upregulate expression of inhibitory B7 molecules and that these contribute to tumor immune evasion (Zou and Chen (2008) "Inhibitory B7 -Family Molecules In The Tumour Microenvironment," Nature Reviews,

Immunology, 8:467-477; see also Thompson et al. (2006) Tumor B7 -HI Is Associated with Poor Prognosis in Renal Cell Carcinoma Patients with Long-term Follow-up," Cancer Res. 66(7):3381-3385) (suggesting that B7- Hl is expressed by renal cell carcinoma (RCC) tumor cells and is associated with poor prognosis)).

PD-1

Programmed Death -1 ("PD-1") is a receptor of B7-H1 and B7-DC. PD-1 is a 50-55 kDa type I membrane protein member of the extended

CD28/CTLA4 family of T cell regulators (Ishida, Y. et al. (1992) "Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death," EMBO J. 11 :3887-3895; United States Patent Application Publication No. 2007/0202100;

2008/0311117; 2009/00110667; United States Patents Nos. 6,808,710; 7,101,550; 7,488,802; 7,635,757; 7,722,868; PCT Publication No. WO PD-1 is expressed on activated T cells, B cells, and monocytes (Agata, Y. et al. (1996) "Expression Of The PD-1 Antigen On The Surface Of Stimulated Mouse T And B Lymphocytes," Int. Immunol. 8(5):765-772; Yamazaki, T. et al. (2002) "Expression Of Programmed Death 1 Ligands By Murine T Cells AndAPC," J. Immunol. 169:5538-5545) and at low levels in natural killer (NK) T cells (Nishimura, H. et al. (2000) "Facilitation Of Beta Selection And Modification Of Positive Selection In The Thymus Of PD-1 -Deficient Mice," J. Exp. Med. 191 :891-898; Martin-Orozco, N. et al. (2007) "Inhibitory Costimulation And Anti-tumor Immunity," Semin. Cancer Biol. 17(4):288- 298).

The extracellular region of PD- 1 consists of a single

immunoglobulin (Ig)V domain with 23% identity to the equivalent domain in CTLA4 (Martin-Orozco, N. et al. (2007) "Inhibitory Costimulation And Anti-tumor Immunity," Semin. Cancer Biol. 17(4):288-298). The extracellular IgV domain is followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif, which suggests that PD-1 negatively regulates TCR signals (Ishida, Y. et al. (1992) "Induced

Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death," EMBO J. 11 :3887-3895; Blank, C. et al. (Epub 2006 Dec 29) "Contribution Of The PD-Ll/PD-1 Pathway To T-Cell Exhaustion: An Update On Implications For Chronic Infections And Tumor Evasion Cancer," Immunol. Immunother. 56(5):739- 745).

The Interactions of B7-H1 and PD-1

B7-H1 (PD-L1) is a member of the B7 family of co- stimulatory molecules and is primarily expressed on immune cells such as B cells, dendritic cells, macrophages and T cells. The binding of B7-H1 to its receptor, programmed death 1 (PD-1) expressed on activated T cells (and/or to its cognate B7 molecule, B7.1) delivers an inhibitory signal to T cells or "Inhibitory Co stimulation And Antitumor Immunity," Semin. Cancer Biol. 17(4):288-298) and functions as a cell death inducer (Ishida, Y. et al. (1992) "Induced Expression OfPD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death " EMBO J. 11 :3887-3895; Subudhi, S.K. et al. (2005) "The Balance Of Immune Responses:

Costimulation Verse Coinhibition " J. Molec. Med. 83: 193-202).

PD-1 has been shown to negatively regulate TCR signaling. Upon ligating its receptor, B7-H1 has been reported to decrease TCR-mediated

proliferation and cytokine production.

Interaction between low concentrations of the PD-1 receptor and the

B7-H1 ligand results in the transmission of an inhibitory signal that strongly inhibits the proliferation of antigen-specific CD8⁺ T cells; at higher concentrations the interactions with PD- 1 do not inhibit T-cell proliferation but markedly reduce the production of multiple cytokines (Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily;' Nature Rev. Immunol. 2: 116-126). T-cell proliferation and cytokine production by both resting and previously activated CD4 and CD8 T cells, and even naive T cells from umbilical-cord blood, are inhibited by soluble B7-Hl-Ig fusion proteins coupled to beads with an anti-CD3 mAb (Freeman, G.J. et al. (2000) "Engagement Of The PD-1 Immunoinhibitory Receptor By A Novel B7 Family Member Leads To Negative Regulation Of Lymphocyte Activation " J. Exp. Med. 192: 1-9; Latchman, Y. et al. (2001) "PD-L2 Is A Second Ligand For PD-1 And Inhibits T Cell Activation " Nature Immunol. 2:261-268; Carter, L. et al. (2002) "PD-l. -PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2 " Eur. J. Immunol. 32(3):634-643; Sharpe, A.H. et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2: 116-126).

B7-H1 - PD-1 interactions lead to cell cycle arrest in G0-G1 but do not increase cell death (Latchman, Y. et al. (2001) "PD-L2 Is A Second Ligand For PD-1 And Inhibits T Cell Activation," Nature Immunol. 2:261- 268; Carter, L. et al. (2002) "PD-l. PD-L inhibitory pathway affects both antagonize the B7 - CD28 signal when antigenic stimulation is weak or limiting, and plays a key role in down-regulating T-cell responses.

The signal transduction mediated by B7-H1 and PD-1 is complex. Both molecules additionally bind to other proteins. B7-H1 is capable of binding to B7-1 (CD80) (Butte, M.J. et al. (2008) "Interaction ofPD-Ll and 57-1," Molecular Immunol. 45:3567-3572); PD-1 is capable of binding to B7-DC (PD-L2) (Lazar-Molnar, E. et al. (2008) "Crystal Structure Of The Complex Between Programmed Death-1 (PD-1) And Its Ligand PD-L2," Proc. Natl. Acad. Sci. (USA) 105(30): 10483-10488). B7-1 interacts with CD28 to deliver a co-stimulatory signal for T-cell activation that is important in the early stages of immune response (Elloso, M.M. et al. (1999) "Expression and Contribution ofB7-l (CD80) and B7-2 (CD86) in the Early Immune Response to Leishmania major Infection," J. Immunol. 162:6708- 6715). B7-DC is a strong stimulator of T cells, enhancing T cell proliferation and IFN-γ production. However, it also exhibits an inhibitory effect on the immune response via its interaction with PD-1 (Ishiwata, K. et al. (Epub January 10, 2010) "Costimulator Responses Induced by Nippostrongylus brasiliensis," J. Immunol 184:2086-2094). B7-DC also is also believed to regulate respiratory immunity by binding to repulsive guidance molecule b (RGMb) (Xia, et al., "RGMb is a novel binding partner or PD-L2 and its engagement with PD-L2 promotes respiratory tolerance", J. Experimental Med., 211(5):943-959 (2014), WO 2014/022758).

Microbes and tumors appear to have exploited PD-1 and B7-H1 to evade eradication by the immune system. Differences in binding affinities to the various receptors and ligands that interact with PD-1 and B7-H1 have been proposed to provide distinct functional outcomes of blockade of PD- 1 and B7-H1 in disease models (Butte, M.J. et al. (2008) "Interaction ofPD- Ll

Molecular Immunol. 45:3567-3572). The PD-1 pathway has also been implicated as playing a key role in the impairment of immune function during chronic infection ("T cell exhaustion"), and a blockade of

PD- 1 function is able to restore many T cell functions (Rodriquez-Garcia, M. The role of B7-H1 and PD-1 in inhibiting T cell activation and proliferation has suggested that these biomolecules might serve as therapeutic targets for treatments of inflammation and cancer. In contrast to normal tissues, which show minimal surface expression of B7-H1, B7-H1 expression has been found to be abundant on many murine and human cancers, and may be further up-regulated upon IFN-γ stimulation. Thus, B7- Hl has been noted to play an important role in tumor immune evasion. See Blank and Gajewski (2004) "Interaction ofPD-Ll on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications or tumor immunotherapy " Cancer Immunol Immunother. 54:307-314. PD- 1 expression is upregulated during chronic infections, such as viral infections (Golden-Mason, et al., Upregulation of PD-1 Expression on Circulating and Intrahepatic Hepatitis C Virus-Specific CD8+ T Cells Associated with Reversible Immune Dysfunction, J. Virol., 81(17):9249-9258 (2007)).

Accordingly, agents that modulate the interaction of PD-1 with B7-H1 have been suggested to have utility in up- or down-modulating the immune response (see, United States Patents Nos. 7,029,674; 7,488,802; United States Patent Application Publications Nos. 2007/0122378; 2009/0076250; 2009/0110667; 2009/0263865; 2009/0297518; PCT Publication No. WO 2006/133396). The use of anti-PD-1 antibodies to treat infections and tumors and up-modulate an adaptive immune response has been proposed and demonstrated clinically (see, United States Patent Application Publication Nos. 2010/0040614; 2010/0028330; 2004/0241745; 2008/0311117;

2009/0217401 ; United States Patents Nos. 7,521,051; 7,563,869; 7,595,048; PCT Publications Nos. WO 2004/056875; WO 2008/083174). Likewise, the use of anti-B7-Hl antibodies to treat infections and tumors and enhance an adaptive immune response has been proposed and demonstrated clinically (United States Patent Application Publication Nos. 2009/0055944;

2009/0274666; 2009/0317368; United States Patents Nos. 6,803,192;

7,794,710; PCT Publications Nos. WO 01/39722; WO 02/086083).

The presence of either or both tumor infiltrating lymphocytes Spot In The Ranks Of Cancer Therapy," J. Exp. Med. 209(2):201-209), as targeting this pathway leads to important changes in enhancing immune responses to the tumor effected by such IFN-gamma and TILs as well as other factors.

The inflammatory milieu of the tumor microenvironment can cause the up-regulation of B7-H1 on both the surface of tumors (Zou, W et al. (2008) "Inhibitory B7 -Family Molecules In The Tumour Microenvironment," Nat. Rev. Immunol. 8(6):467-771) and the surfaces of CD68⁺ Tumor Associated Macrophages ("TAMs") (Kuang, D.M. et al. (2009) "Activated Monocytes In Peritumoral Stroma Of Hepatocellular Carcinoma Foster Immune Privilege And Disease Progression Through PD-L1," J. Exp. Med. 206(6): 1327-1337), further impairing anti-tumor T cell responses, and thereby correlating with poor prognosis and outcome (Gao, Q. et al. (2009) "Over expression OfPD-Ll Significantly Associates With Tumor

Aggressiveness And Postoperative Recurrence In Human Hepatocellular Carcinoma," Clin. Cancer Res. 15(3):971-999; Shi, F. et al. (2011) "PD-i And PD-L1 Upregulation Promotes CD8( + ) T-Cell Apoptosis And Post- Operative Recurrence In Hepatocellular Carcinoma Patients," Int. J. Cancer 128(4):887-896).

TAMs provide a link between inflammation and cancer.

Macrophages are immune system cells derived from activated blood monocytes. They are primarily recognized as participating in inflammatory responses induced by pathogens or tissue damage by acting to remove (i. e. , phagocytose) pathogens, dead cells, cellular debris, and various components of the extra-cellular matrix (ECM). Macrophages have been found to constitute an important constituent in the tumor microenvironment and to represent up to 50% of the tumor mass. TAMs may be tumor infiltrating (occasionally referred to as Tumor Infiltrating Macrophages, or TIMs) or on the periphery of the tumor, and the location may be relevant to patient prognosis, diagnosis and/or treatment.

In addition to mediating phagocytosis, macrophages secrete pro- Macrophages In Development And Disease," Nat. Rev. Immunol. 9:259- 270). As such, the presence of macrophages within a tumor appears to assist the growth of the tumor. A number of studies provide evidence that the presence of tumor-associated macrophages within the tumor is a negative prognostic factor of survival (Farinha, P. et al. (2005) "Analysis Of Multiple Biomarkers Shows That Lymphoma- Associated Macrophage (LAM) Content Is An Independent Predictor Of Survival In Follicular Lymphoma (FL)," Blood 106:2169-2174; Dave, S.S. et al. (2004) "Prediction Of Survival In Follicular Lymphoma Based On Molecular Features Of Tumor-Infiltrating Immune Cells;' N. Engl. J. Med. 351 :2159-2169; Solinas, G. et al. (2009) "Tumor-Associated Macrophages (TAM) As Major Players Of The Cancer- Related Inflammation " J. Leukoc. Biol. 86(5): 1065-1073).

Further, since B7-H1 protein is highly expressed by cancer cells, but limited to the macrophage lineage of cells in normal tissues (Dong, H. (2003) "B7-H1 Pathway And Its Role In The Evasion Of Tumor Immunity " J. Mol. Med. 81 :281-287), detection of its presence on a cell (such as by such cell's binding to anti-B7-Hl antibodies or fragments) is generally considered indicative and diagnostic of a cancer cell. Antibodies that bind to B7-H1 have found particular utility in the diagnosis of cancer (see, United States Provisional Patent Application No. 61/477,414, herein incorporated by reference).

B7-H4

B7-H4 is another member of the B7 family that is a negative regulator of immune cell responses. The B7-H4 protein possesses 282 amino acid residues, which have been categorized as comprising an amino terminal extracellular domain, a large hydrophobic transmembrane domain and a very short intracellular domain (consisting of only 2 amino acid residues). Like other B7 family members, B7-H4 possesses a pair of Ig-like regions in its extracellular domain. The B7-H4 protein has an overall structure of a type I transmembrane protein. The protein has minimal (about 25%) homology with other B7 family members (Zang, X. et al. (2003) B7x: A Widely amino acid identity, suggesting an important evolutionarily conserved function.

The receptor for B7-H4 has not been cloned. B7-H4 has been shown not to bind to known CD28 family members such as CD28, CTLA-4, ICOS, and PD- 1 (Sica, et al., Immunity, 18:849-861 (2003)), and these are therefore not potential receptors for B7-H4. Functional studies using B7-H4 transfectants and B7-H4-Ig fusion proteins demonstrate that B7-H4 delivers a signal that inhibits TCR-mediated CD4+ and CD8+ T cell proliferation, cell-cycle progression and IL-2 production. B7-1 costimulation cannot overcome B7-H4-Ig-induced inhibition. In agreement with the in vitro activity, B7-H4 knock-out mice develop autoimmunity. The broad and inducible expression of B7-H4, together with functional studies, suggests that B7-H4 serves to downregulate immune responses in peripheral tissues.

In contrast to other B7 members, B7-H4 mRNA is widely expressed. Its expression has been found in the brain, heart, kidney, liver, lung, ovary, pancreas, placenta, prostate, skeletal muscle, skin, small intestine, spleen, stomach, testis, thymus, thymus, and uterus (Sica, G.L. et al. (2003) "57-H4, A Molecule Of The B7 Family, Negatively Regulates T Cell Immunity " Immunityl8:849-861 ; Zang, X. et al. (2003) B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation " Proc. Natl. Acad. Sci. (USA) 100: 10388- 10392; Prasad, D.V. et al. (2003) B7S1, A Novel B7 Family Member That Negatively Regulates T Cell Activation " Immunity 18:863-873 ; Prasad, D.V. et al. (2003) B7S1, A Novel B7 Family Member That Negatively Regulates T Cell Activation," Immunity 18:863-873).

Despite the widespread expression of Β7-Η4 mRNA, the presence of

Β7-Η4 protein on the surface of normal cells seems to be limited (Sica, G.L. et al. (2003) "57-H4, A Molecule Of The B7 Family, Negatively Regulates T Cell Immunity," Immunityl8:849-861 ; Choi, Ι.Η. et al. (2003) "Genomic Organization And Expression Analysis OfB7-H4, An Immune Inhibitory Molecule Of The B7 Family," J. Immunol. 171 :4650-4654). Although freshly isolated human T cells, B cells, monocytes, and dendritic cells do not (IFN-γ), phorbol 12-myristate 13-acetate (PMA), or ionomycin (Sica, G.L. et al. (2003) "B7-H4, A Molecule Of The B7 Family, Negatively Regulates T Cell Immunity," Immunityl8:849-861). The finding of such a wide distribution of B7-H4 expression suggests that the function of B7-H4 is quite distinct from that of other inhibitory B7 molecules (see, Zang, X. et al. (2003) B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation;' Proc. Natl. Acad. Sci. (USA) 100: 10388-10392).

Relatively high levels of B7-H4 protein expression have been found in microenvironments of numerous tumor types, for example, human ovarian cancers (Choi, I.H. et al. (2003) "Genomic Organization And Expression Analysis 0/B7-H4, An Immune Inhibitory Molecule Of The B7 Family " J. Immunol. 171:4650-4654; Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203(4):871-881; Bignotti, E. et al. (2006) "Differential Gene Expression Profiles Between Tumor Biopsies And Short Term Primary Cultures Of Ovarian Serous Carcinomas: Identification Of Novel Molecular Biomarkers For Early Diagnosis And Therapy " Gynecol. Oncol. 103:405-416; Tringler, B. et al. (2006) "B7-H4 Overexpression In Ovarian Tumors," Gynecol. Oncol. 100:44-52; Simon, I. et al. (2006) "B7- h4 Is A Novel Membrane-Bound Protein And A Candidate Serum And Tissue Biomarker For Ovarian Cancer," Cancer Res. 66: 1570-1575; Salceda, S. et al. (2005) "The Immunomodulatory Protein B7-H4 Is Overexpressed In Breast And Ovarian Cancers And Promotes Epithelial Cell Transformation," Exp. Cell Res. 306: 128-141), non-small-cell lung cancer (Sun, Y. et al. (2006) "B7-H3 And B7-H4 Expression In Non-Small-Cell Lung Cancer "

Lung Cancer 53: 143-151), ductal and lobular breast cancer (Salceda, S. et al. (2005) "The Immunomodulatory Protein B7-H4 Is Overexpressed In Breast And Ovarian Cancers And Promotes Epithelial Cell Transformation," Exp. Cell Res. 306: 128-141 ; Tringler, B. et al. (2005) "B7-H4 Is Highly

Expressed In Ductal And Lobular Breast Cancer " Clin. Cancer Res.

11: 1842-1848), and renal cell carcinoma (Krambeck, A.E. et al. (2006) "B7- In addition to being expressed on the tumor, B7-H4 has also been shown to be over-expressed in TAMs, including those present in ovarian tumors (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203(4):871-881; Kryczek, I. et al. (2007) "Relationship Between B7-H4, Regulatory T Cells, And Patient Outcome In Human Ovarian Carcinoma," Cancer Res. 67(18):8900-8905) and is present in tumor vasculature. Regulatory T cells (Tregs) induce upregulation of Β7-Η4 on TAMs via IL-6 and IL-10; this is thought to be one of the mechanisms by which Tregs contribute to immune suppression. (Kryczek, J.I., J. Immunol., 177(l):40-44 (2006)). Β7-Η4 expression has also been observed in tubule epithelial cells of diseased kidneys (Chen, Y., Kidney Int., 70(12):2092-9 (2006) Epub 2006 Oct 18.)

The high levels of Β7-Η4 expression found in numerous tumor tissues, for example, human ovarian cancers, points to a key role for Β7-Η4 in mediating immune suppression. TAMs expressing Β7-Η4 have been found to suppress tumor-associated antigen-specific T cell immunity (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian Carcinoma," J. Exp. Med. 203(4):871-881). The intensity of Β7-Η4 expression in TAMs correlates significantly with Treg cell numbers in the tumor. Furthermore, Β7-Η4 expressed on TAMs, but not tumor cell expressed Β7-Η4, is associated with poor patient outcome (Kryczek, I. et al. (2006) "B7-H4 Expression Identifies A Novel Suppressive Macrophage Population In Human Ovarian

Carcinoma," J. Exp. Med. 203(4):871-881). Previously published data also showed that TAMs spontaneously produce chemokine CCL22 that mediates Treg cell trafficking into the tumor, and Treg cell-induced Β7-Η4 expression on antigen-presenting cells (APC), including TAMs themselves (Kryczek, I. et al. (2006) "Cutting Edge: Induction OfB7-H4 On APCs Through IL-10: Novel Suppressive Mode For Regulatory T Cells," J. Immunol. 177(1) :40- 44). Taken together, such findings suggest that TAMs expressing Β7-Η4 Tumor Heterogeneity

Cancer cells acquire a characteristic set of functional capabilities during their development, albeit through various mechanisms. Such capabilities include evading apoptosis, self-sufficiency in growth signals, and insensitivity to anti-growth signals, tissue invasion/metastasis, and limitless explicative potential and sustained angiogenesis. The term "cancer cell" is meant to encompass both pre-malignant and malignant cancer cells. In some embodiments, cancer refers to a benign tumor, which has remained localized. In other embodiments, cancer refers to a malignant tumor, which has invaded and destroyed neighboring body structures and spread to distant sites.

Current approaches to the treatment of cancer attempt to exploit the abnormal biology of tumor cells to selectively attack such cells while sparing normal cells. Thus, a wide array of tumor antigens, such as Her2/neu, CEA, PSA, Bladder tumor antigen, thyroglobulin, alpha- fetoprotein, CA125, CA19.9, CA15.3, have been used as targets for anticancer therapies

(Polanski, M. et al. (2006) "A List Of Candidate Cancer Biomarkers For Targeted Proteomics," Biomarker Insights 2: 1-48; see also, Yoneda, A. et al. (2012) "Breast And Ovarian Cancers: A Survey And Possible Roles For The Cell Surface Heparan Sulfate Proteoglycans," J. Histochem. Cytochem. 60(1):9-21 ; Willier, S. et al. (2011) "Defining The Role Of TRIP6 In Cell Physiology And Cancer," Biol Cell. 103(12):573-591; Gokmen-Polar, Y. et al. (2011) "Biomarkers For Breast Cancer Stem Cells: The Challenges Ahead," Biomark. Med. 5(5):661-671; FitzGerald, D.J. et al. (2011), "Treatment Of Hematologic Malignancies With Immunotoxins And

Antibody-Drug Conjugates " Cancer Res. 71(20):6300-6309; Balkwill, F.R. (2011) "The Chemokine System And Cancer," J Pathol. 226(2): 148-157; Hasan, A. (2011) "Therapeutic Targeting OfB7-Hl In Breast Cancer," Expert Opin. Ther. Targets. 15(10): 1211-1225; Strasser, A. et al. (2011) "Deciphering The Rules Of Programmed Cell Death To Improve Therapy Of Cancer And Other Diseases," EMBO J. 30(18):3667-3683; Yim, K.L. et al. (2011) "Targeted Drug Therapies And Cancer," Recent Results Cancer Res. Expert Opin. Biol. Ther. l l(7):875-892; George, B. et al. (2011) "Predictive And Prognostic Markers In Colorectal Cancer," Curr. Oncol. Rep.

13(3):206-215; Padmanabhan, P. et al. (2011) "Molecular Targeting Of Breast Cancer: Imaging And Therapy," Curr. Pharm. Biotechnol. 12(4):528- 538).

Efforts to treat cancer by exploiting the presence of tumor antigens has, however, not been as effective as had been initially anticipated due to the presence of micro-heterogeneity amongst the cells of individual tumors. Such intra-tumor microheterogeneity can lead to an under- appreciation of the nature of the tumor landscape revealed from single tumor-biopsy samples and presents a major challenge to personalized-medicine and the use of biomarkers in cancer therapy (Gerlinger, M. et al. (2012) "Intratumor Heterogeneity And Branched Evolution Revealed By Multiregion

Sequencing N. Engl. J. Med. 366:883-892).

The microheterogeneity of tumors is discussed in Kuwabara, M. et al.

(1996) {"Molecular Microheterogeneity Of Tumor Marker Substances And Its Significance Of Biological Recognition," Nihon Rinsho. 54(6): 1580- 1586); Vaughan, H.A. et al. (2004) ("Immunohistochemical And Molecular Analysis Of Human Melanomas For Expression Of The Human Cancer- Testis Antigens NY-ESO-I And IAGE-1," Clin. Cancer Res. 10(24):8396- 8404); Gonzalez- Garcia, I. et al. (2002) ("Metapopulation Dynamics And Spatial Heterogeneity In Cancer," Proc. Natl. Acad. Sci. (USA)

99(20): 13085-13089); Klein, C.A. et al. (2002) ("Genetic Heterogeneity Of Single Disseminated Tumour Cells In Minimal Residual Cancer," Lancet. 2002 Aug 31 ;360(9334):683-689); Kuczyk, M. et al. (1999) ("The Need For Micro dissectional Tumor Cell Preparation During The Molecular Genetic Analysis Of Prostate Cancer World J. Urol. 17(2): 115-122); and Benjamin, D. et al. (1992) ("Human B-Cell TNF-Beta Microheterogeneity,"

Lymphokine Cytokine Res. l l(l):45-54).

The existence of intra-tumor microheterogeneity suggests that single region biopsies may thus not be sufficient to provide oncologists with a true inconvenience. Additionally, such multiple biopsies may not be practicable in many situations.

Cancer and the RAS-RAF-MEK-ERK Pathway

Cancers arise due to the accumulation of mutations in critical genes that alter normal programs of cell proliferation, differentiation and death (Davies, H. et al. (2002) "Mutations Of The BRAF Gene In Human Cancer," Nature 417:949-954). The mitogen-activated protein kinases ("MAPKs") have been found to be key regulators of embryogenesis, cell differentiation, cell proliferation, and apoptosis and have therefore been targeted as potentially involved in oncogenesis (Lee, J.T. Jr. et al. (2002) "The

Raf/MEK/ERK Signal Transduction Cascade As A Target For

Chemotherapeutic Intervention In Leukemia," Leukemia 16:486-507. The serine/threonine MAP kinases are activated in response to upstream receptor tyrosine kinases and/or cytokine receptors that associate with heterotrimeric G proteins so as to trigger downstream pathways (Lee, J.T. Jr. et al. (2002) "The Raf/MEK ERK Signal Transduction Cascade As A Target For

Chemotherapeutic Intervention In Leukemia," Leukemia 16:486-507). The most extensively studied MAP kinase pathway is the Raf-MEK-ERK cascade. This pathway regulates the expression of B7-H1 (Yamamoto, R. et al. (Epub 2009 Aug 1) "B7-H1 Expression Is Regulated By MEK/ERK Signaling Pathway In Anaplastic Large Cell Lymphoma And Hodgkin Lymphoma;' Cancer Sci. 100(11):2093-2100).

The Raf-MEK-ERK cascade is a signal transduction pathway which relays extracellular signals from the cell membrane to the nucleus via an ordered series of consecutive phosphorylation events (Madhunapantula,

S.R.V. et al. (2008) "Is B-Rafa Good Therapeutic Target for Melanoma and Other Malignancies?" Cancer Res 68:5-8). Raf is a family of protein kinases which acts to phosphorylate and thereby activate the MAP/ERK family of kinases (MEKs) (Kyriakis, J.M. et al. (1992) "Raf-1 Activates MAP Kinase- Kinase," Nature 358:417-421; Dent, P. et al. (1992) "Activation OfMitogen- Activated Protein Kinase Kinase By V-Rafln NIH 3T3 Cells And in vitro," frequently activated by mutation in cancer (Madhunapantula, S.R.V. et al. (2008) "Is B-Raf a Good Therapeutic Target for Melanoma and Other Malignancies?" Cancer Res 68:5-8). Mutations in BRAF have been found in 66% of malignant melanomas and at lower frequency in a wide range of human cancers (Davies, H. et al. (2002) "Mutations Of The BRAF Gene In Human Cancer " Nature 417:949-954). One mutation, which changes valine to glutamic acid at codon 600 (V600E) in exon 15 is prevalent in -90% of those melanoma tumors having BRAF mutations. The mutated protein exhibits more a kinase activity that is more than 10-fold higher than normal BRAF (Davies, H. et al. (2002) "Mutations Of The BRAF Gene In Human Cancer," Nature 417:949-954; Hong, D.S. et al. (Epub 21 Feb 2012) "BRAF(V600) Inhibitor GSK2118436 Targeted Inhibition of Mutant BRAF in Cancer Patients Does Not Impair Overall Immune Competency," Clin. Cancer Res. 18:2326-2335). Such abnormally high activation of the MAP kinase pathway can inhibit cellular growth in a wide variety of normal and cancer cells by promoting cellular senescence (Michaloglou, C. et al. (2005) "BRAFE600-Associated Senescence -Like Cell Cycle Arrest Of Human Naevi," Nature 436:720-724).

The MAP/ERK family of kinases (MEKs), in turn, activate an extracellular signal-regulated kinase (ERK) (Seger, R. et al. (1992) "Human T-Cell Mitogen-Activated Protein Kinase Kinases Are Related To Yeast Signal Transduction Kinases," J. Biol. Chem 267:25628-25631 ; Crews, C. et al. (1992) "The Primary Structure OfMEK, A Protein Kinase That

Phosphorylates The ERK Gene Product," Science 258:478-480; Kosako, H. et al. (1992) "Xenopus MAP Kinase Activator Is A

Serine/Threonine/Tyrosine Kinase Activated By Threonine

Phosphorylation," EMBO J. 11 :2903-2908; McCubrey, J.A. et al. (2012) "Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR Cascade Inhibitors: How Mutations Can Result in Therapy Resistance and How to Overcome

Resistance," Oncotarget; 3 : 1068- 1111). ERK exhibits proliferative effects when activated (Boulton, T.G. et al. (1990) "An Insulin-Stimulated Protein In Response To Insulin And NGF ," Cell 65:663-675; Rossomando, AJ. et al. (1989) "Evidence That pp42, A Major Tyrosine Kinase Target Protein, Is A Mitogen- Activated Serine/Threonine Protein Kinase " Proc. Natl. Acad. Sci. (U.S.A.) 86:6940-6943; Payne, D.M. et al. (1991) "Identification Of The Regulatory Phosphorylation Sites In pp42/Mito gen- Activated Protein Kinase (MAP Kinase):' EMBO J. 10:885-892). The primary cytoplasmic target of ERK is p90RSK, also known as the ribosomal protein S6 kinase, but a wide array of other targets are known to exist.

The Raf-MEK-ERK signal transduction cascade is primarily activated in response to various extracellular growth factors which are able to initiate intracellular signaling. This mitogenic signal most often occurs at the level of a ligand-receptor interaction, followed by downstream signaling, which ultimately causes altered regulation of the genes responsible for oncogenesis (Lee, J.T. Jr. et al. (2002) "The Raf/MEK/ERK Signal

Transduction Cascade As A Target For Chemother apeutic Intervention In Leukemia," Leukemia 16:486-507). The Raf-MEK-ERK pathway is deregulated in over 90% of malignant melanomas. In over 40% of cases this is due to activating mutations in the serine -threonine kinase BRAF, and in a further 20% this is caused by mutations in NRAS (Ferguson, J. et al. (2012) "Combination ofMEK and SRC Inhibition Suppresses Melanoma Cell Growth And Invasion," Oncogene 32(l):86-96).

Although BRAF mutations are found in a wide range of cancers, a substantial proportion of cases have been found to additionally involve mutations in the RAS oncogene (for example, malignant melanoma, colorectal cancer and borderline ovarian cancers (Vogelstein, B. et al. (1988) "Genetic Alterations During Color ectal-Tumour Development " N. Engl. J. Med. 319:525-532; van't Veer, L.J. et al. (1989) "N-ras Mutations In Human Cutaneous Melanoma From Sun-Exposed Body Sites," Mol. Cell. Biol. 9:3114-3116; Caduff, R.F. et al. (1999) "Comparison Of Mutations OfKi- RAS And p53 Immunoreactivity In Borderline And Malignant Epithelial Ovarian Tumours " Am. J. Surg. Pathol. 23:323-328) (1999). regulated kinase (ERK)-MAP kinase pathway can be achieved by mutation at various levels in the pathway and that the pathway is activated in a substantial proportion of cases in these cancer types (Davies, H. et al. (2002) '^■'^■Mutations Of The BRA I^' Gene In Human Cancer," Nature 417:949-954). Additionally, the finding of an association of multiple genes has suggested that multi-drug cocktails be used to treat patients suffering from cancers involving BRAF/RAS mutations (Al-Lazikani, B. et al. (2013) "Unpicking the Combination Lock for Mutant BRAF and RAS Melanomas," Cancer Discovery 3(1): 14-19; Held, M.A. et al. (2013) "Genotype-Selective Combination Therapies For Melanoma Identified By High-Throughput Drug Screening," Cancer Discovery 3:52-67; Arkenau, H.T. et al. (2011)

"Targeting BRAF For Patients With Melanoma," Brit. J. Cancer 104:392- 398). Preclinical studies have shown a synergism between BRAF and MEK inhibitors, with significantly increased apoptosis and prolonged phospho- ERK inhibition compared with BRAF inhibition alone (Paraiso K.H. et al. (2010) "Recovery Of Phospho-ERK Activity Allows Melanoma Cells To Escape From BRAF Inhibitor Therapy," Br. J. Cancer 102(12): 1724-1730; Park, B.J. et al. (Epub 2 Feb 2012) "Dasatinib Synergizes With Both Cytotoxic And Signal Transduction Inhibitors In Heterogeneous Breast Cancer Cell Lines-Lessons For Design Of Combination Targeted Therapy," Cancer Lett. 320(1): 104-110; Straussman, R. et al. (2012) "Tumour Micro- Environment Elicits Innate Resistance To RAF Inhibitors Through HGF Secretion," Nature 487(7408):500-504).

Such an approach differs from the classical approach of combining agents with different molecular mechanisms in order to enhance anti-cancer efficacy (Al-Lazikani, B. et al. (2012) "Combinatorial Drug Therapy For Cancer In The Post-Genomic Era," Nature Biotechnol. 30:679-692).

Unfortunately, in light of the hundreds of potential antineoplastic agents, defining a potential multi-drug regimen requires sieving among vast numbers of possible combinations. The challenge to finding such a regimen is exacerbated by the poor performance of single agent regimens (Al-Lazikani, MAP kinase pathway activation is believed to be central to cancer development by enhancing multiple oncogenic processes (Wan, P.T. et al. (2004) "Mechanism Of Activation Of The RAF-ERK Signaling Pathway By Oncogenic Mutations Of B-RAF," Cell 116:855-867). Therefore, therapies targeting mutant V600E B-Raf activity or other components of the MAP kinase cascade have potential as agents for halting the progression of malignant tumors by slowing tumor growth, preventing angiogenesis, inhibiting invasion and metastasis, inducing tumor cell death, or promoting tumor differentiation (Tuveson, D.A. et al. (2003) "BRAF As A Potential Therapeutic Target In Melanoma And Other Malignancies," Cancer Cell 4:95-98; Gaggioli, C. et al. (2007) "Tumor-Derived Fibronectin Is Involved In Melanoma Cell Invasion And Regulated By V600E B-Raf Signaling Pathway," J. Invest. Dermatol. 127:400-410; Sharma, A. et al. (2005) "Mutant V599EB-Raf Regulates Growth And Vascular Development Of Malignant Melanoma Tumors," Cancer Res. 65:2412-2421; Sharma, A. et al. (2006) "Targeting Mitogen- Activated Protein Kinase/Extracellular Signal- Regulated Kinase Kinase In The Mutant (V600E) B-Raf Signaling Cascade Effectively Inhibits Melanoma Lung Metastases," Cancer Res. 66:8200-8209; Madhunapantula, S.R.V. et al. (2008) "Is B-Raf a Good Therapeutic Target or Melanoma and Other Malignancies!" Cancer Res 68:5-8).

Melanoma

Melanoma is the most dangerous type of skin cancer and is the leading cause of death from skin disease. The prognosis of patients with metastatic melanoma is particularly poor and is not influenced by systemic therapy with cytotoxic drugs (Arkenau, H.T. et al. (2011) "Targeting BRAF For Patients With Melanoma " Brit. J. Cancer 104:392-398). Only 5% of patients with visceral metastases survive for 2 years (Balch, CM. et al. (2001) "Prognostic Factors Analysis of 17,600 Melanoma Patients:

Validation of the American Joint Committee on Cancer Melanoma Staging System " J. Clin. Oncol. 19(16):3622-3634). In a meta-analysis of 42 phase II trials with 2,100 patients, median survival was approximately 6 months, Stage IV Melanoma To Determine Progression-Free And Overall Survival Benchmarks For Future Phase II Trials," J. Clin. Oncol. 26:527-534.

Mutations in the genes of the Raf-MEK-ERK signal transduction cascade have been identified in over 80% of primary melanomas studied (Platz, A. et al. (2008) "Human Cutaneous Melanoma; A Review OfNRAS And BRAF Mutation Frequencies In Relation To Histogenetic Subclass And Body Site," Mol. Oncol. 1:395-405). Such mutations have been documented in all subtypes of melanoma, including cutaneous, mucosal and uveal melanomas (Arkenau, H.T. et al. (2011) "Targeting BRAF For Patients With Melanoma," Brit. J. Cancer 104:392-398). Whereas the presence of BRAF mutations in a primary melanoma does not correlate with disease progression, the presence of such mutations in metastasized melanomas is associated with poorer survival (Long, G.V. et al. (2010) "Clinicopathologic Correlates Of BRAF Mutation Status In 207 Consecutive Patients With Metastatic Melanoma?' J. Clin. Oncol 28: 15s (abstract 8548)).

Although treatment with BRAF inhibitors, such as vemurafenib and dabrafenib, can result in the rapid onset of tumor response in many patients, intrinsic and/or acquired resistance means these are often temporary, with a median time to progression of less than 7 months (Ascierto, P.A. et al. (2012) "Sequencing Of BRAF Inhibitors And Ipilimumab In Patients With Metastatic Melanoma: A Possible Algorithm For Clinical Use," J.

Translational Med. 10: 107 (pp. 1-8). An alternative approach having the potential to circumvent these involves directly targeting MEK, the common NRAS and BRAF downstream effector with Saracatinib (a suppressor of melanoma-cell collagen adhesion and invasion) (Ferguson, J. et al. (2012) "Combination of MEK and SRC Inhibition Suppresses Melanoma Cell Growth And Invasion," Oncogene 32(l):86-96). An alternative approach involves combining BRAF inhibitors (vemurafenib or dabrafenib) and an anti-CTLA-4 antibody (ipilimumab). A retrospective study of such treatment has shown that the order of providing these agents impacts their efficacy; the provision of the BRAF inhibitors followed by treatment with disease progression (Ascierto, P. A. et al. (2012) "Sequencing OfBRAF Inhibitors And Ipilimumab In Patients With Metastatic Melanoma: A Possible Algorithm For Clinical Use," J. Translational Med. 10: 107 (pp. 1- 8).

As indicated above, the RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas, (as well as in many other tumor types). Targeting MEK and RAF (BRAF V600 mutants) as key kinases of this pathway is currently being evaluated in clinical trials.

However, dose-limiting side effects have been observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response. BRAF inhibitors demonstrate potent early responses but exhibit longer term evidence of resistance to treatment and progression with more aggressive tumors.

In addition to dose limitations, the up-regulation of counteracting signaling cascades {e.g. , alternative kinase pathways and/or key immunomodulatory molecules (such as B7-H1, IDO, ICOS, PD-1, etc)) as a direct response to MEK or BRAF inhibition is also believed to play a role in the low response to MEK targeting drugs and resistance/evasion to BRAF inhibitors. Such up-regulation results in resistance to treatment and in the re-emergence of the tumor (progression of disease).

Thus, despite all present advances a need remains for improved methods for characterizing the cells of a tumor and for selecting patients who would be amenable for targeted anti-cancer therapies and combination therapies, and especially such therapies that target the RAS-RAF-MEK-ERK pathway.

Therefore, it is an object of the invention to provide improved methods for characterizing the cells of a tumor and their use in the diagnosis, patient selection, and the treatment of cancer.

SUMMARY OF THE INVENTION

The present disclosure relates to improved methods for characterizing tumors and/or the tumor microenvironment. In particular, the disclosure express cell surface molecules, such as B7-H1, B7-H4 and PD-1, and/or that are capable of physiospecifically or immunospecifically binding to B7-H1, B7-H4 or PD-1, and to distinguish between tumor cells that express such biomarkers and non-tumor cells present within the tumor and/or tumor microenvironment. The disclosure concerns the uses of such methods in the diagnosis, prognosis, selection of patients, and the treatment of cancer and other diseases.

Immunotherapeutic approaches to cancer therapy have shown promise in inducing durable, long lasting responses in cancer patients.

However, these approaches are hindered by the overall immunosuppressive nature of the tumor microenvironment. Thus, positive results have to date only been seen in 20-30% of patients. New approaches are needed to inhibit the mechanisms of immune suppression and identify biomarkers and/or patient selection markers that can distinguish responders from non- responders.

Tumor specific T-cell function is regulated by a myriad of positive and negative co-stimulatory pathways that regulate the immune response by maintaining tolerance and controlling the balance between immunity and immune suppression. Regulation occurs via checkpoint receptors and their ligands expressed on cells throughout the tumor microenvironment such as: the tumors themselves, cytotoxic T cells, regulatory T cells, myeloid derived suppressor cells, dendritic cells and/or macrophages among others.

A first aspect of the present disclosure derives, in part, from the recognition that the presence of either (or both) TILs and IFN-gamma production disrupts co-stimulatory pathways (such as those involving, for example, PD-1 and B7-H4). See Chen, J. et al. (2012) "Upregulation OfB7- Hl Expression Is Associated With Macrophage Infiltration In Hepatocellular Carcinomas," Cancer Immunol. Immunother. 61(1): 101-108). B7-H1+ immunohistochemical stains of tumor biopsies may reflect the fact that the tumor cells are B7-H1+, or it may reflect the fact that B7-H1+ CD68+ TAMs are present within the tumor microenvironment. As such, tumor biopsies responses and/or treatments by overcoming any initial suppressive tumor microenvironment (if present).

Since CD68+ TAMs and TILs can correlate with the presence of B7- H1+ tumors, one aspect of the methods of the present disclosure relate to an assessment of the distinct cellular patterns of B7-H1 expression within a tumor to determine whether detected B7-H1 is being expressed by the tumor cells or by non-tumor cells (such as, for example, B7-H1+ CD68+ TAMs) that have infiltrated into the tumor. The disclosure thus more specifically relates to methods sufficient to accomplish the dual (or differential) detection of the cells of the tumor microenvironment (so as to assess their expression of B7-H1 and other biomarkers indicative of non-tumor cells). Such dual or differential verification provides clarification on which key cellular subsets within the tumor microenvironment are important therapeutic targets or predictive biomarkers for patient response to therapies targeting the immune checkpoint pathways.

B7-H1 expression may occur on the tumor, infiltrating macrophages, or both. Therefore, additional biomarkers can be used to differentiate which cells, tumor or non-tumor, are expressing the B7-H1 within the tumor microenvironment. If there is evidence that B7-H1 is expressed on tumor infiltrating macrophages, B7-H1 negative tumors may be targeted with PD- 1/B7-H1 targeted agents as if they are B7-H1 positive biopsies for diagnosis and/or treatment of the tumor. A lower expression of B7-H1 on TAMs provides an additional or alternative immunosuppressive B7-H1 target for therapeutic intervention to overcome compared to an absence of B7-H1 positive tumors, or alternatively when B7-H1 is broadly expressed across the entire tumor.

B7-H4 expression may also occur on either the tumor, infiltrating macrophages, or both. Therefore, additional biomarkers can be used to differentiate which cells, tumor or non-tumor, are expressing the B7-H4 within the tumor microenvironment. If there is evidence that B7-H4 is expressed on tumor infiltrating macrophages, B7-H4 negative tumors may be macrophages, such as CD14, CD68, CD163 and TLR2, FoxP3 etc, and biomarkers of other tumor infiltrating cells. Biomarkers suitable for use in accordance with the methods of the present disclosure are known in the art (see, e.g. , Kunisch, E. et al. (2004) "Macrophage Specificity Of Three Anti- CD68 Monoclonal Antibodies (KP1, EBM11, And PGM1 ) Widely Used For Immunohistochemistry And Flow Cytometry," Ann Rheum Dis. 63(7):774- 784; Kowal, K. et al. (2011) "CD163 And Its Role In Inflammation," Folia Histochem Cytobiol. 49(3):365-374; Thorp, E. et al. (2011) "The Role Of Macrophages And Dendritic Cells In The Clearance Of Apoptotic Cells In Advanced Atherosclerosis," Eur. J. Immunol. 41(9):2515-2518; Piccoli, A.K. et al. (2011) "Expression Of Complement Regulatory Proteins CD55, CD59, CD35, And CD46 In Rheumatoid Arthritis," Rev. Bras. Reumatol. 51(5):503- 510; Blum, K.A. (2010) "Upcoming Diagnostic And Therapeutic

Developments In Classical Hodgkin's Lymphoma," Hematology Amer. Soc. Hematol. Educ. Program. 2010:93-100; Conroy, H. et al. (2010)

"Inflammation And Cancer: Macrophage Migration Inhibitory Factor (MIF)-The Potential Missing Link " QJM 103(11):831-836; Grieb, G. et al. (2010) "Macrophage Migration Inhibitory Factor (MIF): A Promising Biomarker," Drug News Perspect. 23(4):257-264; Brown, K.E. et al. (2010) "Role OfPD-1 In Regulating Acute Infections," Curr. Opin. Immunol.

22(3):397-401; Jaoude, P.A. (2010) "Biomarkers In The Diagnosis Of Aspiration Syndromes," Expert Rev. Mol. Diagn. 10(3):309-319; Crowe, S.M. et al. (2009) "The Macrophage: The Intersection Between HIV

Infection And Atherosclerosis," J. Leukoc. Biol. 87(4):589-598; Beswick, E.J. et al. (2009) "CD74 In Antigen Presentation, Inflammation, And Cancers Of The Gastrointestinal Tract," World J. Gastroenterol.

15(23):2855-2861 ; Gibbings, D. et al. (2009) "CD4 and CD8: an inside-out coreceptor model for innate immune cells," J. Leukoc. Biol. 86(2):251-259; Andre, S. et al. (2009) "Surveillance Of Antigen-Presenting Cells By CD4+ CD25+ Regulatory T Cells In Autoimmunity: Immunopatho genesis And

Therapeutic Implications," Amer. J. Pathol. 174(5): 1575-1587; Joyce, J.A. Invest. Dermatol. 129(5): 1100-1114; Ford, J.W. et al. (2009) "TREM And TREM-Like Receptors In Inflammation And Disease," Curr. Opin. Immunol. 21(l):38-46; Collot-Teixeira, S. et al. (2007) "CD36 And Macrophages In Atherosclerosis " Cardiovasc Res. 2007 Aug l;75(3):468-477; Thompson, R.H. et al. (2007) "Implications OfB7-Hl Expression In Clear Cell

Carcinoma Of The Kidney For Prognostication And Therapy," Clin. Cancer Res. 13(2 Pt 2):709s-715s; Thompson, R.H. et al. (2006) "Significance of B7-H1 overexpression in kidney cancer," Clin. Genitourin. Cancer. 5(3):206- 211).

The present disclosure contemplates the concurrent or sequential analysis of: (1) B7-H4, B7-H1 or PD-1 expression and (2) the expression of one, two, three or more additional biomarkers, especially biomarkers that are characteristic of non-tumor cells within tumors and/or the tumor

microenvironment.

Antibodies that are immunospecific for biomarkers are known in the art (see citations noted above with respect to such biomarkers), or may be readily obtained using methods known in the art. Although antibodies (or their respective antigen-binding fragments) are the preferred binding molecules of the present disclosure, the disclosure further contemplates the use of protein receptors or receptor ligands as binding molecules. For example, PD-1 protein (or a B7-Hl-binding fragment thereof) may be employed as a B7-Hl-binding molecule. Similarly, the disclosure contemplates the use of B7-H1 protein (or a PD-l-binding fragment thereof) or B7-DC protein (or a PD-l-binding fragment thereof) as PD-l-binding molecules.

The disclosure additionally is directed to the use of fusion proteins possessing all or one or more contiguous fragments of such molecules (for example PD-1, B7-H4 or B7-H1) in the characterization of the cells of a tumor and/or tumor microenvironment. In one embodiment, such a fusion protein comprises all or one or more fragments of both B7-H1 and PD-1 and binds to both molecules. In an alternative embodiment, such a fusion protein CD14, CD68, CD163, TLR2, etc.). In a further alternative embodiment, such a fusion protein comprises all or one or more fragments of B7-H1 and a molecule (including an antibody or an antigen-binding fragment thereof) that binds a cell marker {e.g. , CD8, melanin, or a macrophage marker (e.g., CD14, CD68, CD163, TLR2, etc.) (see, e.g. , U.S. Patent No. 4,676,980).

The present disclosure also relates to improved methods for selecting patients who would be amenable for B7-H4 and/or PD-1 pathway targeted therapies and combination therapies, and for treating such patients. The disclosure also pertains to improved PD- 1 targeted therapies and

combination therapies for treating patients who have failed treatment with BRAF/MEK inhibitors or other inhibitors of the RAS-RAF-MEK-ERK pathway. The disclosure further pertains to improved PD- 1 targeted therapies and combination therapies to overcome resistance caused by "tumor dormancy" and to prevent the selection/outgrowth of rapidly, progressing, resistant tumors in the presence of various small molecule inhibitors. The present disclosure additionally provides a PD-1 targeted therapy which involves the administration of an immunomodulatory molecule such as a PD-l-binding fusion protein/antibody (e.g., an anti-PD-1 antibody, a B7-DC-Ig, a B7-Hl-Ig, etc.) with a BRAF inhibitor ("BRAFi") or other small molecule as an initial treatment regimen in such selected

HI

patients. The disclosure particularly provides a B7-DC-Ig that binds PD-1

B7-H1 T cells (chronically stimulated / exhausted T cells) but is substantially less capable or substantially incapable of binding to PD-1⁺ B7-

Hl⁺ cells (normal activated T cells).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the scoring of B7-H1 expression via

immunohistochemical methods of tumors using a scaled score that ranges from 0 (negative) to 3 (intense positive stain).

Figure 2 shows the extent of correlations between B7-H1 expression on tumors and PD-1+ TILs from individual patients with a variety of cancers. B7-H1 and PD-1 were detected in tumor biopsies via IHC. Figure 4 shows necrotic tissue that picks up the brown DAB stain used for B7-H1 detection non-specifically along with the presence of B7- H1+ CD68+ tumor associated macrophages.

Figure 5 shows that single stain (B7-H1 stain only)

immunohistochemical analysis of a tumor suggests the presence of B7-H1+ tumor cells (Left Panel), whereas dual stain (B7-H1 stain and CD68 stain) of the same tumor specimen establish that the detected expression of B7-H1 actually reflects the presence of CD68+ B7-H1+ macrophages.

Figure 6 shows an example where the tumor is B7-H1+ and the surrounding macrophages are B7-H1-. The dual CD68/B7-H1 stain confirms that the few tumor cells are B7-H1+. The disclosed examples show that B7- Hl is expressed only on the tumor, on the tumor and macrophages, or only on the macrophages in the presence of a B7-H1 negative tumor.

Figure 7 shows a sustained reduction in the percentage of PD-1 (HI) CD4+ and PD-1 (HI) CD8+ T cells remaining in the periphery from a melanoma BRAFm patient following therapy with a PD- 1 binding agent.

Figure 8 shows the H&E stains from three fresh biopsy specimens taken from a BRAFm melanoma patient who had failed BRAFi/MEKi therapy and then subsequently received treatment with a PD-1 binding agent. The pre-treatment biopsy was performed prior to therapy, the first post treatment biopsy was performed on Cycle 1, Day 15, following 1 dose of the PD-1 binding molecule. The second post treatment biopsy (C2D15) was taken on Cycle 2, Day 15 following three doses of the PD-1 binding molecule. This biopsy contains fibrous tissue with pockets of lymphocyte infiltrates. The presence of tumor cells was confirmed with S100 stain.

Figure 9 shows the results of immunohistochemical staining for B7- Hl, PD-1, CD8, CD4 and FoxP3. of biopsy samples of Figure 8, along with an archival specimen taken from the same patient prior to BRAFi/MEKi therapy.

Figure 10 shows that a sustained increase in TILs was observed following treatment with a PD-1 -binding molecule for tumors that had Figure 11, Panels A-D show multiple tumor biopsies from a BRAF mutant melanoma cancer patient. Prior to BRAFi/MEKi therapy (Panel A) B7-H1 expression was scored as 1; after such therapy but prior to therapy with a PD-1 binding molecule (Panel B), B7-H1 expression was scored as 3. Post-treatment (Panel C; Panel D), B7-H1 expression remained scored as 3 and as 2. Following treatment with a PD-1 binding molecule, B7-H1 expression was detected on remaining tumor cells, shed membrane and/or lymphocytes.

Figures 12A-12C are photoimages of a biopsy of a tumor taken from a BRAF mutant melanoma patient stained with B7-H1, PD-1 and CD8. The images reveal that there is a heterogeneous distribution of cells expressing B7-H1, CD8 and PD-1 markers across tumor tissue samples (Figure 12A), not all CD8+ T cells express PD-1 (Figure 12A), expression of tumor cells expressing B7-H1 tumor cells are located in the same area as CD8+ T cells (Figure 12A-12B) and the B7-H1 membranous expression is entirely on tumor cells in this biopsy (Figure 12C).

Figure 13 shows that high baseline LDH levels, or levels that rapidly increase above the upper level of normal (ULN) is prognostic of patients that will not successfully respond to PD-1 -targeted immunotherapy.

Figure 14 shows that the baseline absolute lymphocyte count (ALC) is a prognostic biomarker of successful response to PD-l-targeted immunotherapy.

Figure 15 shows the correlation between clinical outcome and baseline TIL levels.

Figures 16A-16B show the correlation between clinical outcome and polyfunctional T cell populations (CD8+ (Figure 16A) and CD4+ (Figure 16B)).

Figure 17 summarizes the preferred prognostic biomarker criteria of the present disclosure for patient selection for PD-1 targeted immunotherapy.

Figures 18A-18B show tumor biopsy stains of tumors of a patient having TILs (patient 0505 (CR); Figure 18A) and a patient lacking TILs the survival (Figure 19A) or tumor volume (Figure 19B) of mice having subcutaneous syngeneic CT26 colon carcinoma (5 independent studies, n = 10 mice/group/study). Figure 19C shows that the tumor cells were rejected following re-challenge.

Figure 20, Panels A-H, show the results of immunophenotype analysis conducted on Day 15 and Day 24 post- inoculation on mice having subcutaneous syngeneic CT26 colon carcinoma after administration of murine B7-DC IgG fusion.

Figure 21, Panels A-B, shows the results of CT scans of a melanoma patient in the 30 mg/kg dose-escalation cohort exhibiting a Partial Response (PR). The CT scans (of the lung) were performed prior to Cycle 1 (Figure 21, Panel A) and at the end of Cycle 4 (Figure 21, Panel B).

Figure 22, Panels A-B, shows the results of CT scans of a melanoma patient in the 30 mg/kg dose-escalation cohort exhibiting sustained (>20 months) Stable Disease (SD). The CT scans (of the neck) were performed prior to Cycle 1 (Figure 22, Panel A) and at the end of Cycle 6 (Figure 22, Panel B).

Figure 23, Panels A-B, shows the results of CT scans of a melanoma patient in the 30 mg/kg dose-escalation cohort exhibiting Mixed Response (MR) meaning that reduction in tumor volumes were observed for some lesions while increased tumor volumes were observed at other lesions. The CT scans (of the liver) were performed prior to Cycle 1 (Figure 23, Panel A) and at the end of Cycle 2 (Figure 23, Panel B).

Figures 24A-24B show the effect of the human B7-DC-Ig fusion on the levels of absolute lymphocyte, T cell, CD4⁺ T cell , CD8⁺ T cell , and

PD-l^LO T cell counts at 4 hours post-dose (Figure 24 A), and at 2-weeks post- dose (Figure 24B).

Figures 25A-25E show the changes in peripheral blood and the tumor microenvironment after treatment with human B7-DC-Ig Fusion molecules. Figure 25A: changes in the number of polyfunctional CD4⁺ T cells/ ml of blood; Figure 25B: changes in the number of polyfunctional CD8⁺ T cells/ specimens; Figure 25E: changes in TBX21 and FOXP3 gene expression (normalized to the expression of housekeeping genes) in paired tumor biopsy specimens.

Figure 26 shows changes in tumor CXCL9 gene expression in patients who left the trial after fewer than 4 cycles (black circles), patients who remained on the trial for 4 or more ("4+") (cycles (gray squares), and a clinical responder patient (gray triangles).

Figure 27 shows the correlation between tumor CXCL9 gene expression and CD8 TIL density in biopsy specimens (pretreatment, gray circle' post treatment, black triangle).

Figures 28 A-28E show the numbers of lymphocytes /ml of blood (Figure 28A), LDH fold over the upper limit of normal (Figure 28B), expression of tumor B7-H1 (Figure 28C), average number of CD8+ TIL cells per hpf (Figure 28D) and average number of PD-1+ TIL cells per hpf (Figure 28E) for Clinical Responders of treatment with human B7-DC-Ig Fusion molecules.

Figures 29A-29C show IHC images of a biopsy specimen from a melanoma metastasis on neck stained for B7-H1 (Figure 29A), CD8 (Figure 29B) or PD-1 (Figure 29C). White circles indicate cells that were counted as positive for the indicated marker.

Figures 30A-30B show changes in the ratio of CD8+ TIL cells to PD- 1+ TIL cells of paired tumor biopsy specimens from the 10-30 mg/kg cohorts, pre-treatment vs. post-treatment with a B7-DC Ig fusion molecule (Figure 30A) or across three treatment cycles (Figure 30B).

Figure 31 (Panels A-D) show IHC staining of B7-H1 (Panels A and

B) and CD8 (Panels C and D) pre-treatment (Panels A and C) and post- treatment with a B7-DC Ig fusion molecule (Panels B and D).

Figures 32A-32B show the normalized gene expression of a series of biomarkers in patients receiving human B7-DC-Ig Fusion therapy (black triangle - clinical responder; square - patients who stayed on trial for 4+ cycles; gray circle - patients who came off the clinical trial more rapidly due Fusion therapy.

Figure 34 shows the correlation between bDNA and IHC analyses of the gene expression of CXCL9 in patients receiving human B7-DC-Ig Fusion therapy.

Figures 35A-35C show the changes in gene expression of biomarkers in a clinical responder patient (patient 0402) receiving human B7-DC-Ig Fusion therapy over the course of the trial.

Figure 36 shows the changes in gene expression of biomarkers in a patient who remained in a human B7-DC-Ig Fusion therapy clinical trial 4+ cycles (patient 0506).

Figures 37A-37B show the changes in gene expression of biomarkers in a patient who remained in a human B7-DC-Ig Fusion therapy clinical trial 4+ cycles (patient 0609).

Figures 38A-38C show the effect of the administration of human B7- DC-Ig Fusion molecules on the number of polyfunctional CD4 T cells

(Figure 38A), polyfunctional CD8 T cells (Figure 38B), and GzmB+ Effector cells and EMRA cells (Figure 38C), per mL of blood as a function of the number of treatment cycles.

Figures 39A and 39B are micrographs of PD-L1 (B7-H1) and B7-H4 (CD68) in a set of serial tissue sections of a melanoma under low (39 A) and high (39B) magnification.

Figures 40A and 40B are micrographs of PD-L1 (B7-H1) and B7-H4 (CD68) in a set of serial tissue sections of a renal cell carcinoma under low (40A) and high (40B) magnification.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Programmed Death -1 ("PD-1") is a receptor of B7-H1 and B7-DC. PD-1 is a 50-55 kDa type I membrane protein member of the extended CD28/CTLA4 family of T cell regulators (Ishida, Y. et al. (1992) "Induced Expression Of PD-1, A Novel Member Of The Immunoglobulin Gene Superfamily, Upon Programmed Cell Death," EMBO J. 11 :3887-3895; 7,101,550; 7,488,802; 7,635,757; 7,722,868; PCT Publication No. WO 01/14557).

The amino acid sequence of human PD-1 is (SEQ ID NO:l):

QIPQAPWPV VWAVLQLGWR PGWFLDSPDR PWNPPTFSPA LLWTEGDNA TFTCSFSNTS ESFVLNWYRM SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFH SV VRARRNDSGT YLCGAI SLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGWGGLLGS LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW REKTPEPPVP CVPEQTEYAT IVFPSG GTS SPARRGSADG PRSAQPLRPE DGHCSWPL

The amino acid sequence of human B7-H1 is (SEQ ID NO:2):

RIFAVFIF TYWHLLNAFT VTVPKDLYW EYGSN TIEC KFPVEKQLDL AALIVYWE E

DKNI IQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRC ISYGG

ADYKRI TVKV NAPYNKINQR ILWDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT

TTNSKREEKL FNVTSTLRIN TTTNE IFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH

LVILGAILLC LGVALTFIFR LRKGR DVK KCGIQDTNSK KQSDTHLEET

The amino acid sequence of human B7-DC is (SEQ ID NO:3):

IFLLL LSL ELQLHQIAAL FTVTVPKELY I IEHGSNVTL ECNFDTGSHV NLGAITASLQ

KVENDTSPHR ERATLLEEQL PLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVK

ASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSV PANTSHSRTP EGLYQVTSVL

RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQ EPRTHPT WLLHIFIPFC IIAFIFIATV

IALRKQLCQK LYSSKDTTKR PVTTTKREVN ΞΑΙ

B7-H4 is member of the B7 family that is a negative regulator of T cell responses (U.S. Published Application Nos. 2012/0177645 and 2012/0276095).

The amino acid sequence of human B7-H4 is (SEQ ID NO:4):

ASLGQILFW SIISIIIILA GAIAL I IGFG ISGRHSITVT TVASAGNIGE DGILSCTFEP

DIKLSDIVIQ WLKEGVLGLV HEFKEGKDEL SEQDE FRGR TAVFADQVIV GNASLRLKNV

QLTDAGTYKC YIITSKGKGN ANLEYKTGAF S PEVNVDYN ASSETLRCEA PRWFPQPTVV

WASQVDQGAN FSEVSNTSFE LNSENVT KV VSVLYNVTIN NTYSC IEND IAKATGDIKV

TESEIKRRSH LQLLNSKASL CVSSFFAISW ALLPLSPYL LK

The amino acid sequence of another human B7-H4 is (SEQ ID

NO:5):

MASLGQILFW SIISIIIILA GAIAL I IGFG ISGRHSITVT TVASAGNIGE DGIQSCTFEP DIKLSDIVIQ WLKEGVLGLV HEFKEGKDEL SEQDEMFRGR TAVFADQVIV GNASLRLKNV QLTDAGTYKC YIITSKGKGN ANLEYKTGAF SMPEVNVDYN ASSETLRCEA PRWFPQPTVV WASQVDQGAN FSEVSNTSFE LNSENVTMKV VSVLYNVTIN NTYSCMIEND IAKATGDIKV benign. In contrast, a "non-tumor cell" is a normal cell (which may be quiescent or activated) that is located within a tumor microenvironment, including but not limited to Tumor Infiltrating Lymphocytes (TILs), leucocytes, macrophages, and/or other cells of the immune system, and/or stromal cells, and/or fibroblasts (e.g., cancer or tumor associated fibroblasts). The term "cell(s) of a tumor" is employed to refer to tumor cells and non- tumor cells located within a tumor or a tumor environment. The subject (e.g. , patient) and the tumors to be characterized in accordance with the present disclosure may be of any mammalian species (e.g. , human, or primate, canine, feline, bovine, ovine, equine, porcine, rodent species (e.g. , murine), etc.). The disclosure particularly concerns the characterization of human tumor cells as well as the characterization of human tumor microenvironments. The tumor cells of relevance to the present disclosure include, but are not limited to, tumor cells of the following cancers:

leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblasts, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and

myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia;

monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as, but not limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma

(hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget' s disease, and inflammatory breast cancer; adrenal cancer, including but not limited to, pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers including but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma; vaginal cancers, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget' s disease; cervical cancers including, but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers including, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor;

esophageal cancers including, but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma,

liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers including, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancers including, but not limited to, adenocarcinoma; cholangiocarcinomas including, but not limited to, cancer; testicular cancers including, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers including, but not limited to, squamous cell carcinoma; basal cancers;

salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers including, but not limited to, squamous cell cancer, and verrucous; skin cancers including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers including, but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/ or uterer); Wilms' tumor; bladder cancers including, but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, and gastic (for a review of such disorders, see Fishman et ah , 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia and Murphy et al. , 1997, Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A., Inc., United States of America).

As used herein, the term "characterizing" is intended to refer to assessing a patient, tissue sample or cell for the expression of a biomarker and its presentation on the surface of or within a cell. In accordance with the principles of the present disclosure, such characterization is mediated using molecules that physiospecifically bind, or that immunospecifically bind, to such expressed and presented molecules. "physiospecifically binding" to one another. A molecule may be capable of physiospecifically binding to more than one other molecule. A molecule is said to be able to "immunospecifically bind" a second molecule if such binding exhibits the specificity and affinity of an antibody to its cognate antigen. Antibodies are said to be capable of "immunospecifically binding" to a target region or conformation ("epitope") of an antigen (and in particular, the antigens: B7-H1 or PD-1) if such binding involves the antigen recognition site of the immunoglobulin molecule. An antibody that immunospecifically binds to a particular antigen may bind to other antigens with lower affinity if the other antigen has some sequence or conformational similarity that is recognized by the antigen recognition site as determined by, e.g. , immunoassays, BIACORE® assays, or other assays known in the art, but would not bind to a totally unrelated antigen. Preferably, however, antibodies (and their antigen binding fragments) will not cross-react with other antigens. Antibodies may also bind to other molecules in a way that is not immunospecific, such as to FcR receptors, by virtue of binding domains in other regions/domains of the molecule that do not involve the antigen recognition site, such as the Fc region.

In one embodiment, such binding molecules will be "homogeneic," (i.e. , molecules of the same species as that of the tumor being characterized, such as the use of human, chimeric or humanized antibodies for the characterization of the cells of a human tumor, or the use of human PD-1 protein to detect human B7-H1 on the surface of tumor or non-tumor cells). Alternatively, such binding molecules may be "heterogeneic," (i.e. , molecules of a species that differs from that of the tumor being characterized, such as the use of a murine monoclonal antibody for the characterization of a human tumor).

The term "biomarker" is intended to denote a molecule whose expression and presentation on the surface of a cell is characteristic of a particular cell type, or an attribute of a cell or tissue sample that is characteristic of a particular cell or tissue type. As indicated above, B7-H1 is Also of particular concern to the present disclosure are biomarkers that are prognostic for the selection of patients for subsequent treatment with PD- 1 targeted therapy. The preferred "prognostic biomarkers" of the present disclosure include:

Peripheral PD-1^HI Levels: The level of PD- 1^HI cells is a prognostic biomarker of immune function and response to PD-1 -targeted immunotherapy (PD-1^HI cells are discussed in: Onabajo, O.O. et al. (2013) "Rhesus Macaque Lymph Node PD-l(Hi)CD4( +) T Cells Express High Levels 0/CXCR5 And IL-21 And Display A CCR7(Lo)ICOS(+ )Bcl6(+ ) T- Follicular Helper (Tfh) Cell Phenotype," PLoS One. 8(3):e59758;

Myklebust, J.H. et al. (Epub 2013 Jan 7) "High PD-1 Expression And Suppressed Cytokine Signaling Distinguish T Cells Infiltrating Follicular Lymphoma Tumors From Peripheral T Cells," Blood 121(8): 1367-1376; Zhu, Z. et al. (Epub 2012 Nov 30) "High-Avidity T Cells Are Preferentially Tolerized In The Tumor Microenvironment," Cancer Res. 73(2):595-604; Yokosuka, T. et al. (Epub 2012 May 28) "Programmed Cell Death 1 Forms Negative Costimulatory Microc lusters That Directly Inhibit T Cell Receptor Signaling By Recruiting Phosphatase SHP2," J. Exp. Med. 209(6): 1201- 1217; Zelinskyy, G. et al. (2011) "Virus-Specific CD8+ T Cells Upregulate Programmed Death-1 Expression During Acute Friend Retrovirus Infection But Are Highly Cytotoxic And Control Virus Replication," J. Immunol. 187(7):3730-3737; Prendergast, A. et al. (2012) "Factors Influencing T Cell Activation And Programmed Death 1 Expression In HIV-infected Children," AIDS Res. Hum. Retroviruses 28(5):465-468; Duraiswamy, J. et al. (2011) "Phenotype, Function, And Gene Expression Profiles Of Programmed Death-1 (Hi) CD8 T Cells In Healthy Human Adults," J. Immunol.

186(7):4200-4212; Hamel, K.M. et al. (2010) "B7-H1 Expression On Non-B And Non-T Cells Promotes Distinct Effects On T- And B-Cell Responses In Autoimmune Arthritis," Eur. J. Immunol. 40(11):3117-3127; Blackburn, S.D. et al. (2010) "Tissue-Specific Differences In PD-1 And PD-L1 Expression During Chronic Viral Infection: Implications For CD8 T-Cell Exhaustion," (2009) "Impact Of Epitope Escape On PD-1 Expression And CD8 T-Cell Exhaustion During Chronic Infection," J. Virol. 83(9):4386-4394; Holm, M. et al. (2008) "PD-1 Predicts CD4 Loss Rate In Chronic HIV-1 Infection Better Than HIV RNA And CD38 But Not In Cryopreserved Samples," Curr. HIV Res. 6(l):49-58; Okazaki, T. et al. (2007) "PD-1 And PD-1 Ligands: From Discovery To Clinical Application," Int. Immunol. 19(7):813-824).

Released Lactate Dehydrogenase: Lactate dehydrogenase (LDH) is released into the serum from dying cells, and is a marker of rapid disease progression in cancer, particularly melanoma (Brown, J.E. et al. (Epub 2012 Sep 4) "Serum Lactate Dehydrogenase Is Prognostic For Survival In Patients With Bone Metastases From Breast Cancer: A Retrospective Analysis In Bisphosphonate-Treated Patients," Clin. Cancer Res.

18(22):6348-6355; Armstrong, A.J. et al. (Epub 2012 Aug 13) "Serum Lactate Dehydrogenase Predicts For Overall Survival Benefit In Patients With Metastatic Renal Cell Carcinoma Treated With Inhibition Of

Mammalian Target Of Rapamycin," J. Clin. Oncol. 30(27):3402-3407; Cetin, B. et al. (2012) "Prognostic Factors For Overall Survival In Patients With Metastatic Colorectal Carcinoma Treated With Vascular Endothelial Growth Factor-Targeting Agents," Asian Pac. J. Cancer Prev. 13(3): 1059- 1063; Cairo, M.S. et al. (Epub 2012 Jan 3) "Advanced Stage, Increased Lactate Dehydrogenase, And Primary Site, But Not Adolescent Age (> 15 Years), Are Associated With An Increased Risk Of Treatment Failure In Children And Adolescents With Mature B-Cell Non-Hodgkin's Lymphoma: Results Of The FAB LMB 96 Study," J. Clin. Oncol. 30(4):387-393; Chau, N.G. et al. (2011) "Early Mortality And Overall Survival In Oncology Phase I Trial Participants: Can We Improve Patient Selection! " BMC Cancer. 11:426; Van Cutsem, E. et al. (2011) "Randomized, Placebo-Controlled, Phase III Study Of Oxaliplatin, Fluorouracil, And Leucovorin With Or Without PTK787/ZK 222584 In Patients With Previously Treated Metastatic Colorectal Adenocarcinoma," J. Clin. Oncol. 29(15):2004-2010; Brunetto, A.T. et al. (2010) "A Study Of The Pattern Of Hospital Admissions In A 60 Years Of Age With Acute Lymphoblastic Leukemia In First Relapse," Leuk. Lymphoma. 50(7): 1126-1131; Agarwala, S.S. et al. (2009) "LDH Correlation With Survival In Advanced Melanoma From Two Large, Randomised Trials (Oblimersen GM301 And EORTC 18951)," Eur. J.

Cancer. 45(10): 1807-1814; Arkenau, Η.Τ. et al. (2009) "Prospective Validation Of A Prognostic Score To Improve Patient Selection For

Oncology Phase I Trials " J. Clin. Oncol. 27(16):2692-2696; van Imhoff, G.W. et al. (2005) "Impact Of Three Courses Of Intensified CHOP Prior To High-Dose Sequential Therapy Followed By Autologous Stem-Cell

Transplantation As First-Line Treatment In Poor-Risk, Aggressive Non- Hodgkin 's Lymphoma: Comparative Analysis Of Dutch-Belgian Hemato- Oncology Cooperative Group Studies 27 And 40," J. Clin. Oncol.

23(16):3793-3801).

ALC Level: Absolute lymphocyte counts (ALC) and the rate of decline of ALC over time in the peripheral blood can be markers for the ability to mount an immune responsive (immune competency) (Lad, D.P. et al. (Epub 2012 Oct 16) "Regulatory T-Cells In B-Cell Chronic Lymphocytic Leukemia: Their Role In Disease Progression And Autoimmune Cytopenias," Leuk. Lymphoma. doi: 10.3109/10428194.2012.728287; Decker, T. et al. (Epub 2012 Jul 4) "Increased Number Of Regulatory T Cells (T-Regs) In The Peripheral Blood Of Patients With Her-2/Neu-Positive Early Breast Cancer," J. Cancer Res. Clin. Oncol. 138(11): 1945-1950; Koh, Y.W. et al. (Epub 2012 May 15) "The Ratio Of The Absolute Lymphocyte Count To The Absolute Monocyte Count Is Associated With Prognosis In Hodgkin 's Lymphoma: Correlation With Tumor-Associated Macrophages," Oncologist 17(6):871-880; Mitrovic, Z. et al. (Epub 2012 Apr 10) "The Prognostic Significance Of Lymphopenia In Peripheral T-C ell And Natural Killer/T-Cell Lymphomas: A Study Of 826 Cases From The International Peripheral T- Cell Lymphoma Project," Am. J. Hematol. 87(8):790-794; Droeser, R. et al. (2012) "Differential Pattern And Prognostic Significance Of CD4+, FOXP3+ And IL-17+ Tumor Infiltrating Lymphocytes In Ductal And Rituximab, Cyclophosphamide, Adriamycin, Vincristine And Prednisone," Kim, Y.R. et al. (2011) "Lymphopenia Is An Important Prognostic Factor In Peripheral T-Cell Lymphoma (NOS) Treated With Anthracycline-Containing Chemotherapy," J. Hematol. Oncol. 4:34; Mocikova H. (2010) "Prognostic Significance Of Absolute Lymphocyte Count And Lymphocyte Subsets In Lymphomas," Prague Med. Rep. 111(1):5-11; Porrata, L.F. et al. (2009) "Absolute Lymphocyte Count At The Time Of First Relapse Predicts Survival In Patients With Diffuse Large B-Cell Lymphoma," Am. J. Hematol.

84(2):93-97; Oki, Y. et al. (2008) "Low Absolute Lymphocyte Count Is A Poor Prognostic Marker In Patients With Diffuse Large B-Cell Lymphoma And Suggests Patients' Survival Benefit From Rituximab," Eur. J. Haematol. 81(6):448-453; Palmer, S. et al. (2008) "Prognostic Importance Of T And NK-Cells In A Consecutive Series Of Newly Diagnosed Patients With Chronic Lymphocytic Leukaemia," Br. J. Haematol. 141(5):607-614).

Baseline TIL Level: Fresh tumor biopsies taken at baseline

(prior to enrollment) can be used to measure the number of tumor infiltrating lymphocytes (TILs) in a tumor (Ascierto, P.A. et al. (2013) "The Additional Facet Of Immunoscore: Immunoprofiling As A Possible Predictive Tool For Cancer Treatment," J. Transl. Med. 11 :54; Donskov, F. (2013)

"Immunomonitoring And Prognostic Relevance Of Neutrophils In Clinical Trials " Semin. Cancer Biol, dokpii: S1044-579X(13)00013-8.

10.1016/j.semcancer.2013.02.001 ; Judd, N.P. et al. (2012) "Comparative Analysis Of Tumor- Infiltrating Lymphocytes In A Syngeneic Mouse Model Of Oral Cancer," Otolaryngol. Head Neck Surg. 147(3):493-500; Ji, R.R. et al. (2012) "An Immune-Active Tumor Microenvironment Favors Clinical

Response To Ipilimumab," Cancer Immunol. Immunother. 61(7): 1019-1031, Hamid, O. et al. (2011) "A Prospective Phase II Trial Exploring The Association Between Tumor Microenvironment Biomarkers And Clinical Activity Of Ipilimumab In Advanced Melanoma," J. Transl. Med. 9:204; Huang, R.R. et al. (2011) "CTLA4 Blockade Induces Frequent Tumor

Infiltration By Activated Lymphocytes Regardless Of Clinical Responses In 220; Atzpodien, J. et al. (2008) "Peripheral Blood Neutrophils As

Independent Immunologic Predictor Of Response And Long-Term Survival Upon Immunotherapy In Metastatic Renal-Cell Carcinoma," Cancer Biother. Radiopharm. 23(1): 129-134; Romano, F. et al. (2006) "Preoperative IL-2 Immunotherapy Enhances Tumor Infiltrating Lymphocytes (TILs ) In Gastric Cancer Patients " Hepatogastroenterology 53(70):634-638).

As used herein, a "PD-1 targeted therapy" is a therapy that involves the administration of molecules that physiospecifically or

immunospecifically bind PD-1 or any of its ligands (e.g. , B7-H1, B7-DC, etc.). More preferably, such molecules physiospecifically bind PD-1 and comprise, for example, anti-PD-1 antibodies, anti-PD-1 antibody antigen- binding fragments, and B7-DC (or B7-H1) fusion proteins (such as a B7-DC- Ig fusion or a B7-Hl-Ig fusion). As used herein, PD-1 targeted therapies in which the mechanism of action is dependent on blocking the interaction between the ligand (B7-H1 or B7-DC) and the receptor (PD-1) are denoted as "ligand dependent." Blocking or neutralizing anti-PD- 1 antibodies are examples of molecules with ligand dependent activity. PD-1 targeted therapies that are able to bind PD-1+ cells and modulate PD-1 levels or cellular activity in the absence of PD-1 ligands are denoted as having "ligand independent activities." Agonistic anti-PD-1 antibodies and B7-DC fusion proteins (such as a B7-DC-Ig) are examples of molecules with ligand independent activity whereby the ability to modulate PD-1⁺ cells directly has been clearly demonstrated. Ligand dependent and ligand independent activities are not mutually exclusive and a single PD- 1 targeted therapy may demonstrate both activities.

Such molecules can be produced by screening hybridoma lines for those that produce antibody that are immunospecific for human PD-1, and then optionally screening amongst such lines for those exhibiting modulating activity (e.g. , neutralizing activity, agonizing activity, internalizing activity, altered signal transducing activity, etc.). In an alternative embodiment, the disclosure provides for the use of PD-1 ligands that physiospecifically bind (also known as Nivolumab or BMS-936558), MK3475 (also referred to as lambrolizumab and pembrolizumab), and CT-011 (Pardoll, D.M. (April 2012) "The Blockade Of Immune Checkpoints In Cancer Immunotherapy," Nature Reviews Cancer 12:252-264).

As used herein, a "B7-H4 targeted therapy" is a therapy that involves the administration of molecules that physiospecifically or

immunospecifically bind B7-H4 or any of its receptors. More preferably, such molecules physiospecifically bind B7-H4 and comprise, for example, anti-B7-H4 antibodies, and anti-B7-H4 antibody antigen-binding fragments. As used herein, B7-H4 targeted therapies in which the mechanism of action is dependent on blocking the interaction between the ligand (B7-H4) and a receptor thereof are denoted as "ligand dependent." Blocking or neutralizing anti-B7-H4 antibodies are examples of molecules with ligand dependent activity. B7-H4 targeted therapies that are able to bind B7-H4 receptor+ cells and modulate B7-H4 receptor levels or cellular activity in the absence of B7-H4 are denoted as having "ligand independent activities."

Antagonistic anti-B7-H4 receptor antibodies and soluble, antagonistic B7-H4 proteins (such the extracellular domain of B7-H4) that can bind to B7-H4 receptors without activating signal transduction through the receptor are examples of molecules with ligand independent activity. Ligand dependent and ligand independent activities are not mutually exclusive and a single B7- H4 targeted therapy may demonstrate both activities.

Such molecules can be produced by screening hybridoma lines for those that produce antibody that are immunospecific for human B7-H4 or a receptor thereof, and then optionally screening amongst such lines for those exhibiting modulating activity (e.g. , neutralizing activity, agonizing activity, internalizing activity, altered signal transducing activity, etc.). In an alternative embodiment, the disclosure provides for the use of B7-H4 ligands that physiospecifically bind to human B7-H4 receptors. Exemplary molecules are known in the art. See, for example, WO 2013/025779 which provides anti-B7-H4 antibodies and WO 2008/083239 which provides B7- recognition site. Natural antibodies and monoclonal antibodies possess two heavy chains and two light chains, each of which contains a variable domain and one or more constant domains. The term "variable region" is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region comprises a "hypervariable region" whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from a "Complementarity Determining Region" or "CDR" (i.e. , typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (i.e. , residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96- 101 (H3) in the heavy chain variable domain; Chothia, C. et al. (1987) "Canonical Structures For The Hypervariable Regions Of

Immunoglobulins," J. Mol. Biol. 196:901-917). "Framework Region" or "FR" residues are those variable domain residues other than the

hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi- specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldermans, S. et al. (2001) "Recognition Of Antigens By Single-Domain Antibody Fragments: The Superfluous Luxury Of Paired Domains," Trends Biochem. Sci. 26:230-235; Nuttall, S.D. et al. (2000) "Immunoglobulin VH Domains And Beyond: Design And Selection Of Single-Domain Binding And Targeting Reagents," Cur. Pharm. Biotech. 1:253-263; Reichmann, L. et al. (1999) "Single domain antibodies:

comparison of camel VH and camelised human VH domains," J. Immunol. Methods 231(l-2):25-38; PCT Publication Nos. WO 94/04678 and WO

94/25591; U.S. Patent No. 6,005,079), single-chain Fvs (scFv) (see, e.g., see anti-idiotypic (anti-Id) antibodies (including, e.g. , anti-Id and anti-anti-Id antibodies to antibodies of the disclosure). In particular, such antibodies include immunoglobulin molecules of any type (e.g. , IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. , Igd, IgG₂, IgG₃, IgG₄, IgA] and IgA₂) or subclass.

As used herein, the term "antigen binding fragment" of an antibody refers to one or more portions of an antibody that contain three light chain CDRs and three corresponding heavy chain CDRs and optionally the framework residues that comprise the antibody's "variable region" antigen recognition site, and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab, F(ab')₂, Fv, single chain (ScFv),and mutants thereof, naturally occurring variants, and fusion proteins comprising the antibody' s "variable region" antigen recognition site and a heterologous protein (e.g. , a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.). As used herein, the term "fragment" refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

A humanized or chimeric antibody of the disclosure may comprise substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i. e. , donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus may be selected with respect to the proposed function of the antibody, in particular the effector function which may be required. In some

embodiments, the constant domains of the antibodies of the disclosure are (or comprise) human IgA, IgD, IgE, IgG or IgM domains. In a specific embodiment, human IgG constant domains, especially of the IgGl and IgG3 isotypes are used, when the humanized antibodies of the disclosure is intended for therapeutic uses and antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement- dependent cytotoxicity (CDC) activity are needed. For example, PD-1 is highly expressed on T cells as well as rare peripheral T cell lymphomas such as Angioimmunoblastic T-cell lymphoma (AITL). Anti-PD-1 antibodies with ADCC or CDC activity are particularly relevant as therapeutic agents for treating such cancers. In alternative embodiments, IgG2 and IgG4 isotypes are used when the antibody of the disclosure is intended for therapeutic purposes and antibody effector function is not required. For example, if you want to increase the activity of T cells by targeting PD-1 on the surface of T cells, then effector functions that would kill the T cell are undesirable. The disclosure encompasses Fc constant domains comprising one or more amino acid modifications which alter antibody effector functions such as those disclosed in U.S. Patent Application Publication Nos. 2005/0037000 and 2005/0064514.

The therapeutic antibodies used in the methods of the present disclosure may be monospecific. Of particular interest, however, are bispecific antibodies, trispecific antibodies or antibodies of greater multispecificity that exhibit specificity to one, two or more targets in addition to B7-H1, B7-H4 or PD-1. For example, such antibodies may bind to multiple cell antigens or cellular molecules (e.g., CD4, CD8, CD25, CTLA4, melanin, or a macrophage marker (e.g. , CD14, CD68, CD163, TLR2, etc.). Such bispecific antibodies, trispecific antibodies or antibodies of greater multispecificity may bind, for example, to both B7-H1 and PD-1.

Antibodies or fragments thereof with increased in vivo half-lives can multifunctional linker either through site-specific conjugation of the PEG to the N- or C- terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used. The degree of conjugation will be closely monitored by SDS- PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies. Unreacted PEG can be separated from antibody-PEG conjugates by, e.g. , size exclusion or ion-exchange chromatography.

The antibodies of the disclosure may also be modified by the methods and coupling agents described by Davis et al. (See U.S. Patent No. 4,179,337) in order to provide compositions that can be injected into the mammalian circulatory system with substantially no immunogenic response.

The present disclosure also encompasses antibodies (and more preferably, humanized antibodies) and antigen-binding fragments thereof that are recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a heterologous molecule (i.e. , an unrelated molecule). The fusion does not necessarily need to be direct, but may occur through linker sequences. The Fc portion of the fusion the fusion protein may be varied by isotype or subclass, may be a chimeric or hybrid, and/or may be modified, for example to improve effector functions, control of half-life, tissue accessibility, augment biophysical characteristics such as stability, and improve efficiency of production (and less costly). Many modifications useful in construction of disclosed fusion proteins and methods for making them are known in the art, see for example Mueller, et al., Mol. Immun., 34(6):441-452 (1997), Swann, et al., Cur. Opin. Immun., 20:493-499 (2008), and Presta, Cur. Opin. Immun. 20:460-470 (2008). In some embodiments the Fc region is the native IgGl, IgG2, or IgG4 Fc region. In some embodiments the Fc region is a hybrid, for example a chimeric consisting of IgG2/IgG4 Fc constant regions. Modifications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to changing expression host), and IgGl with altered pH-dependent binding to FcRn. The Fc region may include the entire hinge region, or less than the entire hinge region.

Any of the molecules of the present disclosure can be fused to marker sequences, such as a peptide, to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et ah , 1984 Cell, 37:767) and the "flag" tag (Knappik et al , 1994 Biotechniques, 17(4):754-761).

As used herein, the term "combination therapy" refers to a treatment of a disease or a method for achieving a desired physiological change, such as increased or decreased response of the immune system to an antigen or immunogen, such as an increase or decrease in the number or activity of one or more cells, or cell types, that are involved in such response, wherein said treatment or method comprises administering to an animal, such as a mammal, especially a human being, a sufficient amount of two or more chemical agents or components of said therapy to effectively treat a disease or to produce said physiological change, wherein said chemical agents or components are administered together, such as part of the same composition, or administered separately and independently at the same time or at different times (i.e., administration of each agent or component is separated by a finite period of time from one or more of the agents or components). In some embodiments, administration of said one or more agents or components in combination achieves a result greater than that of any of said agents or components when administered alone or in isolation.

II. Methods For Selecting and Treating Patients Amenable For PD-1 or B7-H4 Targeted Therapies And Combination Therapies

However, these approaches are hindered by the finding that certain patients are refractile to therapy, and thus fail to exhibit a positive or sustained A. Characterization of Tumor and Non-Tumor Cells

Methods for characterizing tumors and/or for characterizing the tumor microenvironment are provided. In particular, the disclosure pertains to improved methods for characterizing tumors so as to assess the extent to which the tumor cells and/or tumor infiltrating cells or tumor associated cells express cell surface molecules, such as B7-H1, PD-1 and B7-H4, and to distinguish between tumor cells that express such biomarkers and non-tumor cells present within the tumor and/or within the tumor microenvironment. The disclosure concerns the uses of such methods in the diagnosis and the treatment of cancer and other diseases.

Therefore, methods for characterizing a cell of a tumor are provided. The methods can include determining whether a cell of the tumor expresses B7-H1; and determining whether the cell of the tumor that expresses B7-H1 is a tumor cell or a non-tumor cell. In some embodiments, a method for characterizing a cell of a tumor includes determining whether a cell of a tumor expresses B7-H4; and determining whether the cell of the tumor that express B7-H4 is a tumor cell or a non-tumor cell.

Methods for assessing the amenability of subject to a proposed anticancer therapy are also provided. The methods can include, for example, characterizing cells of a tumor of said patient by determining whether the cells of the tumor express B7-H1 or B7-H4; and determining whether the cells of the tumor that express B7-H1 or B7-H4 are tumor cells or non-tumor cells.

Methods for assessing the efficacy of an anti-cancer therapy provided to a subject are also disclosed. The methods can include, for example, characterizing cells of a tumor of the patient during the course of the therapy or after the completion thereof, wherein said characterization can include determining whether the cells of the tumor express B7-H1 or B7-H4; and determining whether said cells of said tumor that express B7-H1 or B7- H4 are tumor cells or non-tumor cells.

Methods for selecting patients for anti-cancer therapy based on biologic therapies, and/or other therapeutic interventions (e.g. radiation, cryoablation, surgical resection of the tumor etc.) in order to see if expression patterns of B7-H1, B7-H4 or PD-1 within the tumor

microenvironment have changed and whether or not cells expressing markers are tumor cells or non-tumor cells.

The methods typically include detecting B7-H1, PD-1, and/or B7- H4 alone or in combination with one or more biomarkers of non-tumor cells. Suitable methods of detection are known in the art and discussed above. For example, some of the disclosed methods include a step of contacting the cell of the tumor with: (A) a molecule that immunospecifically or

physiospecifically binds B7-H1; and (B) a molecule that immunospecifically binds to a biomarker that is characteristic of a non-tumor cell. In some embodiments, the contacting (A) and (B) are conducted concurrently. In some embodiments, the contacting (A) and (B) are conducted sequentially. In some preferred embodiments, the molecule that immunospecifically or physiospecifically binds B7-H1 is an anti-B7-Hl antibody or an antigen- binding fragment thereof. In another embodiment the molecule that immunospecifically or physiospecifically binds B7-H1 includes PD-1 or a B7-Hl-binding portion thereof.

Some of the methods include, for example, contacting said cell of the tumor with: (A) a molecule that immunospecifically or physiospecifically binds B7-H4; and (B) a molecule that immunospecifically binds to a biomarker that is characteristic of a non-tumor cell. In some embodiments, the contacting (A) and (B) are conducted concurrently. In some

embodiments, the contacting (A) and (B) are conducted sequentially. In some embodiments the molecule that immunospecifically or

physiospecifically binds B7-H4 is an anti-B7-H4 antibody or an antigen- binding fragment thereof.

In some embodiments, the disclosed methods include contacting the cell of the tumor with more than one molecule, each of which

immunospecifically binds to a different biomarker that is characteristic of a FoxP3. Furthermore, CD3 can be used to differentiate between lymphocytes that expression B7-H1 and those that do not. It will also be appreciated that these are markers that are characteristic of immune cells and can be expressed on neoplastic cells in leukemia / lymphoma. Therefore, in some embodiments, these markers are used to identify non-tumor cells in cancer samples from non-hematological cancers. In preferred embodiments, these are biomarkers of non-tumor cells present in solid tumors.

In preferred embodiments, the molecule that immunospecifically or physiospecifically binds B7-H1, PD-1, B7-H4, and biomarkers of non-tumor cells to detect the B7-H1, PD-1, B7-H4, or biomarker of non-tumor cells is an antibody or antigen-binding fragment thereof. As discussed above, in some embodiments, the antibody is detectably-labeled. In other

embodiments, a antibody the immunospecifically or physiospecifically binds B7-H1, PD-1, B7-H4, or biomarker of non-tumor cells is detected using a second antibody that immunospecifically or physiospecifically binds to the first antibody. The second antibody can be detectably-labeled. In a preferred embodiment, detection of the molecule, such as an antibody, is carried out by immunohistochemistry or immunocytochemistry. The detectable label can be a fluorophore.

In some embodiments, the method of detection is an in vitro method.

In another embodiment, the in vitro method comprises immunohistochemical staining, in situ hybridization; or flow cytometry.

In another embodiment, the method of detection is an in vivo method. In one implementation, the in vivo method comprises computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

Suitable tumors that can be analyzed according the disclosed methods are discussed above and elsewhere herein. In preferred embodiments, the tumor is a solid tumor. In particular embodiments, tumor is a tumor of an adrenal cancer, a bladder cancer, a bone and connective tissue sarcoma, a brain tumor, a breast cancer, a colon or rectal cancer, an esophageal cancer, cancer, a stomach cancer, a testicular or penile cancer, a thyroid cancer, or a vaginal, ovarian, uterine, or cervical cancer, or a gastric cancer.

In some embodiments, the non-tumor cell is a macrophage, lymphocyte, leukocyte, stromal cell, or cancer associated fibroblast.

B7-H1 expression may also occur on either the tumor, infiltrating macrophages, or both. If there is evidence that B7-H1 is expressed on tumor infiltrating macrophages, subjects with B7-H1 negative tumors can be selected for treatment with PD-1/B7-H1 targeted agents as if they are B7-H1 positive biopsies for diagnosis and/or treatment of the tumor. A lower expression of B7-H1 on TAMs provides an additional or alternative immunosuppressive B7-H1 target for therapeutic intervention to overcome compared to an absence of B7-H1 positive tumors, or alternatively when B7- Hl is broadly expressed across the entire tumor. For example, subjects can be selected for treatment with a PD-1 therapy if the tumor cells, non-tumor cells (particularly TAMs), or both tumor and non-tumor cells of the tumor are found to express B7-H1. In some embodiments, subjects that have B7- Hl positive tumor cells, B7-H1 positive TAMs, or both are treated with a PD-1 therapy that blocks PD-1 dependent signaling, but is not cytotoxic to cells expressing B7-H1. In other embodiments, subjects that have B7-H1 positive tumor cells, and preferably B7-H1 negative TAMs are treated with a PD-1 therapy that is cytotoxic to cells expressing B7-H1.

B7-H4 expression may also occur on either the tumor, infiltrating macrophages, or both. Therefore, additional biomarkers can be used to differentiate which cells, tumor or non-tumor, are expressing the B7-H4 within the tumor microenvironment. If there is evidence that B7-H4 is expressed on tumor infiltrating macrophages, B7-H4 negative tumors may be targeted with B7-H4 targeted agents as if they are B7-H4 positive biopsies for diagnosis and/or treatment of the tumor. For example, subjects can be selected for treatment with a B7-H4 therapy if the tumor cells, non-tumor cells (particularly TAMs), or both tumor and non-tumor cells of the tumor are found to express B7-H4. In some embodiments, subjects that have B7- cells expressing B7-H4. In some embodiments, subjects that have B7-H4 positive tumor cells and B7-H4 negative TAMs are treated with a B7-H4 therapy that is cytotoxic to cells expressing B7-H4. In some embodiments, subjects that have B7-H4 negative tumor cells and B7-H4 positive TAMs are treated with a B7-H4 therapy that blocks B7-H4 dependent signaling but is not cytotoxic to cells expressing B7-H4.

B. Prognostic Markers

As indicated above, the present disclosure relates to improved methods for selecting patients who would be amenable for PD- 1 targeted therapies and combination therapies. In particular, the disclosure pertains to improved PD- 1 targeted therapies and combination therapies for treating patients who have failed treatment with BRAF/MEK inhibitors or other inhibitors of the RAS-RAF-MEK-ERK pathway. The disclosure further pertains to improved PD- 1 targeted therapies and combination therapies to overcome resistance caused by "tumor dormancy" and to prevent the selection/outgrowth of rapidly, progressing, resistant tumors in the presence of various small molecule inhibitors.

Therefore, prognostic markers capable of providing improved patient selection criteria for PD-1 targeted therapy or other immunotherapies are provided. In order for patients to respond to immunotherapy, such as PD- 1 targeted therapy, they should typically demonstrate a threshold level of immune competency (i.e., a level of immune competency sufficient to allow the immune system to mount a response when an immune stimulatory molecule is administered). Based on clinical observations, a number of such prognostic markers have been identified that can be used for patient selection (i. e. , for the identification of "immune responders" or for defining inclusion/exclusion criteria).

1. Peripheral PD-1^HI T Cell Levels

PD-1 is up-regulated following antigen exposure via TCR engagement and activation of the transcription factor NFAT (Oestreich et al. (2008) "NFATcl Regulates PD-1 Expression Upon T Cell Activation," J. Immunol. 184(l):476-487). As shown herein, the population of CD4 or

+ HI

CD8 cells that are PD-lⁿⁱ T cells can be reduced following treatment with PD- 1 targeted therapy in a dose-dependent manner, and reductions are can be sustained through at least the first cycle of treatment. Thus, the level of

HI

peripheral PD- 1 T cells is a prognostic biomarker for immune

responsiveness to PD-1 targeted therapy.

A finding that the level of PD-1^HI T cells does not show an acute reduction immediately following PD- 1 targeted therapy, or if there is initial reduction, that the levels are not sustained and subsequently rebound above baseline, is prognostic of inability to respond to PD-1 targeted therapy. The ability to maintain a sustained decrease in PD-1 levels in the periphery is thus a prognostic marker for response to PD-1 targeted therapy. In contrast, patients who exhibit an early and sustained decrease in the level of their PD-

HI

lⁿⁱ T cells are more likely to exhibit a successful ultimate clinical response (see, e.g. , Table 1).

2. LDH

Lactate dehydrogenase (LDH) is released into the serum from dying cells and is a marker of rapid disease progression in cancer, particularly melanoma. High baseline LDH or rapidly increasing LDH is a biomarker for rising antigen exposure, which leads to increased T cell exhaustion. High baseline LDH is also associated with up-regulation of PD-1 expression. Rapid T cell exhaustion and PD-1 expression may counteract the activity of a PD-1 targeting therapy. Patients with high LDH levels, or levels that rapidly increase above the upper level of normal (ULN), particularly early in the course of therapy, do not respond well to immunotherapy (such as a PD- 1 targeted therapy). Therefore, cancer patients selected for immunotherapy, and particularly immunotherapy involving such as PD- 1 targeted therapy, can have low baseline LDH levels (or be enrolled for treatment before LDH levels reach high levels). LDH levels suitable for immunotherapy treatment are those generally that are < 2-fold greater than the ULN, and/or which are not increasing rapidly. A level of lactate dehydrogenase that is more than should be monitored to make sure they are stable, or more preferably, decreasing. Patients who exhibit rapid increases in LDH levels following the start of treatment (i. e. , within the first couple of months) may be considered for alternative therapy.

3. Combined LDH Release and PD-1^HI T Cell Levels A correlation exists between of the level of LDH release and the level of PD-1^HI cells, such that a synergistic prognostic benefit is obtained by evaluating both such prognostic markers.

4. ALC Levels:

Absolute lymphocyte counts (ALC) in the peripheral blood are a marker for the ability to mount an immune responsive (immune

competency). In some embodiments, patients that demonstrate the ability to respond to PD-1 targeting therapy have high ALC counts (> 1000 cells/ 'μί), and high ALC counts correlated with improved immune function following PD- 1 targeted therapy, as indicated by high intracellular cytokine markers (IFNy, TNFa and IL2) and/or the expression of immune effector genes (TNFRSF9, TNFRSF4, ICOS, KLRG1, CXCL10, CCR2, CXCL9, granzymeA and granzymeB).

In other embodiments, a rapid decline in ALC over a short period of time is an indicator that the subject may respond poorly to PD-1 targeted therapy, and may require a combination therapy, for example a therapy including a PD- 1 targeted therapy and a second agent that boosts the immune system (e.g., IL-2). A rapid decline in in absolute lymphocyte count over a short period of time can be, for example, a persistent decline of >40 of the ALC count over the course of 2 treatment cycles. In a particular

embodiment, each treatment cycle is about 1 month (e.g., 2 treatment cycles can equal two months). A persistent decline excludes acute drops that can be observed immediately or shortly after dosing, which subsequently rebound.

Patients demonstrating a low ALC may be treated with an agent to boost lymphocyte counts prior to immune therapy. Suitable pre-treatment introduced into the patient by adoptive cell therapy (ACT) prior to immune therapy. ALCs should be monitored with treatment as a metric for improved immune function and responsiveness. No increase in ALCs with treatment over time may indicate that the patient is not responding to immune therapy and not a good candidate for such therapy. A baseline absolute lymphocyte count that is less than approximately 1000 cells/ μL can be predictive of a patient's enhanced suitability for treatment with a PD-1 targeted combination therapy, particularly where the combination therapy causes an increase in ALC levels.

5. Baseline TIL Levels

Fresh tumor biopsies taken at baseline (prior to enrollment) can be used to measure the number of tumor infiltrating lymphocytes (TILs) in a tumor. Patients that demonstrate a high number of TILs (e.g. , greater than 100 per high powered microscope field (hpf)) are most likely to demonstrate a clinical response to immune therapy, such as PD-1 targeting therapy. A baseline tumor infiltrating lymphocyte count that is less than approximately 100 cells/hpf is predictive of a patient's enhanced suitability for treatment with a PD-1 targeted combination therapy. TILs can be identified by immunohistochemistry of tumor sections that is capable of differentiating lymphocytes from tumor cells (e.g. , staining sections for CD8 or PD-1). Post therapy tumor biopsies can be used to monitor TILs in response to immune therapy, and an increase in TILs is representative of a response to immune therapy.

C. Exemplary Methods of Detection, Selection and

Treatment

The methods of characterizing tumors discussed above alone can be applied to enhance the selection of patients for specific therapies, and to monitor the efficacy of therapies. Therefore, any of the disclosed methods can be coupled to steps of treatment and/or monitoring.

For example, in some embodiments, the methods relate to selecting patients who would be amenable for PD-1 and/or B7-H4 targeted therapies response to the tumor. Accordingly, prognostic markers/patient selection criteria for PD-1 and/or B7-H4 targeted therapy are provided.

As discussed above prognostic markers/patient selection criteria for PD- 1 targeted therapy for the rescue of patients who have failed treatment with BRAF/MEK inhibitors or other inhibitors of the RAS-RAF-MEK-ERK pathway are provided. Such PD-1 targeted therapy can overcome treatment resistance caused by "tumor dormancy" and prevent the selection/outgrowth of rapidly, progressing, resistant tumors in the presence of various small molecule inhibitors. In one embodiment, this can involve unique monotherapy approaches following treatment failure and/or combinatorial approaches that are more potent and allow for lower concentrations of the combination drug being used (for example, combination therapy including a PD-1 targeted therapy in combination with a BRAF /MEK (or other kinase) inhibitors to enhance overall tumor responses and efficacy, particularly PD- 1 targeted therapy with ligand independent activity).

Therefore improved PD- 1 targeted therapies and combination therapies for treating patients who have failed treatment (and preventing treatment failure that is frequently observed) with BRAF/MEK inhibitors or other inhibitors of the RAS-RAF-MEK-ERK pathway are also provided and discussed in more detail below. Improved PD-1 targeted therapies and combination therapies to overcome treatment resistance caused by "tumor dormancy" and to prevent the selection/outgrowth of rapidly, progressing, resistant tumors in the presence of various small molecule inhibitors are also provided and discussed in more detail below.

Accordingly, the disclosure includes solutions to the problem of identifying and/or selecting immune competent patients most likely to respond to PD-1 and/or B7-H4 targeted therapy and to the problem of monitoring the response of such patients over time using immune competency prognostic markers. Furthermore, the disclosure includes solutions to the problem of providing rescue and combination treatments to overcome/prevent tumor resistance to cancer therapy (particularly cancer the efficacy for RAS-RAF-MEK-ERK pathway inhibitors (and other agents that induce homeostatic proliferation of immune cells) as well as to enhance the immunomodulatory effects of PD-1/B7-H4 targeted molecules (fusion proteins and antibodies etc.). The disclosures addresses these problems by combining existing therapeutic approaches with PD-1 and/or B7-H4 targeted therapies (and other therapeutics that target key-co-stimulatory pathway molecules that may be up-regulated following treatment with such inhibitors).

For example, in a particular embodiment, a PD-1 targeted therapy includes the administration of an immunomodulatory molecule such as a PD- 1 -binding fusion protein/antibody (e.g. , an anti-PD-1 antibody, a B7-DC-Ig, a B7-Hl-Ig, etc.) with a BRAF inhibitor ("BRAFi") or other small molecule up-front (i.e. , as an initial treatment regimen), and particularly as long as:

(A) the small molecules do not impair T cell/immune responses; (B) the treatment is used in patients with appropriate mutations for target molecules (e.g., BRAF mutants and PD-1 positive). Such an approach is a synergistic/additive effect strategy in addition to the "rescue" strategy proposed above. In some cases (e.g. , those involving MEKi), the small molecule(s) may enhance T cell responses leading to further synergy/additive effects with the upfront combination, using the correct staging. The present disclosure is thus also directed to the solution of the problem of enhancing immune responses through up-front combination therapies.

The present disclosure thus derives in part, from the discovery that in patients refractory to treatment with BRAF/MEK inhibitors or other inhibitors of the RAS-RAF-MEK-ERK pathway, tumors that are refractory to treatment with such inhibitors or with other inhibitors of the RAS-RAF- MEK-ERK pathway can be effectively treated by targeting key

immunomodulatory molecules that are up-regulated in the tumor environment. Such treatment can involve monotherapy approaches (i. e. , treatment with a single drug) following treatment failure and/or embodiment, agents that target PD-1 are employed to rescue patients who have failed prior MEK/BRAF inhibitor treatment and/or prior ipilimumab (anti-CTLA-4 antibody) treatment. In particular, combination or rescue treatments utilizing PD- 1 targeted therapies in combination with (or following) drug treatments targeting the RAS-RAF-MEK-ERK pathway

(and/or other kinases) provides a means to enhance overall responsiveness to tumors and/or rescue tumors that have become resistant to such treatments.

In a particular embodiment, a method for determining whether a cancer patient suffers from a cancer having enhanced suitability for treatment with a PD-1 targeted monotherapy or a PD-1 targeted combination therapy includes evaluating tissue or fluid of the patient to ascertain the level of a prognostic biomarker correlative of immune system responsiveness. The method can include providing the patient with the PD- 1 targeted

monotherapy or the PD- 1 targeted combination therapy in response to the determination.

In some embodiments, the evaluating includes removing the tissue or fluid from the patient, and/or wherein the evaluation of the tissue or fluid of the patient includes immunohistochemical staining, in situ hybridization; gene expression analysis (e.g., bDNA, qRT-PCR, or microarray analysis), or flow cytometry (including, for example, FACS assays that assess cell surface expression and/or FACS assays that assess intracellular expression).

In some embodiments, the prognostic marker is the baseline:

(A) percentage of CD4+ or CD8+ T cells that are PD- 1^HI cells;

(B) the concentration of serum lactate dehydrogenase;

(C) the absolute lymphocyte count and/or the rapid decline in absolute lymphocyte count over a short period of time;

(D) the frequency of CD8+ or PD-1+ tumor infiltrating

lymphocytes; or

(E) gene expression of CD8A, FCGR3A, CTLA4, PD1, FASLG, CCL3, CXCL9, CXCL10, or GZMA in a tumor biopsy specimen. Therefore, when identifying patients who are likely to respond to immunotherapy, it can be best to identify a subject whose tumors are not rapidly progressing, and where a tumor marker (be it LDH, CA125, or any other) is not significantly high.

In some embodiments, the methods include evaluating the level of at least two of the prognostic biomarkers, or three, four or more of the prognostic biomarkers.

In a particular embodiment at least one of the prognostic biomarkers is the level of peripheral CD4⁺ or CD8⁺ cells that are PD-1^HI cells, and wherein:

(A) a sustained reduction in the level of PD-1^HI T cells following PD-1 targeted therapy is prognostic of an ability to respond to PD-1 targeted therapy; and

(B) a finding that any reduction in the level of PD-1 HI T cells following PD-1 targeted therapy is not sustained and subsequently rebounds above baseline is prognostic of an inability to respond to PD- 1 targeted therapy.

In a preferred embodiment, PD- 1 target therapy includes administering the subject a B7-DC-Ig fusion protein.

In another particular embodiment, at least one of the prognostic biomarkers is lactate dehydrogenase, and wherein:

(A) a level of lactate dehydrogenase that is within, or less than two-fold greater than, the upper level of normal (ULN) is predictive of the patient's enhanced suitability for treatment with a PD-1 targeted monotherapy or combination therapy; and

(B) a level of lactate dehydrogenase that is more than two-fold greater than the upper level of normal (ULN) is predictive of the patient's reduced suitability for treatment with a PD-1 targeted monotherapy or combination therapy.

Preferably, the tumor in such embodiments is a melanoma. (A) a baseline absolute lymphocyte count that is equal to or greater than approximately 950 e\ /μL· is predictive of the patient's enhanced suitability for treatment with a PD-1 targeted monotherapy; and

(B) a baseline absolute lymphocyte count that is less than said baseline absolute lymphocyte count is predictive of the patient's enhanced suitability for treatment with a PD-1 targeted monotherapy or combination therapy. and/or

(C) a decline of absolute lymphocyte count by about >40 over the course of 2 treatment cycles (or 2 months) is predictive of the patient's enhanced suitability for treatment with a PD-1 targeted combination therapy.

(D) a decline of absolute lymphocyte count by no more than about 40% over the course of 2 treatment cycles (or 2 months) is predictive of the patient's enhanced suitability for treatment with a PD- 1 targeted monotherapy or combination therapy.

In some embodiments the absolute lymphocyte count is determined using an intracellular cytokine lymphocyte marker selected from the group consisting of: IFNy, TNFa and IL2.

In another preferred embodiment, at least one of the prognostic biomarkers is a baseline tumor infiltrating lymphocyte count, and wherein:

(A) a baseline tumor infiltrating lymphocyte count that is equal to or greater than approximately 50-100 cells per high powered microscope field is predictive of the patient's enhanced suitability for treatment with immunotherapy, such as a PD- 1 targeted monotherapy or combination therapy; and

(B) a baseline tumor infiltrating lymphocyte count that is less than the baseline tumor infiltrating lymphocyte count of approximately 50-100 cells per high powered microscope field is predictive of the patient' s enhanced suitability for In a particular embodiment, the prognostic biomarker is gene expression of CD8A, FCGR3A, CTLA4, PDl, FASLG, CCL3, CXCL9, CXCL10, or GZMA in a tumor biopsy specimen.

Suitable immunotherapies including PD-1 therapies, and particularly PD-1 combination therapies are discussed in more detail below. However, in some particular embodiments,

(I) the PD- 1 targeted monotherapy or combination therapy

includes administration of an anti-PD-1 antibody, a PD-1- binding fragment of an antibody, or a B7-DC-Ig fusion molecule, and/or

(II) (A) the PD-1 targeted combination therapy enhances the activity of a RAS-RAF-MEK-ERK inhibitor, or

(B) includes the administration of an agent that targets a co- stimulatory pathway molecule that is up-regulated following treatment with a RAS-RAF-MEK-ERK inhibitor, or

(C) includes administration of cyclophosphamide,

carboplatin, paclitaxel, docetaxel or doxorubicin, or

(D) includes the administration of a BRAFi or other small molecule, as an initial treatment regimen.

Methods of determining patient suitability for participation in a trial for the safety and/or efficacy of a PD-1 targeted cancer therapy are also provided. The method can include determining whether tissue or fluid of a candidate patient for the trial possesses:

(A) a level of peripheral CD4⁺ or CD8⁺ T cells that are PD- 1 ^HI cells;

(B) a concentration of serum lactate dehydrogenase, and/or the rate of decline in absolute lymphocyte count over a short period of time;

(C) a baseline absolute lymphocyte count;

(D) a baseline tumor infiltrating lymphocyte count; and/or that is correlative of immune system responsiveness.

In some methods, tumor cells of a subject are analyzed for expression of both B7-H1 and B7-H4 according to one or more of the methods disclosed herein. If the tumor cells of the subject express both B7- HI and B7-H4 the subject can be selected for B7-H4 targeted therapy alone or in combination with PD- 1 targeted therapy and/or administration of a second therapeutic agent such as cyclophosphamide.

Suitable tumors that can be analyzed according the disclosed methods are discussed above and elsewhere herein. In preferred embodiments, the tumor is a solid tumor. In particular embodiments, tumor is a tumor of an adrenal cancer, a bladder cancer, a bone and connective tissue sarcoma, a brain tumor, a breast cancer, a colon or rectal cancer, an esophageal cancer, an eye cancer, a kidney cancer, a leukemia, a lymphoma, a multiple myeloma, a liver cancer, a lung cancer, a pancreatic cancer, a pharyngeal cancer, a pituitary cancer, an oral cancer, a salivary gland cancer, a skin cancer, a stomach cancer, a testicular or penile cancer, a thyroid cancer, or a vaginal, ovarian, uterine, or cervical cancer, or a gastric cancer.

D. Exemplary Applications of Detection, Diagnosis, and

Selection

1. Inclusion Criteria for PD-1 Targeted Therapy

Patients most likely to demonstrate immune responsiveness to PD- 1 targeted therapy (or clinical response to such therapy), exhibit a combination of at least two, and most preferably all three, of the pre-treatment prognostic markers described above, i.e. , low baseline LDH levels (within ULN), ALCs > 1000 e\ /μL·, and >100 TILs per hpf. Therefore, inclusion criteria for

PD-1 targeted therapy, such as a molecule that binds PD-1 (e.g. , soluble B7- DC fused to an Ig (B7-DC-Ig, or soluble B7-H1 fused to an Ig), should include these markers of immune competency. Patients that demonstrate all three criteria (i.e. , ALC >1000); CD8 TILs >100/hpf; and LDH (within ULN)) are particularly suitable for such PD-1 targeted therapy. In a preferred embodiment, the PD- 1 targeted therapy can be combined with not demonstrate one or more of the immune competency markers are better suited to combination therapy that combines PD- 1 targeted therapy with an immune stimulator capable of improving one or more immune competency markers, such as IL2 or GMCSF.

2. The Immune Front

B7-H1 expression by tumors is associated with immune evasion by the tumor. Furthermore, γ-interferon (IFNy) expression by TILs in the tumor microenvironment up-regulates B7-H1 expression by tumor cells. Therefore, B7-H1 expression in tumor biopsies has been used as a selection criterion for PD-1 targeted therapy wherein B7-H1 expression must be present for a PD-1 targeted therapy to function, particularly as it relates to therapies that block PD-1 binding to its ligands. However, one aspect of the present disclosure relates to the recognition that B7-H1 expression by tumor cells represents an active and evolving immune response rather than a required pre-disposition for PD-1 targeted therapy. In patients that respond to PD-1 targeted therapy, B7-H1 expression in the tumor is co-localized with infiltrating TILs (as determined by CD8 or PD-1 staining), with localized expression of IFNy by TILs leading to B7-H1 expression. Therefore, co-localization of CD8 TILs and B7-H1 in the tumor represents an "immune front," which is necessary for response to immune therapy.

Conversely, and contrary to prior reports, measuring B7-H1 expression in the tumor alone, without measuring infiltrating TILs can be misleading, and B7-H1 expression by the tumor without TILs can be a poor prognostic marker for patient response. This is particularly relevant for therapies where the mechanism of action is not dependent on the presence of the ligand and whereby the therapy can modulate PD- 1 expression on TILs directly (a ligand independent mechanism).

When evaluating B7-H1 expression and TILs in the tumor microenvironment, it is desired to stain multiple serial tumor sections, since the expression of markers and the infiltration of TILs can be highly heterogenous within the tumor. Furthermore, when studying B7-H1 associated macrophages (TAMs). Expression of B7-H1 on TAMs (or other cell types) can be differentiated using cell surface markers that are specific for different cell types. For example, B7-H1 expression on TAMs can be determined by dual staining for B7-H1 in conjunction with a TAM- specific marker such as CD68.

III. Methods of Detection

Any of the methods described herein can include one or more steps of detecting PD-1, B7-H1, B7-H4, any of the prognostic biomarkers discussed herein, or any combination thereof. For example, B7-Hl-binding molecules, B7-H4 binding molecules or PD-1 -binding molecules can used for the in vitro or in vivo analysis of B7-H1, B7-H4 or PD-1 expression, respectively, by tumor and/or non-tumor cells in conjunction with agents that specifically detect other cell biomarkers in order to differentiate and characterize specific cells types.

In some embodiments, the prognostic biomarkers are assessed immunohistochemically by staining or using FACS, etc. using one or a plurality of binding molecules specific for the prognostic biomarker of

HI

interest (e.g. , peripheral PD-1 levels, LDH release, baseline ALC levels, baseline TIL levels, etc.).

Detection of such biomarkers can be facilitated by coupling a biomarker- specific molecule (e.g. , a soluble molecule that binds to ALC or TIL cells or that is specific for LDH or PD-1, or a fusion molecule thereof) to a detectable substance, or detecting said biomarker- specific molecule with a detectable secondary antibody. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the biomarker- specific molecule or secondary antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for antibody to detectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent material such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive material such as, but not limited to, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt (⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga), germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ^mIn, ^mIn), iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu), manganese (⁵⁴Mn),

99 103 32

molybdenum ( Mo), palladium ( Pd), phosphorous ( P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe), ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn); positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The molecules may be attached to solid supports, which are particularly useful for immunoassays of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

1. In vitro Methods

In some embodiments, the characterization of the cellular expression of the desired biomarkers {e.g. , B7-H1, B7-H4, or PD-1, a macrophage biomarker, etc.) will be accomplished in vitro using an antibody (or antigen binding fragment thereof) to human B7-H1, B7-H4 or PD-1 in conjunction with antibodies that bind to one or more additional biomarkers. The synthesis of one or more of such biomarkers, assessing the presence of one or more of such biomarkers on tumor and/or non-tumor cells, etc.

In an alternative embodiment, the evaluation of the biomarker(s) will be accomplished using histochemical stains, fluorescence in situ

hybridization (FISH), FACS, bright-field in situ hybridization techniques (such as chromogenic in situ hybridization (CISH) and silver-enhanced in situ hybridization (SISH)), etc. Methods for conducting such analyses that may adapted to the present disclosure are well known in the art (see, e.g. , Penault-Llorca, F. et al. (2009) "Emerging Technologies For Assessing HER2 Amplification;' Amer. J. Clin. Pathol. 132(4):539-548; Houghton, O. et al. (2009) "The Expression And Diagnostic Utility OfP63 In The Female Genital Tract," Adv. Anat. Pathol. 16(5):316-321; Paner, G.P. (2008) "Best Practice In Diagnostic Immunohistochemistry: Prostate Carcinoma And Its Mimics In Needle Core Biopsies," Arch. Pathol. Lab. Med. 132(9): 1388- 1396; Beasley, M.B. (2008) "Immunohistochemistry Of Pulmonary And

Pleural Neoplasia," Arch. Pathol. Lab. Med. 132(7): 1062-1072; Erratum in: Arch. Pathol. Lab Med. (2008) 132(9): 1384; Olsen, J. et al. (2008) "Acute Leukemia Immunohistochemistry: A Systematic Diagnostic Approach " Arch. Pathol. Lab. Med. 132(3):462-475; Hoei-Hansen, C.E. et al. (2007) "Current Approaches For Detection Of Carcinoma In Situ Testis," Int. J. Androl.

30(4):398-405; Gold, J.S. et al. (2006) "Combined Surgical And Molecular Therapy: The Gastrointestinal Stromal Tumor Model " Ann. Surg.

244(2): 176-184; Bogen, S.A. et al. (2004) "Recent Trends And Advances In Immunodiagnostics Of Solid Tumors," BioDrugs 18(6):387-398; Fedor, H.L. et al. (2005) "Practical Methods For Tissue Microarray Construction " Methods Molec. Med. 103:89-101 ; van de Rijn, M. et al. (2004)

"Applications Of Microarrays To Histopathology," Histopathology 44(2):97- 108; or Kajima, H. et al. (2003) "Histopathology And Tumor Markers," Rinsho Byori. 2003 Dec;51(12): 1203-1215). Most preferably, such in vitro characterization is an immunohistochemical (IHC) or physiohistochemical analysis (e.g. , staining) of biopsied cells for cells that express one or more of used will determine the quantity of imaging moiety needed to produce diagnostic images. In vivo tumor imaging is described in S.W. Burchiel et al. , "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

In some embodiments, the evaluation of the biomarker(s) will be accomplished in vitro using an antibody (or an antigen binding fragment thereof) that, for the evaluation of peripheral PD-l/LDH, immunospecifically or physiospecifically binds to LDH or PD-1 ; for the evaluation of ALC

Levels, immunospecifically or physiospecifically binds to IFNy, TNFa, IL2, TNFRSF9, TNFRSF4, ICO, KLRG1, CXCL10, CCR2, CXCL9, granzyme A and granzyme B; and/or for the evaluation of baseline TIL levels, immunospecifically or physiospecifically binds to CD8 or PD-1.

2. In vivo Methods

One aspect of the disclosure relates to the use of antibodies and fragments, and particularly such antibodies and fragments that bind to human B7-H1, B7-H4 or PD-1 in conjunction with antibodies that bind to one or more additional biomarkers, as reagents for IHC analysis in cells in vivo. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In vivo tumor imaging is described in S.W. Burchiel et al. , "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the infection, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject using methods known in the art in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the disclosure include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et ah, U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Methods of administering the molecules of the disclosure for in vivo diagnostic use include, but are not limited to, parenteral administration (e.g. , intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g. , intranasal and oral routes). In a specific embodiment, the antibodies of the disclosure are administered

intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. See, e.g. , U.S. Patent Nos. 6,019,968; 5,985, 20;

5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO administered with a pharmaceutically acceptable carrier. As used herein, the term "carrier" refers to a diluent, excipient, or vehicle. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In one embodiment, monitoring of a disease, disorder or infection is carried out by repeating the method for diagnosing the disease, disorder or infection, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject using methods known in the art in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance (Thurston et ah , U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. See, e.g., U.S. Patent Nos. 6,019,968; 5,985, 20;

5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; WO 99/66903; Epstein et al , 1985, Proc. Natl. Acad. Sci. USA, 82: 3688; Hwang et al. , 1980 Proc. Natl. Acad. Sci. USA, 77: 4030-4; U.S. Patent Nos. 4,485,045 and 4,544,545. Typically the molecules are administered with a pharmaceutically acceptable carrier. As used herein, the term "carrier" refers to a diluent, excipient, or vehicle. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

IV. Methods of Treatment

Any of the disclosed methods of detection, diagnosis, or selection can be linked to a method of treatment. The methods typically include administered a subject a PD-1 targeted therapy or a B7-H4 targeted therapy. In preferred embodiments, the PD-1 targeted therapy or B7-H4 targeted therapy is co-administered in combination with a second therapeutic agent.

For example, in some embodiments, the methods include the use of PD- 1 targeted therapies and combination therapies that include such PD- 1 targeted therapies in the treatment of individuals who have been selected based on an evaluation of any one, two or, more preferably, all of the above- described prognostic markers. Such prognostic markers can be used as inclusion criteria for a clinical trial involving PD-1 targeting agents. In a particular embodiment, targeting PD-1 can rescue melanoma patients (including ocular melanoma patients) who have failed MEK/BRAF treatment and/or ipilimumab treatment prior therapies. Similarly, the administration of a PD- 1 targeted therapy can rescue patients suffering from other tumors involving the RAS-RAF-MEK-ERK pathway. Exemplary combinations are discussed in more detail below.

A. PD-1 Targeted Therapeutics to Rescue BRAFi/MEKi Ongoing studies indicate that significant challenges will confront the use of small molecule inhibitor chemotherapy that target solely the RAS- RAF-MEK-ERK pathway. Patients receiving treatments (such as Braf inhibitors (BRAFi) or MEK inhibitors (MEKi)) that target this pathway frequently stop responding to treatment as the tumors become resistant. This resistance is likely the result of selective pressure whereby cells within the tumor mutate and develop resistance to therapy and outgrow the non- resistant cells within the tumor microenvironment. One aspect of the present prevent the initial outgrowth of resistant cells and thus the development of resistance, and provide improved clinical responses. Suitable PD-1 targeted therapies include anti-PD-1 antibodies, anti-PD-1 antibody antigen-binding fragments, and fusion proteins such as a B7-DC-Ig or B7-Hl-Ig.

Furthermore, such patients rapidly progress once taken off treatment as the tumors grow aggressively and this correlates with a decline in immune competence as indicated by any of the prognostic markers for immune competence described above. Therefore, it is desirable to treat such patients with immunomodulatory therapies as early as possible following removal from BRAFi or MEKi treatment before the tumor has had an opportunity to develop further and overrun the immune system and, more preferably, to combine BRAFi/MEKi therapy with such immunotherapy as early as possible following diagnosis in order to prevent the emergence of resistance.

An analysis of real-time data from patient samples indicates that one may identify those patients at high risk of rapid disease progression upon relapse with a BRAF inhibitor (and possibly other therapies) who might not have time to subsequently complete PD-1 targeted therapy (or any other proposed immunomodulatory therapy) because they are immune

compromised, as indicated by any of the above-described prognostic markers for immune competence.

As discussed above, the present disclosure additionally provides a PD-1 targeted therapy in which immune responses of a patient are enhanced through the administration of up-front combination therapies that involve an immunomodulatory molecule, such as a PD-1 -binding fusion

protein/antibody (e.g. , an anti-PD-1 antibody, a B7-DC-Ig, a B7-Hl-Ig, etc.) and a BRAFi or other small molecule, up-front (i.e. , as an initial treatment regimen), and particularly as long as:

(A) the small molecules do not impair T cell/immune responses;

(B) the treatment is used in patients with appropriate mutations for target molecules (e.g., BRAF mutants and PD-1 positive).

Such an approach is a synergistic/additive effect strategy that may be cell responses leading to further synergy/additive effects with the upfront combination, using the correct staging.

B. Combination Therapy Including BRAFi/MEKi and PD-1 Targeted Therapy

As discussed herein, BRAF- mutation positive patients (i.e. , patients having a mutation in their BRAF gene, and particularly the V600E and V600K BRAF mutations) will benefit from combination therapy in which PD- 1 targeted therapies (such as those described above) are provided in conjunction with Braf inhibitor/chemotherapy. Accordingly, PD-1 targeted therapies in conjunction with Braf inhibitor/chemotherapy are also provided. Benefits of such treatments include limiting the outgrowth of resistant tumor cell types, thereby prolonging the efficacy of treatment, improving response and survival. It is particularly desirable for such combination therapy to be provided early in the progression of the disease, since the early

administration of the combined therapy enhances the immune response repertoire and generates a diverse response to the tumor, thereby minimizing its ability to escape treatment via such mutation, selection, etc.

C. PD-1 Targeted Therapy to Overcome Resistance Caused by "Tumor Dormancy"

Long term tumor dormancy can occur when residual cancer cells develop strategies to escape cell death and exist in equilibrium with the immune system of the host (Quesnel, B. "Tumor Dormancy: Long-Term Survival in a Hostile Environment," In: SYSTEMS BIOLOGY OF TUMOR DORMANCY, ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY (H. Enderling et al., Eds.), Springer Science+Business Media, NY; Chapter 9, pp. 181-200; Quesnel, B. (2008) "Tumor Dormancy And Immunoescape," APMIS 116(7-8):685-94; Quesnel, B. (2008) "Dormant Tumor Cells As A Therapeutic Target!," Cancer Lett. 267(1): 10-17; Quesnel, B. (2006) "Cancer Vaccines And Tumor Dormancy: A Long-Term Struggle Between Host Antitumor Immunity And Persistent Cancer Cells!," Expert Rev

Vaccines 5(6):773-781; Hensel, J.A. et al. (Epub 2012 Nov 27) "Clinical Adv. Exp. Med. Biol. 734:73-89; Patel, T. et al. (Epub 2012 Oct 23) "Cancer Stem Cells, Tumor Dormancy, And Metastasis," Front. Endocrinol.

(Lausanne). 3: 125).

Dormant tumor cells frequently express immune checkpoint markers such as B7-H1 (and B7.1) that inhibit effector T cell function by inducing T cell exhaustion, and can be under selective pressure from maintenance treatments involving the administration of cytotoxic drugs or small molecule kinase inhibitors. Continuous drug treatment of tumors containing such cells selects for the growth of drug-resistant cells that are also resistant to autologous immune responses. Accordingly, after variable lengths of time the dormancy ends, and the cancer cells resume their growth, resulting in disease progression. This may be the result of the development of nonspecific resistance mechanisms, such as deregulation of the JAK/STAT and mTORC2/AKT pathways. Furthermore, the resistance mechanisms that have been selected may result in more aggressive tumor sub-clones and in tumor relapses that are more difficult to treat.

There is thus an advantage to combining immunotherapy, such as PD- 1 targeted immunotherapy, with targeted cancer therapy, and in providing such combined treatment early in the cancer treatment regime. The addition of such immunotherapy will enhance immune responses and will act to prevent the establishment of an immune equilibrium capable of protecting dormant tumor cells. Such prevention of immune equilibrium is particularly beneficial where the immunotherapy has ligand independent activity and is able to modulate T cell activity directly. Furthermore, preventing immune equilibrium will limit the ability of the tumor cells to survive and develop resistance and the outgrowth of more aggressive tumor cell populations, thus enhancing the efficacy of cytotoxic drugs or small molecule kinase inhibitors.

Therefore, combination therapies including a PD- 1 targeted immunotherapy and a cancer targeted therapy are provided. Preferably, such combinations are administered at a time and dosage effective to reduce, D. Other Combination Therapies

PD-1 or B7-H4 targeted therapies (or other immunotherapy relevant to enhancing an immune response) can be combined with: (1) agents/drugs that effect tumor proliferation, growth and/or progression by direct effects on the tumor, or (2) agents/drugs that induce homeostatic proliferation on infiltrating and circulating immune cells that are directed against the tumor. The latter approach (2) is based upon the recognition that immune enhancing agents such as PD-1 targeted therapeutics may best be able to reverse tolerance under conditions of homeostatic proliferation. (Lee, J. Y. et al. (2013) "Remembering To Be Tolerant," Science 335:667-668; Schietinger, A. et al. (2012) "Rescued Tolerant CD8 T Cells Are Preprogrammed to Reestablish the Tolerant State ," Science 335:723-727). Furthermore, such approaches would not be limited to tumors whose cells carry BRAF mutations ("BRAF mut tumors") and extends to combination therapies employing other small molecules/biologies etc. with activities not limited to the RAS-RAF-MEK-ERK pathway.

Other combinations of PD-1 or B7-H4 targeted therapy, particularly therapies with ligand independent activity, include combination with radiation therapy, anti-CTLA4 (ipilimumab) and anti-tumor vaccines. The rationale for approaches outlined below re: enhancing overall tumor immunity by combining tumor killing/suppressing treatments with immunotherapy is the same as for the kinase pathway specific inhibitors described above albeit with different mechanisms of tumor targeting.

E. Preferred Combinations And Trials For PD-1 Targeted Therapy

A preferred combination and trial for PD- 1 targeted therapy employs a differentiated approach sufficient to allow for rapid accrual in the competitive space. In such an approach, patients with a high unmet medical need where PD- 1 pathway plays an important role are targeted, with a focus on different indications from those met by anti-PD-1 antibodies (for example: ovarian cancer (especially ovarian cancer that is platinum sensitive, ("NSCLC"), head and neck cancer, melanoma; and in combination with low dose cyclophosphamide therapy ("CTX"). See, for example, WO

2010/027423.

A second preferred combination and trial for PD- 1 targeted therapy employs the first-line standard of care ("SOC") used in early tumor development in combination with chemotherapeutic agents having immunostimulatory activity. Such an approach is particularly desirable in the treatment of ovarian cancer (e.g. , in combination with

carboplatin/paclitaxel), triple negative breast cancer (e.g. in combination with paclitaxel, docetaxel or doxorubicin), NSCLC (e.g. , in combination with carboplatin/paclitaxel).

V. Optimization of Treatment Regimens and Patient Selection

Differential staining between B7-H1 or B7-H4 (and other co- stimulatory proteins etc.) expression on tumors versus TAMs or other immune cells will enhance the understanding of how mechanisms of immune suppression and clinical response are related, and how patients may be treated. Differential staining to verify the presence of TILs, B7-H1+ TAMs and/or B7-H1+ tumors (as well as other co-stimulatory markers such as B7- H4 etc.) provides clarification on which key cellular subsets within the tumor microenvironment may be beneficial therapeutic targets, which are predictive biomarkers for patient response to therapies targeting the immune checkpoint pathways. Such an approach will aid in optimizing treatment regimens, combination approaches and patient selection for the next generation of immunomodulatory therapies, as well as other treatments that affect the tumor microenvironment.

For example, tumors expressing B7-H1 are thought to respond best to an anti-PD- 1 therapeutic. However, if it is determined that B7-H1 expression is due to TAMs, then alternative therapies that mitigate the suppressive effect of TAMs may be more appropriate. Staining for additional markers of suppression on TAMs (e.g. B7-H4, LAG3 etc.) may suggest the administration of other immunomodulatory drugs (e.g. anti-B7- Furthermore, the distinct cellular patterns of B7-H1 expression may be elucidated to further refine the predictive value of this biomarker in clinical trials that target the PD-1 pathway and that are using the B7-H1 as a primary or sole selection marker. For example, a patient thought to be B7- H1+ may be responding but the tumor itself may actually be B7-H1-.

Therefore, if a patient is excluded based on a lack of B7-H1 staining, such excluded patient might be a potentially responsive patient. Alternatively, if a patient is selected for treatment where B7-H1+ staining is from a non-tumor cell, such a patient may not respond to a treatment that is specific for the PD- 1 pathway, such as an anti-B7-Hl, B7-DC-Ig or anti-PD- 1. Such may be the case for IHC where staining is limited to small areas of the tumor sample.

B7-H1 is not heterogeneously expressed throughout the tumor and B7-H1 staining across the tumor can also be highly variable; some areas of the tumor or some tumor sections may be B7-H1+, whereas other areas may be B7-H1- or necrotic. Therefore, it may be important to stain multiple sections or tumors to fully assess the B7-H1 (or B7-H4) status of a patient' s tumor(s) and use additional markers in combination to assess whether such staining relates to tumor or non-tumor cells. Also, staining for B7-H1 can be highly correlated CD8 (and PD-1) i.e. infiltration of CD8+ T cells into areas where B7-H1 is expressed, thus CD8 may be used as a correlative readout. Also, not all CD8 T cells may have positive staining for PD-1 and so: i) CD8 may provide a better assessment of T cell infiltration and potential response, and ii) it is useful to use both stains to get a good sense of the proportion of tumor infiltrating lymphocytes that are PD- 1 high and thus potentially exhausted, and thus whether a patient may respond to an immunomodulatory treatment. It may also be beneficial to monitor other markers, such as those for TAMs, in order to monitor response to therapy. For example, B7-H1 staining of tumor cells may stay high, but a reduction in TAMs may indicate an immune response, and such tumors may benefit from treatments that enhance T cell activity (e.g., IL-2).

Elevated levels of IFN-gamma in tumors and within the tumor correlate with enhanced expression of B7-H1 on tumor cells or within the tumor microenvironment (perhaps a negative feedback, regulatory mechanism). Changes in B7-H1 expression levels may occur following specific chemotherapeutic and biologic therapies, or other therapeutic interventions (e.g. radiation, cryoablation, surgical resection of the tumor etc.) that can also trigger changes that enhance IFN-gamma and induce B7- Hl expression. Indeed, enhanced IFN-gamma levels can be stimulated by a variety of factors, including tumor necrosis, chemotherapeutic tx, radiation, etc. It is precisely these patients who may benefit most from therapies targeting immune checkpoint pathways, such as B7-H1/PD-1.

The present disclosure is also directed to a method for selecting patients for anti-cancer therapy based on characterization of the tumor or tumor microenvironment. In one embodiment, cancer patient tumor samples are characterized following treatment with following specific

chemotherapeutic and biologic therapies, or other therapeutic interventions (e.g. radiation, cryoablation, surgical resection of the tumor etc.) that can trigger changes in B7-H1 expression as indicated above. For example, expression of B7-H1 in melanoma patients has been shown following Brafi treatment, and thus may be responsive to therapies targeting the B7-H1/PD-1 pathway. Therefore, treatment with therapies targeting the B7-H1/PD-1 pathway may be a good option for patients that fail to respond to other therapies, including but not limited to, Braf and MEK inhibitors.

VI. Diagnostic Kits

The disclosure provides a diagnostic kit comprising one or more containers containing a reagent capable of detecting a prognostic biomarker. The kit may also comprise one or more containers containing ingredient(s) for facilitating the characterization of tumor and/or non-tumor cells.

Optionally associated with such container(s) can be instruction protocols and/or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human provided by way of illustration and are not intended to be limiting of the present disclosure unless specified.

Examples

Example 1: Encumbrances to the Immunohistochemical Analysis of Melanoma Tumors

Biopsied tumor sections of different cancer types were evaluated using immunohistochemical staining and microscopy to determine whether a correlation existed between their B7-H1 expression and the average number of tumor-infiltrating lymphocytes (TILs) observed (per field) viewed. Figure 1 illustrates the scoring of B7-H1 expression using a scaled score that ranges from 0 (negative) to 3 (intense positive stain).

From the patient samples tested, a positive correlation between B7- Hl expression on the tumor and average TIL counts greater than 10 TILs per high powered field was observed in the subset of patients (Figure 2). Less than 30% of patients had B7-H1 scores of 2-3.

In contrast, PD-1 and CD8 expression were found to be highly correlated (although the PD-1 immunohistochemical staining may have been detecting both PD- 1 high expressing cells and PD- 1 low expressing cells) (Figure 3). In most cases, a B7-H1 score > 1 correlates with PD-1 TIL avg > 10 and CD8 TIL avg > 100.

Multiple factors may impair the ability of pathologists to correctly interpret B7-H1 staining results for tumors. First, melanoma tumors contain expressed melanin and thus stain darkly irrespective of their B7-H1 staining. Melanin is cytoplasmic and highly blobby/granular. In optimal samples, such staining may be distinguished from the sharp, intense peripheral staining associated with B7-H1 expression. Unfortunately, biopsy samples from melanoma tumors may have been subjected to sub-optimal fixation and processing since B7-H1 stains are acceptable after 48 hrs of formalin fixation, but are optimal only after 144 hr of formalin fixation. Additionally, the DAB chromagen can be picked up non-specifically at the edge of a diffuse stain in a granular pattern. Additionally, such necrotic tissue contains many macrophages (not necessarily TAMs), which express B7-H1 (Figure 4).

Example 2: Dual Staining of Melanoma Tumors Reveals That Some Tumors Which Appear to Be "B7H1+ Tumors" Are in Fact B7-H1-

Tumor-associated B7-H1+CD68+ macrophages (TAMs) have been observed to be present within tumors (Figure 4). Such macrophages can have membraneous or cytoplasmic B7-H1 expression (e.g. lung biopsies can reveal large numbers of infiltrating macrophages that accumulate brown pigment). Macrophages that express B7-H1 and do not express CD68 can be distinguished by their small nuclei as opposed to tumors with large pleomorphic nuclei.

Although the presence of TAMs usually parallel B7-H1 expression in tumors, rare cases are observed in which the tumor presents as a B7-H1- tumor with a few interspersed CD68+/B7H1+ TAMs (Figure 5), or as a

B7H1+ tumor with CD68+/B7H1- TAMs nearby. As illustrated in Figure 5, Left Panel, immunohistochemical analysis appears to show the presence of B7-H1+ tumor cells. However, the same biopsy sample, when stained using a dual CD68/B7-H1 stain shows that the tumor is B7-H1- and that the detected expression of B7-H1 actually reflects the presence of CD68+ B7- H1+ macrophages (Figure 5, Right Panel). Figure 6 shows an example in which a few interspersed melanoma tumor cells express B7-H1. The expression of B7-H1 on the tumor has been confirmed by the dual CD68/B7- Hl stain. Thus, B7-H1 expression may occur on either the tumor, infiltrating macrophages, or both.

Example 3: Dynamic Changes In Tumor Microenvironment Following Treatment

One aspect of the present disclosure reflects the recognition that tumors are not static, but rather respond dynamically to therapy. Such responses result in changes in the tumor microenvironment and changes in the periphery. Figure 7 illustrates this recognition by showing the sustained expression in the tumor and the periphery (Sfanos et al. (2009) "Human prostate-infiltrating CD8+ T lymphocytes are oligoclonal and PD-1+," Prostate 69(15): 1694-1703; and Shi et al. (2011) "PD-1 and PD-L1 upregulation promotes CD8( + ) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients," 128(4):887-896;

therefore, peripheral changes mimic the changes that are occurring in the tumor microenvironment.

The occurrence of such changes is confirmed in the results shown in Figures 8-11 (Panels A-D). A BRAFm melanoma patient who had previously failed BRAFi/MEKi was treated with a PD-1 binding molecule. Figure 8 shows the H&E stains from three fresh biopsies of a metastatic neck lymph node taken from the patient while receiving treatment with a PD-1 binding agent. The pre-treatment biopsy was performed on prior to therapy, the first post treatment biopsy was performed on Cycle 1, Day 15 (C1D15) following 1 dose of the PD-1 binding molecule. The second post treatment biopsy was taken on Cycle 2, Day 15 (C2D15) following three doses of the PD- 1 binding molecule. Each cycle was a month long. This biopsy contains mostly fibrotic/necrotic tumor tissue with distinct pockets of lymphocyte infiltrates at the edges. The presence of tumor cells was confirmed with S100 stain. The biopsies, along with an archival specimen that was taken prior to the patient' s treatment with the BRAFi/MEKi were evaluated via immunohistochemical staining for B7-H1, PD-1, CD8, CD4 and FoxP3 (Figure 9 and Figure 11 (Panels A-D)). An increase in TILs was observed following BRAFi/MEKi therapy, which was sustained following treatment with a PD-l-binding molecule (Figure 10; the PD-1 IHC stain may be detecting PD-1 (HI) and PD-l(LO) T cells; for the C2D15 biopsy, the reduction in tumor volume results in a higher TIL:tumor ratio).

Figure 11 , Panels A-D show multiple tumor biopsies from a cancer patient. Prior to BRAFi/MEKi therapy (Panel A) B7-H1 expression was scored as 1 ; after such therapy but prior to therapy with a PD- 1 binding molecule (Panel B), B7-H1 expression was scored as 3. Post-treatment confirms the occurrence of change in B7-H1 expression within the tumor microenvironment as a consequence of the cancer therapy.

Example 4: Dynamic Changes In Tumor Microenvironment Following

Treatment

As discussed above, one aspect of the present disclosure relates to the recognition that BRAF mutant melanoma patients who have been treated with BRAFi express B7-H1 on their tumors (Figure 11, Panels A-B) and Figure 12A). Figure 12A is a photo image of tumor cells expressing B7-H1 in a biopsy taken from a BRAF mutant melanoma patient who has failed BRAFi therapy. Changes in B7-H1 expression levels may occur following specific chemotherapeutic and biologic therapies, or other therapeutic interventions (e.g. radiation, cryoablation, surgical resection of the tumor etc.) that can also trigger changes that enhance IFN-gamma and induce B7- Hl expression.

B7-H1 is not heterogeneously expressed throughout the tumor, but is co-localized in the area with CD8+ TILs, which may be PD-1+ or PD-1- or change PD-1 expression over time (Figure 12A-12B). As is evident from Figure 12A, not all CD8 T+ cells are staining for PD-1. This fact indicates that, relative to PD-1 alone, the combined use of PD-1 and CD8 as markers for assessing T cell tumor infiltration and responses to tumor therapy provides a more complete assessment of the proportion of tumor infiltrating lymphocytes that are PD-1 high and thus potentially exhausted, and thus whether a patient may respond to an immunomodulatory treatment. In addition, CD8 may be used as a correlative readout of B7-H1 expression, as CD8+ T cells infiltrate into areas in which B7-H1 is expressed.

Figure 12B shows areas of necrosis in the tumor. Figure 12C shows that B7-H1 is expressed on the membrane of the tumor cells.

Example 5: Prognostic Biomarkers of Response to PD-l-Targeted Immunotherapy

Multiple patients (each assigned a four digit identifier to preserve their anonymity) were evaluated for the above-described prognostic A. The Early Reduction in PD-l^m T Cells During Cycle 1 Establishes that the Level of PD-1^HI Cells Is a Prognostic Biomarker of Response to PD-l-Targeted Immunotherapy

The response of multiple patients to PD-1 targeted therapy involving a B7-DC-Ig fusion and CTX, as represented by changes in the level of PD-

HI

1 cells in the periphery was evaluated at -1, 0 and 1, 7, 14 and 28 days post-dose. Day 0 is the day of CTX treatment. Drug is administered on Day 1 (Dl) and Day 15 (D15), comprising a first cycle (CI) of treatment (e.g. , C1D1 and C1D15). A second cycle (C2) of treatment is commenced on Day 28, such that Day 29 is C2D1 and Day 54 is C2D27, etc. The results of this investigation are shown in Table 1. In Table 1, the level of PD-1 HI T cells is presented normalized to the level found at Day -1 (pre-treatment) 100 for

HI

each patient. Differences between PD-1 levels on Day -1 and Day 0 (samples taken prior to treatment with PD- 1 targeted therapy) reflect changes to the PD-1^HI T cell population as a result of low dose CTX treatment.

Table 1

Patient PD-1^HI Cell Level (Normalized Days Post-Dose) Clinical

-1 0 +1 +7 +14 +28 Response

0606 100 100.3 - 59.3 59.3 62.3 PD

0607 100 124.8 - 48.2 32.4 11.3 PD

0608 100 96.2 - 2.7 5.8 2.6 PD

0609 100 109.8 - 49.0 27.2 - IR

As seen in Table 1, most patients exhibited an acute and sustained

HI

reduction in PD-1 T cells following PD-1 targeted treatment. Three cases

HI

in which the levels of PD-1 cells were not maintained below 50% from baseline prior to the second dose of treatment with a PD- 1 targeted agents (i.e. , Day +14) exhibited progressive disease (PD), indicating that the

HI

increase (or rebound) in PD- 1 T cells (or the lack of a sustained reduction

HI

in the level of PD-1 cells) is a prognostic biomarker of poor immune function and failure to respond to PD-1 -targeted immunotherapy. Five (of seven) patients who did not experience complete reduction in PD-1^HI T cells by Day 14 (i.e. , between 30-50% from baseline), but did show a sustained reduction exhibited an immune response (IR) or a clinical response (CR), potentially due to release of antigen from tumor lysis and activation of an immune response.

B. The Level of LDH Release Is a Prognostic Biomarker of

Response to PD-l-Targeted Immunotherapy

Figure 13 shows the LDH levels observed in such patients following 1 cycle of treatment. Levels were obtained at the start of cycle 1 (i.e. , baseline) and prior to the start of cycle 2. Patients who exhibited disease progression ("PD") typically showed increased LDH release, whereas patients who exhibited an immune response or a clinical response typically showed decreased LDH release (cycle 1 compared to cycle 2). As stated above, high baseline LDH and/or rapidly increasing LDH is a biomarker for rising antigen exposure and up-regulation of PD-1 expression. Figure 13 the upper level of normal (ULN) is prognostic of patients that will not successfully respond to PD-1 -targeted immunotherapy.

C. The Combined Prognostic Effect of Determining the Level of LDH Release and the Level of PD-1^HI T Cells

One aspect of the present disclosure reflects the recognition that a correlation aspect between of the level of LDH release and the level of PD- 1^HI cells, such that a synergistic prognostic benefit is obtained by evaluating both such prognostic markers. Among the patients evaluated (Table 1), patient 0403 had the highest baseline LDH levels, patient 0604 had the most rapidly increasing LDH levels, and patient 0606 had the second highest LDH level at CI DO and the highest at C2D0. These patients exhibited the highest percentages of PD-1^HI cells, and all exhibited disease progression in response to the therapy. Accordingly, determination of (i) the level of LDH release and (ii) the level of PD-1 HI cells is particularly prognostic of successful response to PD-1 -targeted immunotherapy. Without intending to be bound thereby, such synergistic correlation is believed to indicate that when LDH levels are high or rapidly increasing, the action of PD-1 -targeted

immunotherapeutics are insufficient to overcome the signals promoting up- regulation of PD-1.

D. The Baseline ALC Level Is A Prognostic Biomarkers of

Response to PD-l-Targeted Immunotherapy

The baseline ALC levels of patients who experienced a clinical response to PD-1 targeted therapy, patients who exhibited an immune response, and patients who experienced progressive disease were evaluated. Figure 14 shows that 8/13 (61.5%) of evaluated immune competent patients having a baseline ALC > 1,000 had improved immune function following such treatment.

E. The Baseline TIL Level Is A Prognostic Biomarkers of Response to PD-l-Targeted Immunotherapy The baseline TIL levels of patients who experienced a clinical response to PD-1 targeted therapy, patients who exhibited an immune treatment, and that 5/7 (71.5%) of evaluated immune competent patients having a baseline TIL > 100 had improved immune function following such treatment. Figure 15 shows that the baseline TIL level is a prognostic biomarker of successful response to PD-1 -targeted immunotherapy.

F. An Increase in Polyfunctional T Cells Is A Prognostic

Biomarkers of Response to PD-l-Targeted

Immunotherapy

Intracellular cytokine staining was used to determine the fractional change in the population of CD8+ T cells in the above patients. Figures 16A and 16B show that polyfunctional T cell populations (CD8+ (Figure 16A) and CD4+ (Figure 16B)) increased in immune responder patients, but decreased in progressive disease patients. Figures 16A-16B show that a change in polyfunctional T cell populations is a prognostic biomarker of successful response to PD-1 -targeted immunotherapy.

G. The Average Change in Effector Versus Exhaustion

Markers Is A Prognostic Biomarkers of Response to PD-l- Targeted Immunotherapy

bDNA analysis was used to measure the changes in effector markers and exhaustion markers in the T cell populations of the above patients. The measured effector markers were: TNFRSF9, TNFRSF4, ICOS, KLRG1, CCR2, CXCL9, CXCL10, granzymeA (GzmA) and granzymeB (GzmB). The measured markers of T cell exhaustion were: PD1, CTLA4, FasL, CCL3, CD40L, LAG3, CD244 and CD160. Figure 17 summarizes the preferred prognostic biomarker criteria of the present disclosure for patient selection for PD-1 targeted immunotherapy.

Example 6: Changes in Immune Function Correlate with Observed Clinical Activity

Figure 18A-18B show a tumor biopsy stain showing the immune front of a patient. Figure 18A shows a tumor biopsy stain of patient 0505 (CR), fresh pre-treatment BRAFm, with TILs. The left panel shows staining for B7-H1; the right panel shows staining for CD8. In contrast, Figure 18B expression of B7-H1+ on tumors, and provide IHC evidence of an "immune front" in the tumor microenvironment. Figure 18A-18B also shows areas of necrosis as well as the variability of B7-H1 staining across the tumor microenvironment relative to the staining of CD8 (i.e. , areas that are B7-H1 positive, areas that are necrotic, and areas that are B7-H1 negative).

Example 7: Clinical and Pharmacodynamic (PD) Results of a Phase 1 Trial with a B7-DC Ig Fusion

Following low-dose cyclophosphamide (CTX) treatment, a murine B7-DC Ig fusion molecule was found to promote the survival, tumor eradication, and long-term anti-tumor immune memory of B ALB/c (immune competent) mice using a subcutaneous syngeneic CT26 colon carcinoma model. Mice received a low-dose treatment with CTX on Day 10 followed by the murine B7-DC Ig fusion twice weekly for four weeks, starting on Day 11. The tumor was found to have been eradicated in 60% of mice treated (at 15 mg/kg). Figure 19A shows the effect of the murine B7-DC Ig fusion on the survival of the mice (5 independent studies, n = 10 mice/group/study). Figure 19B shows the effect of the murine B7-DC Ig fusion on tumor volume. Following inoculation of tumor cells in naive mice, tumors grew in almost all cases. To evaluate long-term immune memory in mice that eradicated tumor following treatment with CTX + murine B7-DC Ig, these mice were re-challenged with CT26 cells. In almost all cases, the CT26 cells were rejected following re-challenge, demonstrating long-term immune antitumor immune (Figure 19C).

In the context of the CT26 model, immunophenotyping was conducted on Day 15 and Day 24 post-inoculation (Figure 20, Panels A-H). In the absence of treatment, high levels of PD- 1 expression were observed on tumor infiltrating lymphocytes (TIL) including CD8+ T cells, CD4+ T helper cells, and Treg (Figure 20, Panels A, B and C). Mice that received CTX + 5 mg/kg murine B7-DC-Ig were observed to have fewer CD8⁺ TIL on Day 15 (Figure 20, Panels E and F); the TIL cells that are present are almost all PD- 1^~. By Day 24, an expanded population of CD8⁺PD-l^{_ LO} cells are observed tumors. Figure 20, Panels D and H, show the effect of the murine B7-DC-Ig on the concentration of CD4⁺ T cells.

In order to further demonstrate the efficacy of PD-1 -targeted therapy, human patients were provided with a human B7-DC Ig fusion molecule

HI

capable of binding to human PD- 1 B7-H1- T cells (i. e. , chronically stimulated / exhausted T cells) but substantially incapable of binding to human PD-1⁺ B7-H1⁺ cells (i.e. , normal activated T cells. Patients were provided with one or more 28 day treatment cycles consisting of 200 mg/m² CTX on Day 0 and human B7-DC Ig fusion on Days 1 and 15. Twenty-six patients with relapsed or refractory solid tumors were enrolled in 5 dose cohorts (0.3, 1, 3, 10, 30 mg/kg). Eighteen melanoma patients were enrolled at a Recommended Phase 2 Dose ("RP2D") of 10 mg/kg. Whole blood was collected at multiple time-points for all patients for flow cytometry assays evaluating receptor targeting and modulation. PBMC were collected pre- treatment and at the end of each cycle and functionally evaluated by flow cytometry for intracellular cytokine staining. Data from 11 patients in the 10-30 mg/kg dose cohorts were evaluated (in one case only GzmB was evaluable). Fresh pre-treatment biopsies were evaluated by IHC (B7-H1, CD8, PD-1) for 34/44 (77%) of patients; paired fresh pre- and post-treatment biopsies were evaluated by IHC for 24/44 (55%) of patients. The enrolled patients had the following demographics: mean age 56, range 27-80; 26 males (59%) /18 females (41%); 43 Caucasian / European heritage (98%) and 1 African American / African heritage (2%); ECOG performance status of 1 (28 patients, 64%) or 0 (16 patients, 36%). No drug-related serious adverse events (SAE) were observed at dose levels up to 30 mg/kg. Four patients (9%) experienced drug-related Grade 3 adverse events (AE): 1 each infusion related reaction, influenza like illness, myalgia, and decreased platelet count. Infusion reactions (chills, nausea, flushing, back pain, rigors) were observed in 31/44 patients (70%) and were found to be manageable with pre-medication and longer (2-3 hour) infusion. The B7-DC Ig was found to have a sustained serum half-life of approximately 10 days, with a tumor volume was demonstrated in CT scans of the lung performed prior to Cycle 1 (Figure 21, Panel A) vs. at the end of Cycle 4 (Figure 21, Panel B). An overall reduction of 48.4% in tumor burden was observed at the end of Cycle 4.

Figure 22, Panels A-B, shows evidence of Sustained Disease in a melanoma patient in the 10 mg/kg dose-escalation cohort. A reduction of tumor volume was demonstrated in CT scans of the neck performed prior to Cycle 1 (Figure 22, Panel A) vs. end of Cycle 6 (Figure 22, Panel B). This lesion was initially palpable and was observed to begin regressing following the first dose of B7-DC Ig.

Figures 23A-23B show evidence of a Mixed Response (MR) in a melanoma patient in the Expansion cohort. A reduction of tumor volume was demonstrated in CT scans of the liver performed prior to Cycle 1 (Figure 23A) vs. end of Cycle 2 (Figure 23B), however other lesions worsened. Three additional patients in the 10 mg/kg cohorts have also presented evidence of mixed response.

Absolute lymphocyte, T cell, CD4⁺ T cell , CD8⁺ T cell , and PD-l^LO T cell counts were found to be lower at 4 hours post-dose (Figure 24A), recover by 24 hours post-dose, and be stable long term (Figure 24B).

Reductions in peripheral blood PD- 1 HI T cells are monitored in real-time by flow cytometry. Dose-response profile is consistent with murine CT26 tumor model findings in periphery and tumor microenvironment. Sustained reductions in peripheral PD- 1 HI T cells were observed in most patients that received 10-30 mg/kg human B7-DC Ig fusion. An inability to control levels of PD-1^HI T cells correlated with rapid disease progression.

Evidence of enhanced immune function in the periphery and tumor microenvironment was observed in all patients evaluated to date in the 10-30 mg/kg dose cohorts who were able to stay on the trial for at least 4 treatment cycles and provide paired PBMC and/or biopsy specimens for these evaluations. Figures 25A-25E show data for all patients meeting these criteria. Patients who came off trial more rapidly for progressive disease evaluated. Increased counts of GzmB effector / EMRA cells were observed in the peripheral blood of all (7/7) patients evaluated. Paired tumor biopsy specimens for gene expression analysis were available for 3 patients in the 10-30 mg/kg cohorts who remained on the trial for at least 4 cycles.

Increased CXCL9 gene expression in tumor was observed in both patients with low baseline TIL cells. One patient exhibited a 12-fold increase in CD8⁺ TIL by IHC. A second patient exhibited a 2.3-fold increase in CD8A gene expression. The third patient (having high baseline TIL) had very high CXCL9 gene expression at all timepoints and exhibited an improved ratio of ThLTreg cells in the tumor microenvironment. Figure 26 shows changes in tumor CXCL9 gene expression.

Gene expression analysis of tumor biopsy specimens from 10-30 mg/kg cohorts shows that tumor CXCL9 gene expression correlated with CD8 TIL density in biopsy specimens (Figure 27). Gene expression of CXCL9 (a chemoattractant for effector T cells) was increased in tumor post- treatment in patients 10-0506 and 20-0609, potentially promoting TIL recruitment. CXCL9 gene expression was found to remain high in clinical responder 20-0402, consistent with high TIL density pre and post-treatment.

Candidate biomarkers were evaluated for all patients in the 10-30 mg/kg dose cohorts. All patients in these cohorts who had completed 4 or more treatment cycles had relatively normal ALC and serum LDH levels at baseline. Confirmed clinical responders (PR / SD > 6 months) also had an inflammatory tumor microenvironment at baseline, as evidenced by a high average number of CD8⁺ and PD-1⁺ TIL cells per high-powered field (hpf) plus membranous B7-H1 expression on tumor cells in areas of high TIL density. As shown in Figures 28A-28E, patients who were Clinical

Responders exhibited adequate numbers of lymphocytes /ml of blood (Figure 28 A), serum LDH no more than 2-fold over the upper limit of normal (Figure 28B), a high expression of tumor B7-H1 (Figure 28C), a high average number of CD8+ TIL cells per hpf (Figure 28D) and a high average number of PD-1+ TIL cells per hpf (Figure 28E). These findings validate the inflammatory tumor microenvironment (patient SD in Cycle 19) (Figures 29A-29C, respectively). The gene expression analysis corroborates the IHC results; this biopsy specimen had the highest levels of CD8A, FCGR3A, CTLA4, PDl, FASLG, CCL3, CXCL9, CXCLIO, and GZMA expression of the evaluated pre-treatment biopsy specimens.

IHC analysis of paired tumor biopsy specimens from the 10-30 mg/kg cohorts shows increased ratio of CD8⁺ TIL to PD-1⁺ TIL in 9/14 cases, including 5/5 evaluated patients who remained on the trial for 4 or more cycles (Figures 30A-30B), a further increase in average number of CD8⁺ TIL/hpf (from 1068 to 1522) in patient 20-0402 (SD in Cycle 20) and the emergence of an "immune front" in patient 10-0506 (B7-H1 score increased from 0 to 3, and average number of CD8⁺ TIL/hpf increased 11.8- fold (from 8 to 94) (Figure 31, Panels A-D).

In summary, the results show that the human B7-DC-Ig fusion had an acceptable safety profile, with no evidence of pneumonitis or GI toxicities. Initial evidence of clinical activity (PR, long-term SD, and MR's) has been obtained, and shows that a dose-dependent reduction in PD-1 HI T cells occurs following treatment with the human B7-DC-Ig fusion. Improvements in immune function were consistently observed in the periphery and tumor microenvironment in patients in the 10-30 mg/kg cohorts who were able to remain on the trial for 4 or more treatment cycles. Baseline ALC and LDH stratify all patients who remained on trial for 4+ cycles (including clinical responders) vs. patients who came off trial in < 4 cycles due to rapid disease progression. Baseline tumor B7-H1 expression, CD8 TIL levels, and PD-1 TIL levels ("immune front") stratify confirmed clinical responders vs. other patients who remained on trial for 4+ cycles (including MR).

Example 8: Evaluation of Gene Expression Using bDNA Analysis

The expression of 19 exhaustion/effector genes and 17 lymphocyte phenotype genes were evaluated by branched DNA (bDNA) analysis using tumor biopsy specimens from 16 patients participating in the clinical trial of Example 3; paired specimens from 8 patients have been analyzed. The goals Inc., Santa Clara, CA). The assay is based on the direct quantification of 3- 80 different RNA target using magnetic beads for multiplexing the RNA targets and branched DNA (bDNA) signal amplification technology (Zhang, A. et al. (2005) "Small Interfering RNA And Gene Expression Analysis Using A Multiplex Branched DNA Assay Without RNA Purification " J. Biomol. Screen. 10(6):549-556; Zheng, Z. et al. (2006) "Sensitive And Quantitative Measurement Of Gene Expression Directly From A Small Amount Of Whole Blood;' Clin. Chem. 52(7): 1294-1302; Sterling, J. et al. (2008) "Current Trends In High-Throughput Screening " Assay Drug Dev. Technol. 6(4):491-504; Young, H.A. (2009) "Cytokine Multiplex Analysis (Chapter 4). Inflammation and Cancer, In: METHODS IN MOLECULAR BIOLOGY 511 :85-105; Lash, G.E. et al. (2010) "Multiplex Cytokine Analysis Technologies," Expert Rev. Vaccines 9(10): 1231-1237; Hicks, S.D. et al. (2012) "Evaluation Of Cell Proliferation, Apoptosis, And DNA-Repair Genes As Potential Biomarkers For Ethanol-Induced CNS Alterations," BMC Neurosci. 13(1): 128, pp. 1-13).

Whole blood and tumor specimens of the clinical trial participants and four healthy control individuals were evaluated using an "exhaustion and effector" marker panel; tumor specimens were also assessed using a

"phenotyping" marker panel. The markers evaluated are listed in Table 2 below:

TR 91 Thp Table 2

Category Gene Description

GATA3

FCGFR3A CD16

CD68

CD163

IRF5

IRF4

ITGAX CDl lc

IL3RA CD123

CLEC4C BDCA-2

NOS2

IDOl

PTGS2

Reagents were purchased from Affymetrix, Inc. (Santa Clara, CA) and experiments were conducted using a BioPlex Luminex 2.0 instrument (BioRad Laboratories, Hercules, CA). Under the assay conditions, the fluorescence intensity of each bead population is linearly proportional to the level of gene expression. The background-corrected fluorescence intensity values were normalized to the expression of the housekeeping

genes:POLR2A, TBP, PPIB and HPRT1, from the same specimen. Analytes were reported to be below the lower limit of quantification ("<LLOQ") if the fluorescence intensity was within 2 standard deviations of background. Analytes within 3 standard deviations of background were highlighted as these values are of lower confidence. Exhaustion / suppression, activation / effector, and housekeeping biomarkers were evaluated from tumor specimens and whole blood; immunophenotype biomarkers were evaluated from tumor specimens. Immune responders exhibited increases in both exhaustion and effector molecules. Most "exhaustion" markers were actually up-regulated in both activated and exhausted cells, however, the analysis cannot distinguish between lower levels of expression in many cells versus high level expression in a smaller number of cells. Increases in these markers are consistent with data showing an increase in the relative population effector/memory T cells (which express most of these markers) versus naive T cells in Cohort 4-6 patients who have stayed on trial 4+ cycles. Reduction across both exhaustion and effector molecules were observed to correlate with rapidly progressing disease. This is consistent with data showing an increase in the relative population naive T cells (which do not express most of these markers) versus effector/memory T cells with progression.

B7-H1 is upregulated, and PD-1 is down-regulated, in patients who came off trial in < 4 cycles due to progressive disease vs. patients who completed 4+ cycles.

Pre-treatment biopsy specimens from the different patients were compared and evaluated for potential patient selection biomarkers (Figures 32A-32B). The pre-dose biopsy specimen from a confirmed clinical responder (patient 0402) (black triangle) had significantly higher CD8⁺ TIL and PD-1⁺ TIL levels than other fresh pre-dose biopsies evaluated to date, either for patients who stayed on trial for 4+ cycles (square)or patients who came off trial more rapidly(gray circle). Among pre-treatment biopsy specimens evaluated to date, patient 0402 had the highest levels of CD8A (CD8a), FCGR3A (FcyRIIIa, CD16), CTLA4, PD-1, FASL, CCL3, CXCL9, CXCL10, and GZMA gene expression. These results further support the conclusion that the presence of a pre-existing inflammatory tumor microenvironment is required for a clinical response to B7-DC-Ig Fusion therapy.

Tumor bDNA and IHC results were compared. A clear correlation was seen between the gene expression and IHC data for CD4, CD8, and PD- 1 (R2 = 0.75, 0.98, 0.88, respectively), but not for FoxP3 or B7-H1 (R2 = Paired pre- and post-treatment biopsy specimens were compared to evaluate changes in the tumor immune microenvironment following B7-DC- Ig Fusion treatment. The pre-treatment biopsy specimen from a confirmed clinical responder (patient 0402) exhibited very high expression of many immune markers in the tumor. Post-treatment biopsy specimens from this patient exhibited stable, strong expression of many immune markers from CI DO to C2D15, as well as evidence for improved T cell function following treatment. The myeloid populations also appeared to change. More specifically, the patient exhibited an increase in TBX21 (Tbet, Thl master transcription factor) while FOXP3 expression was stable, indicating an improved ratio of Thl to Treg cells in the tumor microenvironment. The patient also exhibited an increased expression of CD40L relative to other markers (CD40L is expressed on effector/memory T cells and plays a central role in promoting DC maturation and migration). The patient also exhibited decreased expression of the T cell suppressive molecules, LAG3 and 2B4, relative to other markers as well as an increase in CCR2 expression (CCR2 is expressed in the tumor microenvironment on monocytes, macrophages and Treg), but without a concomitant increase in FOXP3 or CD68 expression. The patient also exhibited an increase in CLEC4C expression, likely indicating increased pDC infiltration. pDC have been reported to promote immune suppression in melanoma via IDO production. An increase in IRF4 expression was observed (IRF4 is expressed in B, Tfh, Thl7, and some myeloid cells and is associated with germinal center formation; signaling through CD40L has been reported to induce IRF4 expression. A slight increase in IDO expression was observed, which is considered suppressive. The results are shown in Figures 35A-35C.

Pre-treatment biopsy specimens from patients who remained on study 4+ cycles (patients 0506 and 0609) exhibited low-level expression of most immune markers. Overall, changes in gene expression suggested improved function following treatment. Specifically, such patients exhibited a pronounced increase in CXCL9. CXCL10 also increased in patient 0506, while increased recruitment of CD8 T cells was evidenced by an increase in CD8A gene expression observed in patient 0609 (2.3 -fold increase in normalized gene expression, largest fold-increase in phenotyping panel) and increased CD8+ TIL (average # / hpf increased from 8 to 94) in patient 0506.

Both patients exhibited a down-regulation of the suppressive T cell marker, CD160. OX40 was increased in patient 0609. OX40 is expressed by both Treg and Teff, and both FOXP3 and CD8 expression are increasing. Expression of the suppressive marker, CLTA4 decreased in patient 0609. Analysis of the phenotyping panel suggested a broad increase in T cells in patient 0609: CD8A is increased most strongly, and a variety of Th transcription factors (RORC, GAT A3, and FoxP3) are also strongly upregulated. NOS2 expression was down-regulated in patient 0609. While NOS2 is an inflammatory mediator, it has also been associated with tumor progression and suppression of effector T cells. CD40L expression was found to decrease in patient 0506, which is considered unfavorable. The results are shown in Figure 36 (patient 0506) and Figures 37A-37B (patient 0609).

In addition, three paired biopsy specimens from patients in lower dose cohorts (0106, 0301, 0302) and four paired biopsy specimens from patients in cohorts 4-6 who came off trial in < 4 cycles (0604, 0605, 0607, 0608) were evaluated. The patterns of change are quite distinct from those presented above:

1. CXCL9 and CXCL10, which recruit effector T cells to the tumor, were among the most strongly down-regulated genes in 6/7 paired biopsies;

2. Lytic molecules GzmA and GzmB were among the most 3. Inhibitory molecules were among the most strongly up- regulated genes; specifically CD160 (3/7 paired biopsies), CTLA4 (3/7 paired biopsies) and 2B4 (2/7 paired biopsies). In the phenotyping panel, the most up-regulated and down-regulated genes were typically myeloid-associated rather than T-cell associated.

However, large changes were often observed in RORC (Thl7 master transcription factor) and IRF4 (which can be expressed by some T cell subsets as well as other populations).

Baseline CXCL9 expression correlated with levels of CD8⁺ TIL cells. Two patients (patients 0506 and 0609) who had low baseline TIL levels exhibited large increases in tumor-CXCL9 expression following treatment. Increased CD8 infiltration was observed in IHC for patient 0506 (avg #/hpf increased from 8 to 94) and by bDNA for 0609 (2.3 -fold increase). In contrast, patients with rapidly progressing disease were found to have stable or declining levels of CXCL9 and CD8A expression in tumor biopsies.

In summary, the B7-DC Ig fusion was found to have an acceptable safety profile, with no evidence of pneumonitis or GI toxicities. Initial evidence of clinical activity (PR, long-term SD, and MR's) was been obtained, and a dose-dependent reduction in PD- 1 HI T cells was found to occur following B7-DC Ig fusion treatment. Baseline ALC and LDH stratify all patients who remained on the trial for 4+ cycles (including clinical responders) vs. patients who came off trial in < 4 cycles due to rapid disease progression. Baseline tumor B7-H1 expression, CD8 TIL levels, and PD-1 TIL levels ("immune front") stratify confirmed clinical responders vs. other patients who remained on the trial for 4+ cycles (including MR).

Improvements in immune function are consistently observed in the periphery and tumor microenvironment, in patients in 10-30 mg/kg cohorts who were able to remain on trial for 4+ treatment cycles. The treatment resulted in expanded populations of polyfunctional and lytic T cells in peripheral blood, enhanced levels of CXCL9 gene expression (as determined by tumor biopsies), and increased ratio of CD8+ to PD-1⁺ lymphocytes in the tumor least 4 treatment cycles days (Cohort 4-6). This is consistent with enhanced immune function in these patients. The IHC and bDNA analyses of pre- treatment biopsy specimens provided similar results for CD8, CD4, and PD- 1, thus validating their use in screening patient candidates.

The evaluation of paired biopsy specimens from a confirmed clinical responder (patient 20-0402) exhibited evidence of improved immune function:

1. increased expression of TBX21 relative to FoxP3 ;

2. increased expression of CD40L relative to other markers;

3. decreased expression of the T cell suppressive molecules, LAG3 and 2B4, relative to other markers evaluated.

The evaluation of paired biopsy specimens from two patients who remained in the clinical trial for 4 or more cycles (patients 0506 and 0609 of Cohorts 4-6) also exhibited evidence of improved function:

1. pronounced increase in CXCL9 in both patients and increased expression of CXCL10 in one patient (patient 0506). These chemokines recruit effector T cells to the tumor. A 12-fold increase in CD8 T cells was observed by IHC in 0506, and 2.3-fold higher CD8A gene expression was observed for 0609;

2. Down-regulation of the suppressive T cell marker CD160 in both patients

The evaluation of paired biopsy specimens from patients in low-dose cohorts (Cohorts 1-3 and Cohorts 4-6 who came off the clinical trial following < 4 cycles due to rapid disease progression) exhibited evidence of a more suppressive tumor immune microenvironment:

1. CXCL9 and/or CXCL10, which recruit effector T cells to the tumor, were strongly down-regulated in 6/7 cases;

2. Lytic molecules GZMA and GZMB were strongly down- regulated in 4/7 cases;

3. Inhibitory molecules 2B4, CD 160, and/or CTLA4 were received 10-30 mg/kg of a B7-DC-Ig Fusion molecule and were able to stay in the clinical trial for at least 4 cycles. In contrast, evidence of increased immune suppression was observed in patients who came off study more rapidly due to disease progression.

Example 9: Evaluation of the Effect of the Human B7-DC-Ig Fusion on T Cell Function Using Flow Cytometry Intracellular Cytokine Staining

Flow cytometry assays were conducted using PMBC specimens collected from the patients in the clinical trial of Example 3 in order to determine the effects of the human B7-DC Ig Fusion on T cell function.

For such assays, 40 mL of whole blood was collected in heparin tubes on Day 0 of each cycle and PBMC were isolated and cryopreserved. The cells were thawed, rested or stimulated with PMA/ionomycin, and stained for subset markers (CD3, CD4, CD8, CD45RA, CD27), effector markers (IFN-γ, TNF-a, IL-2, IL-17, CD107a, GzmB), and viability. The percentages of CD4 and CD8 T cells expressing IFN-γ, TNF-a, and IL-2 were determined. Very little IL-17 or CD107 expression was observed.

In 4 of 5 evaluated cases (10-0506, 20-0501, 20-0602, 30-0405 and 30-0406), patients who remained in the clinical trial for 3 or more cycles were found to have increased levels of polyfunctional T cells (secreting IFN- γ, TNF-α, and IL-2), dual functional T cells (secreting IFN-γ and TNF-a), and monofunctional IFN-γ ^" T cells (Table 3). Increases in functional T cell populations could be observed after one cycle, and further improvements were typically observed during subsequent cycles. One patient (20-0602) was observed to have improved CD4 function but declining CD8 function. In most cases high-level cytokine production by peripheral T cells persisted through a few cycles, with levels subsequently returning approximately to baseline. Table 3

% Change in Cytokine Expression Relative to Baseline

Cycle (C) (Median Fluorescence Intensity, MdFI)

Patient

Day (D) CD4 T Cells CD8 T Cells

TNF-a IFN-γ IL-2 IFN-γ TNF-a

C1D0 32% 11%

C2D0 41% 13%

30-0405

C5D0 60% 23%

C6D0 70% 29%

C1D0 14%

30-0406

C4D0 26%

C1D0 26%

10-0506 C2D0 45%

C4D0 47%

C1D0 34%

20-0602 C2D0 47%

C3D0 41%

C1D0 18% (4769) 22% (4769)

20-0501 C2D0 33% (5832) 38% (5333)

C3D0 46% (6396) 51% (6858)

In contrast, levels of such polyfunctional T cells (secreting IFN-γ, TNF-a, and IL-2), dual functional T cells (secreting IFN-γ and TNF-a), and monofunctional IFN-γ ^" T cells were found to be stable or declining in 4 of 5 patients (20-0403, 30-0601, 20-0604, 20-0606 and 20-0607) who stayed on the clinical trial for fewer than 4 cycles (due to disease progression).

Patients who remained in the clinical trial through at least 4 cycles generally had relative decreases in naive T cells and relative increases in effector/ memory / EMRA populations.

The unstimulated cells also showed higher levels of GzmB than

PMA/I stimulated cells, likely because degranulation had been triggered by stimulation. GzmB expression was therefore evaluated using unstimulated cell samples. GzmB expression on CD8 T cells was high or increasing in natients who remained on trial for 4+ cvcles. Tvnicallv GzmB. which is treatment in patients 0406, 0501, and 0506; this is generally a very small population but may play an important role in anti-tumor immune response (Quezada, S.A. et al. (2010) "Tumor-Reactive CD4(+) T Cells Develop Cytotoxic Activity And Eradicate Large Established Melanoma After Transfer Into Lymphopenic Hosts," J. Exp. Med. 207(3):637-650). Increases in GzmB cells often occurred at much later timepoints in some patients.

In contrast, 3 of 5 patients who came off the clinical trial more rapidly due to disease progression had decreases in GzmB expression over time.

In order to more fully appreciate the results, the absolute numbers of key effector cell populations were calculated using absolute CD8 counts. The number of cells per mL of blood for these populations was calculated pre- treatment and at each post-treatment timepoint; the highest post-treatment rapidly typically had declining absolute numbers of the key effector cell populations at all post-dose timepoints. The flow cytometry analysis of PBMC specimens from 10-30 mg/kg cohorts thus shows increased numbers of polyfunctional (IFN-γ ^" TNF-a⁺ IL-2⁺) CD4⁺ and CD8⁺ T cells and increased numbers of effector and EMRA T cells producing the lytic marker Granzyme B in patients who stayed on trial for 4 or more cycles, including clinical responders (20-0505 evaluable for GzmB only).

In summary, the results show that consistent evidence of improved immune function was seen in patients who received 10-30 mg/kg of a human B7-DC-Ig Fusion molecule and completed at least 4 cycles of therapy. Such evidence included the findings that patients exhibited increases in the frequency of:

(1) polyfunctional (IFN-γ ^" TNF-a⁺ IL-2⁺) T cells (cells that have been shown to correlate with protective immunity during infection and following vaccination);

(2) dual functional (IFN-y⁺ TNF-a⁺) T cells (cells that have been shown to correlate with response to ipilimumab (anti- CTLA- 4 antibody) (Yuan, J. et al. (2008) "CTLA-4 Blockade Enhances Polyfunctional NY-ESO-1 Specific T Cell

Responses In Metastatic Melanoma Patients With Clinical Benefit;' Proc. Natl. Acad. Sci. (U.S.A.) 105(51):20410- 20415);

(3) CD4 and/or CD8 effector T cells expressing the lytic marker GzmB; and

(4) effector / EMRA / memory subsets vs. naive T cells (relative increases).

In contrast, improvements in peripheral T cell function were generally not observed in PBMC specimens from patients that received < 10 mg/kg of the human B7-DC-Ig Fusion molecule or in PBMC specimens from patients who came off the clinical trial more rapidly due to disease progression (patients 0403, 0601, 0603, 0604, and 0607). immunostainers and detected using DAB (3, 3'-diaminobenzidine) HRP substrate.

The results are illustrated in Figure 39A-39B and 40A-40B. Figures 39A-39B are micrographs showing that PD-Ll (B7-H1) and B7-H4 (CD68) are co-expressed in tissue sections of a melanoma. Figures 40A-40B are micrographs showing that PD-Ll (B7-H1) and B7-H4 (CD68) are co- expressed in tissue sections of a renal cell carcinoma.

All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Claims

We claim Claim 1. A method for determining whether a cancer patient suffers from a cancer having enhanced suitability for treatment with a PD-1 targeted monotherapy or a PD-1 targeted combination therapy, wherein said method comprises evaluating tissue or fluid of said patient to ascertain the level of a prognostic biomarker correlative of immune system responsiveness.

Claim 2. The method of claim 1, wherein said method additionally comprises providing said patient with said PD-1 targeted monotherapy or said PD-1 targeted combination therapy in response to said determination.

Claim 3. The method of any of claims 1-2, wherein said evaluating includes removing said tissue or fluid from said patient.

Claim 4. The method of claim 3, wherein said evaluation of said tissue or fluid of said patient comprises histochemical staining, in situ hybridization; gene expression analysis, or flow cytometry.

Claim 5. The method of claim 4, wherein said evaluation of said tissue or fluid of said patient comprises bDNA, qRT-PCR, or microarray analysis.

Claim 6. The method of any of claims 1-5, wherein said prognostic biomarker is the baseline:

(A) percentage of CD4⁺ or CD8⁺ T cells that are PD- 1^HI cells;

(B) the concentration of serum lactate dehydrogenase;

(C) the absolute lymphocyte count;

(D) the frequency of CD8⁺ or PD-1⁺ tumor infiltrating lymphocytes; or (E) gene expression of CD8A, FCGR3A, CTLA4, PD1, FASLG, CCL3, CXCL9, CXCL10, or GZMA in a tumor biopsy specimen.

The method of claim 6, wherein said method comprises evaluating the level of at least two of said prognostic biomarkers.

The method of claim 6, wherein said method comprises evaluating the level of all of said prognostic biomarkers.

The method of any of claims 1-8, wherein said prognostic biomarker is the level of peripheral CD4⁺ or CD8⁺ cells that are PD-1 HI cells, and wherein:

(A) a sustained reduction in the level of PD-1 HI T cells following PD- 1 targeted therapy is prognostic of an ability to respond to PD-1 targeted therapy; and

(B) a finding that any reduction in the level of PD-1^HI T cells following PD-1 targeted therapy is not sustained and subsequently rebounds above baseline is prognostic of inability diminished ability to respond to PD- 1 targeted therapy.

The method of any of claims 1-8, wherein said prognostic biomarker is lactate dehydrogenase, and wherein:

(A) a level of lactate dehydrogenase that is within, or less than two-fold greater than, the upper level of normal (ULN) is predictive of said patient' s enhanced suitability for treatment with a PD-1 targeted monotherapy or combination therapy; and

(B) a level of lactate dehydrogenase that is more than twofold greater than the upper level of normal (ULN) is predictive of said patient's reduced suitability for treatment with a PD- 1 targeted monotherapy or combination therapy.

Claim 11. The method of any of claims 1-8, wherein said prognostic biomarker is absolute lymphocyte count, and wherein:

(A) an absolute lymphocyte count that is equal to or

greater than approximately 950 e\ /μL· is predictive of said patient's enhanced suitability for treatment with a PD-1 targeted monotherapy or combination therapy; and

(B) an absolute lymphocyte count that is less than said absolute lymphocyte count is predictive of said patient's enhanced suitability for treatment with a PD- 1 targeted monotherapy or combination therapy.

Claim 12. The method of any of claims 1-8, wherein said prognostic biomarker is a baseline tumor infiltrating lymphocyte count, and wherein:

(A) a baseline tumor infiltrating lymphocyte count that is equal to or greater than approximately 50-100 CD8⁺ or PD-1⁺ cells per high powered microscope field is predictive of said patient's enhanced suitability for treatment with a PD-1 targeted monotherapy; and

(B) a baseline tumor infiltrating lymphocyte count that is less than said baseline tumor infiltrating lymphocyte count is predictive of said patient's enhanced suitability for treatment with a PD-1 targeted combination therapy.

Claim 13. The method of claim 6, wherein said prognostic biomarker is gene expression of CD8A, FCGR3A, CTLA4, PDl, FASLG, CCL3, CXCL9, CXCL10, or GZMA in a tumor biopsy specimen.

Claim 14. The method of any of claims 1-13, wherein said cancer is an adrenal cancer, a bladder cancer, a bone and connective tissue sarcoma, a brain tumor, a breast cancer, a colon or rectal cancer, an esophageal cancer, an eye cancer, a kidney cancer, a leukemia, a lymphoma, a melanoma, a multiple myeloma, a liver cancer, a lung cancer, a pancreatic cancer, a pharyngeal cancer, a pituitary cancer, an oral cancer, a salivary gland cancer, a skin cancer, a stomach cancer, a testicular or penal cancer, a thyroid cancer, or a vaginal, ovarian, uterine or cervical cancer.

Claim 15. The method of claim 14, wherein said cancer is an ovarian cancer, a triple negative breast cancer, a non-small cell lung carcinoma (NSCLC), a head and neck cancer, a melanoma.

Claim 16. The method of any of claims 1-15, wherein said PD-1 targeted monotherapy or combination therapy comprises administration of an anti-PD-1 antibody, a PD-1 -binding fragment of an antibody, or a B7-DC-Ig fusion molecule.

Claim 17. The method of any of claims 1-15, wherein said PD-1 targeted combination therapy enhances the activity of a RAS-RAF- MEK-ERK inhibitor, or comprises the administration of an agent that targets a co-stimulatory pathway molecule that is up-regulated following treatment with a RAS-RAF-MEK- ERK inhibitor.

Claim 18. The method of any of claims 1-16, wherein said PD-1 targeted combination therapy comprises administration of cyclophosphamide, carboplatin, paclitaxel, docetaxel or doxorubicin.

Claim 19. The method of claim 17, wherein said combination PD-1 targeted therapy comprises the administration of a BRAFi or other small molecule, as an initial treatment regimen.

Claim 20. A method of determining patient suitability for participation in a clinical trial involving a PD- 1 targeted cancer therapy, wherein said method comprises determining whether tissue or fluid of a candidate patient for said clinical trial possesses:

(A) a level of peripheral CD4⁺ or CD8⁺ T cells that are PD-1^HI cells;

(B) a concentration of serum lactate

dehydrogenase;

(C) a baseline absolute lymphocyte count;

(D) a rapid declining absolute lymphocyte count;

(E) a baseline tumor infiltrating lymphocyte count; or

(F) gene expression of CD8A, FCGR3A, CTLA4, PD1, FASLG, CCL3, CXCL9, CXCL10, or GZMA in a tumor biopsy specimen.

that is correlative of immune system responsiveness.

Claim 21. The method of claim 20, wherein said cancer is an adrenal cancer, a bladder cancer, a bone and connective tissue sarcoma, a brain tumor, a breast cancer, a colon or rectal cancer, an esophageal cancer, an eye cancer, a kidney cancer, a leukemia, a lymphoma, a melanoma, a multiple myeloma, a liver cancer, a lung cancer, a pancreatic cancer, a pharyngeal cancer, a pituitary cancer, an oral cancer, a salivary gland cancer, a skin cancer, a stomach cancer, a testicular or penile cancer, a thyroid cancer, or a vaginal, ovarian, uterine or cervical cancer.

A method for characterizing the cells of a tumor comprising determining whether the cells of the tumor express B7-H1 or B7-H4; and determining whether said cells of said tumor that express B7-H1 or B7-H4 are tumor cells or non-tumor cells.

The method of claim 22 wherein the non-tumor cells are tumor-associated macrophages and are detected using CD68.

A method of selecting a patient for treatment based on the expression of B7-H4 or B7-H1 on tumor and non-tumor cells, wherein:

1) the expression of B7-H4 or B7-H1 predominantly on tumor cells is indicative of a patient that will most benefit from treatment comprising a B7-H4 or B7-H1 therapy that is cytotoxic to the B7-H4 or B7-H1 expressing cells; and,

2) the expression of B7-H4 or B7-H1 predominantly on non-tumor cells is indicative of a patient that will most benefit from treatment comprising a B7-H4 or B7-H1 therapy that blocks PD-1 mediated signaling but is not cytotoxic to the B7-H4 or B7-H1 expressing cells.