WO2020030570A1

WO2020030570A1 - Combinations of an ox40 antibody and a tlr4 modulator and uses thereof

Info

Publication number: WO2020030570A1
Application number: PCT/EP2019/070978
Authority: WO
Inventors: Bruce A HUG; Christopher J MATHENY; Elaine Marie PAUL
Original assignee: Glaxosmithkline Intellectual Property Development Limited
Priority date: 2018-08-06
Filing date: 2019-08-05
Publication date: 2020-02-13

Abstract

Disclosed herein are combinations of an OX40 modulator, such as an agonist OX40 binding protein, and a TLR4 modulator, such as a TLR4 agonist, pharmaceutical compositions thereof, uses thereof, and methods of treatment comprising administering said combination, including uses in cancer.

Description

METHOD OF MAKING AN AEROSOL FORMING SUBSTRATE

FIELD OF THE INVENTION

The present invention relates to a method of treating cancer in a mammal and to combinations useful in such treatment. In particular, the present invention relates to combinations of an 0X40 binding protein and one or more TLR4 agonist.

BACKGROUND OF THE INVENTION

Effective treatment of hyperproliferative disorders, including cancer, is a continuing goal in the oncology field. Generally, cancer results from the deregulation of the normal processes that control cell division, differentiation and apoptotic cell death and is characterized by the proliferation of malignant cells which have the potential for unlimited growth, local expansion and systemic metastasis. Deregulation of normal processes includes abnormalities in signal transduction pathways and response to factors that differ from those found in normal cells.

Immunotherapies are one approach to treat hyperproliferative disorders. A major hurdle that scientists and clinicians have encountered in the development of various types of cancer immunotherapies has been to break tolerance to self antigen (cancer) in order to mount a robust anti-tumor response leading to tumor regression. Unlike traditional development of small and large molecule agents that target the tumor, cancer

immunotherapies target cells of the immune system that have the potential to generate a memory pool of effector cells to induce more durable effects and minimize recurrences.

0X40 is a co-stimulatory molecule involved in multiple processes of the immune system. Antigen binding proteins and antibodies that bind 0X40 receptor and modulate 0X40 signaling are known in the art and are disclosed as immunotherapy, for example, for cancer.

Aminoalkyl glucosaminide phosphates (AGPs) are synthetic ligands of Toll-like

Receptor 4 (TLR4). AGPs are known to be useful as vaccine adjuvants and for stimulating cytokine production, activating macrophages, promoting innate immune response, and augmenting antibody production in immunized animals. Though there have been many recent advances in the treatment of cancer, there remains a need for more effective and/or enhanced treatment of an individual suffering the effects of cancer. The combinations and methods herein that relate to combining therapeutic approaches for enhancing anti-tumor immunity address this need.

SUMMARY OF THE INVENTION

Provided herein are combinations of an 0X40 binding protein and one or more TLR4 modulators. Also provided are methods of treating cancer in a human with the compositions of the invention, and uses of the combinations for therapy, such as therapy for cancer. Further provided are methods for modulating the immune response of a subject in need of cancer treatment, such as a human, comprising administering to said subject an effective amount of the combination, e.g., in one or more pharmaceutical compositions.

In some aspects, the disclosure is drawn to a method of treatment of a subject with cancer, the method comprising: administering to the subject an agonist 0X40 binding protein and a TLR4 agonist, wherein the agonist 0X40 binding protein is administered at a dose of about 8 mg to about 24 mg and the TLR4 agonist is administered at a dose of about 5 ng to about 1000 ng. In an embodiment, the TLR4 agonist is administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to an agonist 0X40 binding protein and a TLR4 agonist for simultaneous or sequential use in treating cancer, wherein the agonist 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and the TLR4 agonist is to be administered at a dose of about 5 ng to about 1000 ng. In an embodiment, the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to an agonist 0X40 binding protein for use in treating cancer, wherein the agonist 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and is to be administered simultaneously or sequentially with a TLR4 agonist at a dose of about 5 ng to about 1000 ng. In an embodiment, the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng. In some aspects, the disclosure is drawn to a TLR4 agonist for use in treating cancer, wherein the TLR4 agonist is to be administered at a dose of about 5 ng to about 1000 ng and is to be administered simultaneously or sequentially with an agonist 0X40 binding protein at a dose of about 8 mg to about 24 mg. In an embodiment, the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to use of an agonist 0X40 binding protein in the manufacture of a medicament for treating cancer, wherein the 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and is to be administered simultaneously or sequentially with a TLR4 agonist at a dose of about 5 ng to about 1000 ng. In an embodiment, the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to use of a TLR4 agonist in the manufacture of a medicament for treating cancer, wherein the TLR4 agonist is to be administered at a dose of about 5 ng to about 1000 ng and is to be administered simultaneously or sequentially with an agonist 0X40 binding protein at a dose of about 8 mg to about 24 mg. In an embodiment, the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to a pharmaceutical kit comprising about 8 mg to about 24 mg of an agonist 0X40 binding protein and about 5 ng to 1000 ng of a TLR4 agonist. In an embodiment, the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng.

In some embodiments, the agonist 0X40 binding protein is administered intravenously.

In some embodiments, the TLR4 agonist is administered intravenously.

In some embodiments, the TLR4 agonist is administered subcutaneously.

In some embodiments, the TLR4 agonist is administered intratumorally.

In some embodiments, the agonist 0X40 binding protein is administered intravenously and the TLR4 agonist is administered intravenously. In some embodiments, the agonist 0X40 binding protein is administered at a dose of about 8 mg.

In some embodiments, the agonist 0X40 binding protein is administered at a dose of about 24 mg. In some embodiments, the TLR4 agonist is administered at a dose of about 5 ng to about 1000 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 50 ng to about 250 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 50 ng. In some embodiments, the TLR4 agonist is administered at a dose of about 100 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 150 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 200 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 250 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 300 ng. In some embodiments, the TLR4 agonist is administered at a dose of about 350 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 400 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 450 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 500 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 550 ng. In some embodiments, the TLR4 agonist is administered at a dose of about 600 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 600 ng to about 1000 ng.

In some embodiments, the agonist 0X40 binding protein comprises (a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: l ; (b) a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2; (c) a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; (d) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7; (e) a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8; and (f) a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9.

In some embodiments, the agonist 0X40 binding protein comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:5; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: l l .

In some embodiments, the agonist 0X40 binding protein is a humanized monoclonal antibody.

In some embodiments, the agonist 0X40 binding protein comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:48 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:49.

In some embodiments, the TLR4 agonist is selected from the group consisting of: CRX- 601; CRX-547; CRX-602; and CRX-527.

In some embodiments, the TLR4 agonist is CRX-527.

In some embodiments, the TLR4 agonist is CRX-601.

In some embodiments, the cancer comprises a solid tumor.

In some embodiments, the cancer comprises squamous cell carcinoma of head and neck (SCCHN). In some embodiments, the SCCHN is recurrent, locally advanced, or metastatic.

In some embodiments, the TLR4 agonist and the agonist 0X40 binding protein are both periodically administered. In some embodiments, the TLR4 agonist and the agonist 0X40 binding protein are both administered to the subject every three weeks.

In some embodiments, the TLR4 agonist is administered for a two-week run in period (e.g. , the TLR4 agonist is administered on day 1 and 8) prior to administering the agonist 0X40 binding protein.

In some embodiments, the TLR4 agonist is administered for a two-week run in period (e.g., the TLR4 agonist is administered on day 1 and 8), and following the run in period, the TLR4 agonist and the agonist 0X40 binding protein are both administered to the subject, e.g., every three weeks, e.g. starting on day 15.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a graph showing dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) of TLR4 agonist (CRX-527) in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals.

Figure IB is a graph showing dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) of a rat anti-mouse 0X40 receptor antibody (clone OX-86) in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals; control treatments in Figure 1A are the same as those in FigurelB.

Figure 2 is a graph showing anti-tumor activity (as measured by tumor growth inhibition over time) of a rat anti-mouse 0X40 receptor antibody (clone OX-86), 5 pg of TLR4 agonist (CRX-527), and the combination of both in a CT-26 syngeneic mouse model of colon cancer. Results are the mean of 10 animals.

Figure 3 is a graph showing dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) of a rat anti-mouse 0X40 receptor antibody (clone OX-86), 25 pg of TLR4 agonist (CRX-527), and the combination of both in a CT-26 syngeneic mouse model of colon cancer measured over 38 days. Results are the mean of 10 animals; control treatments in Figure 2 represent identical animals as those in Figure 3.

Figures 4A-4F are graphs showing dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) in individual mice of a control antibody (IgG), rat anti-mouse 0X40 receptor antibody (clone OX-86), 5 or 25 pg of TLR4 agonist (CRX- 527), and the combination of 0X86 and CRX-527 in a group of mice in a CT-26 syngeneic mouse model of colon cancer measured over 42 days. The average group tumor volume for mice remaining on study in Figures 4A-4F were used to generate the plots in Figures 2-3.

Figure 5 is a graph showing dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) of 4, 20, or 100 pg of TLR4 agonist (CRX-601) in a CT-26 syngeneic mouse model of colon cancer.

Figures 6-12 show sequences of the ABPs and antibodies of the invention, e.g., CDRs and VH and VL sequences.

Figures 13-17 show sequences of ABPs and antibodies of the invention, e.g., CDRs and VH and VL sequences.

Figure 18 is a graph showing dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) of the TLR4 agonist CRX-601 dosed intratumoral in a CT- 26 syngeneic mouse tumor model.

Figure 19 is a graph showing survival curves of mice treated with the TLR4 agonist CRX-601 intratumoral dosed intratumoral in a CT-26 syngeneic mouse tumor model. (*p-values < 0.05).

Figure 20 is a graph showing dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) of the TLR4 agonist CRX-601 in a CT-26 syngeneic mouse tumor model. (*p-values < 0.05)

Figure 21 is a graph showing survival curves of mice treated with the TLR4 agonist CRX-601 dosed intravenous in a CT-26 syngeneic mouse tumor model (*p- values < 0.05).

Figure 22 is a graph showing anti-tumor activity (as measured by tumor growth inhibition over time) of 25 pg/mouse of a rat anti-mouse 0X40 antibody clone OX-86, dosed Anti-tumor activity (as measured by tumor growth inhibition over time) of 25 pg/mouse of a rat anti-mouse 0X40 antibody clone OX-86, dosed via intraperitoneal injection twice per week for 6 doses total, 10 pg or 25 pg/mouse of TLR4 agonist CRX- 601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model.

(*p-values < 0.05)

Figure 23 is a graph showing survival curves of mice treated with 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, 10 pg or 25 pg of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (*p-values < 0.05)

Figure 24 is a graph showing anti-tumor activity (as measured by tumor growth inhibition over time) of 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (*p-values <0.05)

Figure 25 shows survival curves of mice treated with 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (*p-values < 0.05)

Figures 26 A-C are graphs showing increase of leukocytes and immune-activation in mice treated with 10 pg of TLR4 agonist CRX-601, 25 pg of a rat anti-mouse 0X40 receptor antibody (clone OX-86), and the combination of both in a CT-26 syngeneic mouse model of colon cancer measured at 8 days post-dosing.

Figures 27 A-B are graphs showing increases of immune-activating cytokines TNF alpha (A) and IL-l2p70 (B) in mice treated with 10 pg of TLR4 agonist CRX-601 , a rat anti mouse OX40R receptor antibody (clone OX-86), and the combination of both in a CT-26 syngeneic mouse model of colon cancer measured at 1 and 8 days post dosing.

Figure 28 is a graph showing anti-tumor activity (as measured by tumor growth inhibition over time) of 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (0.5% Glycerol/4%

Dextrose vehicle used for CRX-601). (*p-values <0.05)

Figure 29 is a graph showing anti-tumor activity (as measured by tumor growth inhibition over time) of 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intratumoral lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (0.5% Glycerol/4%

Dextrose vehicle used for CRX-601). (*p-values <0.05)

Figure 30 is a graph showing survival curves of mice treated with 25 pg/mouse of a rat anti-mouse 0X40 antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (0.5% Glycerol/4% Dextrose vehicle used for CRX-601) (*p-values < 0.05)

Figure 31 is a graph showing survival curves of mice treated with 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intratumoral lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (0.5% Glycerol/4% Dextrose vehicle used for CRX-601) Cp- values < 0.05)

Figure 32 is a graph showing CT-26 tumor re-challenge of tumor- free mice in study 6.

68 days post first dose, tumor-free mice were re-challenged with CT-26 tumor cells. Naive control mice were also included. While tumors grew as expected in the control naive mice, tumors were rejected and no tumors grew in the treatment groups.

Figure 33 is a graph showing anti-tumor activity (as measured by tumor growth inhibition over time) of 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (0.5% Glycerol/4%

Dextrose vehicle used for CRX-601 intravenous dosing.) (*p-values <0.05)

Figure 34 is a graph showing anti-tumor activity (as measured by tumor growth inhibition over time) of 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intratumoral lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (DOPC/CHOL Liposome formulation used for CRX-601 intratumoral dosing) (*p- values <0.05).

Figure 35 is a graph showing survival curves of mice treated with 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (0.5% Glycerol/4% Dextrose vehicle used for CRX-601 intravenous dosing) (*p-values < 0.05).

Figure 36 is a graph showing survival curves of mice treated with 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed via intraperitoneal injection twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. (0.5% Glycerol/4% Dextrose vehicle used for CRX-601 intravenous dosing) (*p-values < 0.05).

Figure 37 is a graph showing CT-26 tumor re-challenge of tumor-free mice in study 7.

80 days post-first dose, tumor-free mice were re-challenged with CT-26 tumor cells in the number of mice noted. Naive control mice were also included. While tumors grew as expected in the control naive mice, tumors were rejected and no tumors grew in the treatment groups. Figure 38 is a graph showing tumor growth of individual mice of Group 7: CRX-601 25 pg/mouse (in 0.5% glycerol/4% dextrose) dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total.

Figure 39 is a graph showing tumor growth of individual mice of Group 8: CRX-601 25 pg/mouse (in 0.5% glycerol/4% dextrose) dosed intratumoral once per week in the left flank tumor for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total.

Figure 40 is a graph showing tumor growth of individual mice of Group 12: CRX-601 25 pg/mouse (in DOPC/CHOL Liposome) dosed intratumoral once per week in the left flank tumor for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Figure 41A-4D are graphs showing survival curves for all treatment groups in Study 8. Mice remaining on study by day 60 were completely tumor-free.

Figure 42A-C are graphs showing upregulation of 0X40 expression induced by CRX601 treatment with a range of concentrations (0.01 - 1000 ng/ml) on human CD4+ T cells (A), dendritic cells (B), and monocytes (C) at 24 hours in in vitro cell culture.

Figure 43 is a graph showing CT-26 tumor growth in Balb/c mice treated with CRX-601 (TLR4 agonist) and/or 0X86 (mouse surrogate for 0X40) agonist.

Figure 44 is a graph showing survival of Balb/c mice implanted with CT-26 tumors and treated with CRX-601 (TLR4 agonist) and/or 0X86 agonist.

Figure 45 is a graph showing Median Receptor Occupancy Profiles for Varying Doses of ANTIBODY 106-222.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and Combinations Improved function of the immune system is a goal of immunotherapy for cancer. While not being bound by theory, it is thought that for the immune system to be activated and effectively cause regression or eliminate tumors, there must be efficient cross-talk among the various compartments of the immune system as well as at the tumor bed. The tumoricidal effect is dependent on one or more steps, e.g., the uptake of antigen by immature dendritic cells and presentation of processed antigen via MHC I and II by mature dendritic cells to naive CD8 (cytotoxic) and CD4 (helper) lymphocytes, respectively, in the draining lymph nodes. Naive T cells express molecules, such as CTLA-4 and CD28, that engage with co-stimulatory molecules of the B7 family on antigen presenting cells (APCs) such as dendritic cells. In order to keep T cells in check during immune surveillance, B7 on APCs preferentially binds to CTLA-4, an inhibitory molecule on T lymphocytes. However, upon engagement of the T cell receptor (TCR) with MHC Class I or II receptors via cognate peptide presentation on APCs, the co stimulatory molecule disengages from CTLA-4 and instead binds to the lower affinity stimulatory molecule CD28, causing T cell activation and proliferation. This expanded population of primed T lymphocytes retains memory of the antigen that was presented to them as they traffic to distant tumor sites. Upon encountering a tumor cell bearing the cognate antigen, they eliminate the tumor via cytolytic mediators such as granzyme B and perforins. This apparently simplistic sequence of events is highly dependent on several cytokines, co-stimulatory molecules and check point modulators to activate and differentiate these primed T lymphocytes to a memory pool of cells that can eliminate the tumor.

Thus, an emerging immunotherapeutic strategy is to target T cell co-stimulatory molecules, e.g., 0X40. 0X40 (e.g., hOX40 or hOX40R) is a tumor necrosis factor receptor family member that is expressed, among other cells, on activated CD4 and CD8 T cells. One of its functions is in the differentiation and long-term survival of these cells. The ligand for 0X40 (OX40L) is expressed by activated antigen-presenting cells. In one embodiment, the ABPs and antibodies of the invention modulate 0X40 and promote growth and/or differentiation of T cells and increase long-term memory T-cell populations, e.g., in overlapping mechanisms as those of OX40L, by“engaging” 0X40. Thus, in another embodiment, the ABPs and antibodies of the invention bind and engage 0X40. In yet another embodiment, the ABPs and antibodies of the invention modulate 0X40. In a further embodiment, the ABPs and antibodies of the invention modulate 0X40 by mimicking OX40L. In another embodiment, the ABPs and antibodies of the invention are agonist antibodies. In another embodiment, the ABPs and antibodies of the invention modulate 0X40 and cause proliferation of T cells. In a further embodiment, the ABPs and antibodies of the invention modulate 0X40 and improve, augment, enhance, or increase proliferation of CD4 T cells. In another embodiment, the ABPs and antibodies of the invention improve, augment, enhance, or increase proliferation of CD8 T cells. In a further embodiment, the ABPs and antibodies of the invention improve, augment, enhance, or increase proliferation of both CD4 and CD8 T cells. In another embodiment, the ABPs and antibodies of the invention enhance T cell function, e.g., of CD4 or CD8 T cells, or both CD4 and CD8 T cells. In a further embodiment, the ABPs and antibodies of the invention enhance effector T cell function. In another embodiment, the ABPs and antibodies of the invention improve, augment, enhance, or increase long term survival of CD8 T cells. In further embodiments, any of the preceding effects occur in a tumor microenvironment.

Of equal importance is the blockade of a potentially robust immunosuppressive response at the tumor site by mediators produced both by T regulatory cells (Tregs) as well as the tumor itself (e.g., Transforming Growth Factor (TGF-b) and interleukin- 10 (IL-10)). An important immune pathogenesis of cancer can be the involvement of Tregs that are found in tumor beds and sites of inflammation. In general, Treg cells occur naturally in circulation and help the immune system to return to a quiet, although vigilant state, after encountering and eliminating external pathogens. Treg cells help to maintain tolerance to self antigens and are naturally suppressive in function, and they phenotypically characterized as CD4+, CD25+, FOXP3+ cells. In order to break tolerance to effectively treat certain cancers, one mode of therapy is to eliminate Tregs preferentially at tumor sites. Targeting and eliminating Tregs leading to an anti-tumor response has been more successful in tumors that are immunogenic compared to those that are poorly

immunogenic. Many tumors secrete cytokines, e.g., TGF-b that may hamper the immune response by causing precursor CD4+25+ cells to acquire the FOXP3+ phenotype and function as Tregs. “Modulate” as used herein, for example, with regard to a receptor or other target means to change any natural or existing function of the receptor, for example it means affecting binding of natural or artificial ligands to the receptor or target; it includes initiating any partial or full conformational changes or signaling through the receptor or target, and also includes preventing partial or full binding of the receptor or target with its natural or artificial ligands. Also included in the case of membrane bound receptors or targets are any changes in the way the receptor or target interacts with other proteins or molecules in the membrane or change in any localization (or co-localization with other molecules) within membrane compartments as compared to its natural or unchanged state.

Modulators are, therefore, compounds or ligands or molecules that modulate a target or receptor.“Modulate” includes agonizing, e.g., signaling, as well as antagonizing, or blocking signaling or interactions with a ligand or compound or molecule that happen in the unchanged or unmodulated state. Thus, modulators may be agonists or antagonists. Further, one of skill in the art will recognize that not all modulators will have absolute selectivity for one target or receptor, but are still considered a modulator for that target or receptor; for example, a TLR4 modulator may also engage another TLR, but still be considered a TLR4 modulator. Other modulators are known to have multiple

specificities, such as TLR7/8 modulators that modulate both TLR7 and TLR8. Molecules with such known double or multiple specificities are considered a modulator of each of its target; that is, a TLR7/8 modulator is a TLR7 modulator as used herein and likewise a TLR7/8 modulator is a TLR8 modulator as used herein.

As used herein the term“agonist” refers to an antigen binding protein, including but not limited to an antibody, which upon contact with a co-signaling receptor causes one or more of the following (1) stimulates or activates the receptor, (2) enhances, increases or promotes, induces or prolongs an activity, function or presence of the receptor (3) mimics one or more functions of a natural ligand or molecule that interacts with a target or receptor and includes initiating one or more signaling events through the receptor, mimicking one or more functions of a natural ligand, or initiating one or more partial or full conformational changes that are seen in known functioning or signaling through the receptor and/or (4) enhances, increases, promotes or induces the expression of the receptor. Agonist activity can be measured in vitro by various assays know in the art such as, but not limited to, measurement of cell signaling, cell proliferation, immune cell activation markers, cytokine production. Agonist activity can also be measured in vivo by various assays that measure surrogate end points such as, but not limited to the measurement of T cell proliferation or cytokine production. Thus, in one embodiment, the 0X40 binding protein or antibody inhibits the suppressive effect of Treg cells on other T cells, e.g., within the tumor environment.

Accumulating evidence suggests that the ratio of Tregs to T effector cells in the tumor correlates with anti-tumor response. Therefore, in one embodiment, the 0X40 binding proteins or antibodies of the invention modulate 0X40 to augment T effector number and function and inhibit Treg function.

Enhancing, augmenting, improving, increasing, and otherwise changing the anti-tumor effect of 0X40 is an object of the invention. Described herein are combinations of an 0X40 binding protein such as an antibody and another compound, such as a TLR modulator described herein.

Thus, as used herein the term“combination of the invention” refers to a combination comprising an 0X40 binding protein, such as an antibody, and a TLR4 modulator, such as an AGP, each of which may be administered separately or simultaneously as described herein.

As used herein, the terms“cancer”,“neoplasm”, and“tumor”, are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation or undergone cellular changes that result in aberrant or unregulated growth or hyperproliferation. Such changes or malignant transformations usually make such cells pathological to the host organism, thus precancers or pre-cancerous cells that are or could become pathological and require or could benefit from intervention are also intended to be included. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well- established techniques, such as histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and/or which is detectable because of the expression of one or more cancer- specific antigens in a sample obtainable from a patient. In other words, the terms herein include cells, neoplasms, cancers, and tumors of any stage, including what a clinician refers to as precancer, tumors, in situ growths, as well as late stage metastatic growths. Tumors may be hematopoietic tumor, for example, tumors of blood cells or the like, meaning liquid tumors. Specific examples of clinical conditions based on such a tumor include leukemia such as chronic myelocytic leukemia or acute myelocytic leukemia; myeloma such as multiple myeloma; lymphoma and the like.

As used herein, the term,“agent”, means a substance that produces a desired effect in a tissue, system, animal, mammal, human, or other subject. Accordingly, the term,“anti neoplastic agent”, means a substance producing an anti-neoplastic effect in a tissue, system, animal, mammal, human, or other subject. The term,“agent”, may be a single compound or a combination or composition of two or more compounds.

By the term“treating” and derivatives thereof as used herein, is meant therapeutic therapy. In reference to a particular condition, treating means: (1) to ameliorate the condition or one or more of the biological manifestations of the condition (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the condition or (b) one or more of the biological manifestations of the condition; (3) to alleviate one or more of the symptoms, effects or side effects associated with the condition or one or more of the symptoms, effects or side effects associated with the condition or treatment thereof; or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition.

As used herein,“prevention” means the prophylactic administration of a drug, such as an agent, to substantially diminish the likelihood or severity of a condition or biological manifestation thereof, or to delay the onset of such condition or biological manifestation thereof. The skilled artisan will appreciate that“prevention” is not an absolute term. Prophylactic therapy is appropriate, for example, when a subject is considered at high risk for developing cancer, such as when a subject has a strong family history of cancer or when a subject has been exposed to a carcinogen.

As used herein, the term“effective amount” means that amount of a drug or agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term

“therapeutically effective amount” means any amount that, as compared to a

corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. One or both of an 0X40 binding protein and/or a TLR4 modulator can be administered to a subject or used in an effective amount (such as a therapeutically effective amount), e.g., in the methods and uses described herein.

By the term“combination” and grammatical variations thereof, as used herein, means either simultaneous administration or any manner of separate sequential administration of a therapeutically effective amount of Compound A (an OX-40 ABP) and Compound B (a TLR4 agonist) or a pharmaceutically acceptable salt thereof. Furthermore, it does not matter if the compounds are administered in the same dosage form, e.g. , one compound may be administered intravenously and the other compound may be administered intratumorally.

The term“combination kit”, as used herein, means the pharmaceutical composition or compositions that are used to administer Compound A, or a pharmaceutically acceptable salt thereof, and Compound B, or a pharmaceutically acceptable salt thereof, according to the invention. When both compounds are administered simultaneously, the combination kit can contain Compound A, or a pharmaceutically acceptable salt thereof, and

Compound B, or a pharmaceutically acceptable salt thereof, in a single pharmaceutical composition, such as a tablet, or in separate pharmaceutical compositions. When the compounds are not administered simultaneously, the combination kit will contain Compound A, or a pharmaceutically acceptable salt thereof, and Compound B, or a pharmaceutically acceptable salt thereof, in separate pharmaceutical compositions. The combination kit can comprise Compound A, or a pharmaceutically acceptable salt thereof, and Compound B, or a pharmaceutically acceptable salt thereof, in separate pharmaceutical compositions in a single package or in separate pharmaceutical compositions in separate packages.

In one embodiment, the invention provides a combination kit comprising the

components:

Compound A ( e.g ., an OX-40 binding protein, e.g., at a dose of about 8 mg to about 24 mg, at a dose of about 8 mg, or at a dose of about 24 mg), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier; and

Compound B (e.g., a TLR4 agonist, e.g., at a dose of about 50 ng to about 250 ng), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier.

In another embodiment, the combination kit comprises the following components:

Compound A (e.g., an OX-40 binding protein, e.g., at a dose of about 8 mg to about 24 mg, at a dose of about 8 mg, or at a dose of about 24 mg), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier; and

Compound B (e.g., a TLR4 agonist, e.g., at a dose of about 50 ng to about 250 ng), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier, wherein the components are provided in a form which is suitable for sequential, separate and/or simultaneous administration.

In yet another embodiment, the combination kit comprises: a first container comprising Compound A (e.g., an OX-40 binding protein, e.g., at a dose of about 8 mg to about 24 mg, at a dose of about 8 mg, or at a dose of about 24 mg), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier; and a second container comprising Compound B ( e.g ., a TLR4 agonist, e.g., at a dose of about 50 ng to about 250 ng), or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier, and a container means for containing said first and second containers.

The“combination kit” can also be provided by instruction, such as dosage and administration instructions. Such dosage and administration instructions can be of the kind that is provided to a doctor, for example by a drug product label, or they can be of the kind that is provided by a doctor, such as instructions to a patient.

As used herein, the term“Compound A²” means a monoclonal antibody to human OX-40 or the antigen binding portion thereof. Suitably Compound A² means a humanized monoclonal antibody having a heavy chain variable region as set forth in SEQ ID NO:5 and a light chain variable region as set forth in SEQ ID NO: 1 1. Suitably, Compound A² can mean a humanized monoclonal antibody having a heavy chain as set forth in SEQ ID NO:48 and a light chain as set forth in SEQ ID NO:49.

As used herein, the term“Compound B²” means a TLR4 agonist of Formula I or Formula la. Suitably, Compound B² means the TLR4 agonist CRX-601. Suitably, Compound B² means the TLR4 agonist CRX-527.

Suitably, the combinations of this invention are administered within a“specified period”.

The term“specified period” and grammatical variations thereof, as used herein, means the interval of time between the administration of one of Compound A² and Compound B² and the other of Compound A² and Compound B². ETnless otherwise defined, the specified period can include simultaneous administration. ETnless otherwise defined, the specified period refers to administration of Compound A² and Compound B² during a single day.

Suitably, if the compounds are administered within a specified period and not administered simultaneously, they are both administered within about 24 hours of each other - in this case, the specified period will be about 24 hours.; suitably they will both be administered within about 12 hours of each other - in this case, the specified period will be about 12 hours; suitably they will both be administered within about 1 1 hours of each other - in this case, the specified period will be about 11 hours; suitably they will both be administered within about 10 hours of each other - in this case, the specified period will be about 10 hours; suitably they will both be administered within about 9 hours of each other - in this case, the specified period will be about 9 hours; suitably they will both be administered within about 8 hours of each other - in this case, the specified period will be about 8 hours; suitably they will both be administered within about 7 hours of each other - in this case, the specified period will be about 7 hours; suitably they will both be administered within about 6 hours of each other - in this case, the specified period will be about 6 hours; suitably they will both be administered within about 5 hours of each other - in this case, the specified period will be about 5 hours; suitably they will both be administered within about 4 hours of each other - in this case, the specified period will be about 4 hours; suitably they will both be administered within about 3 hours of each other - in this case, the specified period will be about 3 hours; suitably they will be administered within about 2 hours of each other - in this case, the specified period will be about 2 hours..

Suitably, when the combination of the invention is administered for a“specified period”, the compounds will be co-administered for a“duration of time”.

The term“duration of time” and grammatical variations thereof, as used herein means that both compounds of the invention are administered for an indicated number of consecutive days. Unless otherwise defined, the number of consecutive days does not have to commence with the start of treatment or terminate with the end of treatment, it is only required that the number of consecutive days occur at some point during the course of treatment.

Regarding a specified period of administration: suitably, both compounds will be administered within a specified period for at least one day - in this case, the duration of time will be at least one day; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 3 consecutive days - in this case, the duration of time will be at least 3 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 5 consecutive days - in this case, the duration of time will be at least 5 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 7 consecutive days - in this case, the duration of time will be at least 7 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 14 consecutive days - in this case, the duration of time will be at least 14 days; suitably, during the course to treatment, both compounds will be administered within a specified period for at least 30 consecutive days - in this case, the duration of time will be at least 30 days.

In embodiments provided herein, both compounds are administered within a specified period ( e.g ., within one day, e.g., at least an hour between administration of the two compounds) which specified period occurs every three weeks.

Suitably, if the compounds are not administered during a specified period, they are administered sequentially. By the term“sequential administration”, and grammatical derivates thereof, as used herein is meant that one of Compound A² and Compound B² is administered once a day for two or more consecutive days and the other of Compound A² and Compound B² is subsequently administered once a day for two or more consecutive days. Also, contemplated herein is a drug holiday utilized between the sequential administration of one of Compound A² and Compound B² and the other of Compound A² and Compound B². As used herein, a drug holiday is a period of days after the sequential administration of one of Compound A² and Compound B² and before the administration of the other of Compound A² and Compound B² where neither Compound A² nor Compound B² is administered. Suitably the drug holiday will be a period of days selected from: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days and 14 days.

Regarding sequential administration: suitably, one of Compound A² and Compound B² is administered for from 1 to 30 consecutive days, followed by an optional drug holiday, followed by administration of the other of Compound A² and Compound B² for from 1 to 30 consecutive days. Suitably, one of Compound A² and Compound B² is administered for from 1 to 21 consecutive days, followed by an optional drug holiday, followed by administration of the other of Compound A² and Compound B² for from 1 to 21 consecutive days. Suitably, one of Compound A² and Compound B² is administered for from 1 to 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of the other of Compound A² and Compound B² for from 1 to 14 consecutive days. Suitably, one of Compound A² and Compound B² is administered for from 1 to 7 consecutive days, followed by a drug holiday of from 1 to 10 days, followed by administration of the other of Compound A² and Compound B² for from 1 to 7 consecutive days.

Suitably, Compound B² will be administered first in the sequence, followed by an optional drug holiday, followed by administration of Compound A². Suitably,

Compound B² is administered for from 3 to 21 consecutive days, followed by an optional drug holiday, followed by administration of Compound A² for from 3 to 21 consecutive days. Suitably, Compound B² is administered for from 3 to 21 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of

Compound A² for from 3 to 21 consecutive days. Suitably, Compound B² is

administered for from 3 to 21 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of Compound A² for from 3 to 21 consecutive days. Suitably, Compound B² is administered for 21 consecutive days, followed by an optional drug holiday, followed by administration of Compound A² for 14 consecutive days. Suitably, Compound B² is administered for 14 consecutive days, followed by a drug holiday of from 1 to 14 days, followed by administration of Compound A² for 14 consecutive days. Suitably, Compound B² is administered for 7 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of

Compound A² for 7 consecutive days. Suitably, Compound B² is administered for 3 consecutive days, followed by a drug holiday of from 3 to 14 days, followed by administration of Compound A² for 7 consecutive days. Suitably, Compound B² is administered for 3 consecutive days, followed by a drug holiday of from 3 to 10 days, followed by administration of Compound A² for 3 consecutive days.

It is understood that a“specified period” of administration and a“sequential”

administration can be followed by repeat dosing or can be followed by an alternate dosing protocol, and a drug holiday may precede the repeat dosing or alternate dosing protocol. The methods of the present invention may also be employed with other therapeutic methods of cancer treatment.

While it is possible that, for use in therapy, therapeutically effective amounts of the combinations of the present invention may be administered as the raw chemical, it is preferable to present the combinations as a pharmaceutical composition or

compositions. Accordingly, the invention further provides pharmaceutical compositions, which include Compound A² and/or Compound B², and one or more pharmaceutically acceptable carriers. The combinations of the present invention are as described above. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of pharmaceutical formulation, and not deleterious to the recipient thereof. In accordance with another aspect of the invention there is also provided a process for the preparation of a pharmaceutical formulation including admixing Compound A² and/or Compound B² with one or more

pharmaceutically acceptable carriers. As indicated above, such elements of the pharmaceutical combination utilized may be presented in separate pharmaceutical compositions or formulated together in one pharmaceutical formulation.

Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. As is known to those skilled in the art, the amount of active ingredient per dose will depend on the condition being treated, the route of administration and the age, weight and condition of the

patient. Preferred unit dosage formulations are those containing a daily dose or sub-dose, or an appropriate fraction thereof, of an active ingredient. Furthermore, such

pharmaceutical formulations may be prepared by any of the methods well known in the pharmacy art.

Compound A² and Compound B² may be administered by any appropriate

route. Suitable routes include oral, rectal, nasal, topical (including buccal and

sublingual), intratumorally, vaginal, and parenteral (including subcutaneous,

intramuscular, intravenous, intradermal, intrathecal, and epidural). It will be appreciated that the preferred route may vary with, for example, the condition of the recipient of the combination and the cancer to be treated. It will also be appreciated that each of the agents administered may be administered by the same or different routes and that Compound A² and Compound B² may be compounded together in a pharmaceutical composition/formulation.

The administration of a therapeutically effective amount of the combinations of the invention (or therapeutically effective amounts of each of the components of the combination) are advantageous over the individual component compounds in that the combinations provide one or more of the following improved properties when compared to the individual administration of a therapeutically effective amount of a component compound: i) a greater anti-cancer effect than the most active single agent; ii) synergistic or highly synergistic anti-cancer activity; iii) a dosing protocol that provides enhanced anti-cancer activity with reduced side effect profile; iv) a reduction in the toxic effect profile, v) an increase in the therapeutic window; or vi) an increase in the bioavailability of one or both of the component compounds.

The invention further provides pharmaceutical compositions, which include one or more of the components herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The combination of the invention may comprise two pharmaceutical compositions, one comprising an ABP or antibody of the invention, and the other comprising a TLR4 modulator, each of which may have the same or different carriers, diluents or excipients. The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of pharmaceutical formulation, and not deleterious to the recipient thereof. In one embodiment of the invention, the formulation may be aqueous or liposomal. In one embodiment, the liposomal formulation may be a DOPC/CHOL Liposome formulation.

The components of the combination of the invention, and pharmaceutical compositions comprising such components may be administered in any order, and in different routes; the components and pharmaceutical compositions comprising the same may be administered simultaneously.

In accordance with another aspect of the invention there is also provided a process for the preparation of a pharmaceutical composition including admixing a component of the combination of the invention and one or more pharmaceutically acceptable carriers, diluents or excipients.

The components of the invention may be administered by any appropriate route. For some components, suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal, and epidural). The preferred route may vary with, for example, the condition of the recipient of the combination and the cancer to be treated. Each of the agents administered may be administered by the same or different routes, and the components may be compounded together or in separate pharmaceutical compositions.

In one embodiment, one or more components of a combination of the invention are administered intravenously. In another embodiment, one or more components of a combination of the invention are administered intratumorally. In another embodiment, one or more components of a combination of the invention are administered systemically, e.g., intravenously, and one or more other components of a combination of the invention are administered intratumorally. In another embodiment, all of the components of a combination of the invention are administered systemically, e.g., intravenously. In an alternative embodiment, all of the components of the combination of the invention are administered intratumorally. In any of the embodiments, e.g., in this paragraph, the components of the invention are administered as one or more pharmaceutical

compositions.

In some embodiments, the two compounds are administered intravenously. In some embodiments, the 0X40 ABP is administered by intravenous infusion. In some embodiments, the TLR4 agonist is administered by intravenous injection. In some embodiments, the 0X40 ABP is administered by intravenous infusion and the TLR4 agonist is administered by intravenous injection. In some embodiments, the TLR4 agonist is administered by intratumoral injection.

Antigen Binding Proteins that bind 0X40

“Antigen Binding Protein (ABP)” means a protein that binds an antigen, and includes antibodies or engineered molecules that function in similar ways to antibodies. Such alternative antibody formats include triabody, tetrabody, miniantibody, and a minibody, Also included are alternative scaffolds in which the one or more CDRs of any molecules in accordance with the disclosure can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer (see, e.g., U.S. Patent Application Publication Nos. 2005/0053973, 2005/0089932, 2005/0164301) or an EGF domain. An ABP also includes antigen binding fragments of such antibodies or other molecules. Further, an ABP may comprise the VH regions of the invention formatted into a full length antibody, a (Fab')2 fragment, a Fab fragment, a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs, etc.), when paired with an appropriate light chain. The ABP may comprise an antibody that is an IgGl, IgG2, IgG3, or IgG4; or IgM; IgA, IgE or IgD or a modified variant thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. The ABP may also be a chimeric antibody of the type described in WO86/01533, which comprises an antigen binding region and a non-immunoglobulin region.

Thus, herein an ABP of the invention or an 0X40 binding protein is one that binds 0X40, and in some embodiments, does one or more of the following: modulate signaling through 0X40, modulates the function of 0X40, agonize 0X40 signaling, stimulate 0X40 function, or co-stimulate 0X40 signaling. Example 1 of FT.S. Patent 9,006,399 discloses an 0X40 binding assay. One of skill in the art would readily recognize a variety of other well-known assays to establish such functions.

The term“antibody” as used herein refers to molecules with an antigen binding domain, and optionally an immunoglobulin-like domain or fragment thereof and includes monoclonal (for example IgG, IgM, IgA, IgD or IgE and modified variants thereof), recombinant, polyclonal, chimeric, humanized, biparatopic, bispecific and

heteroconjugate antibodies, or a closed conformation multispecific antibody. An “antibody” includes xenogeneic, allogeneic, syngeneic, or other modified forms thereof. An antibody may be isolated or purified. An antibody may also be recombinant, i.e., produced by recombinant means; for example, an antibody that is 90% identical to a reference antibody may be generated by mutagenesis of certain residues using recombinant molecular biology techniques known in the art. Thus, the antibodies of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody or formatted into a full length recombinant antibody, a (Fab')2 fragment, a Fab fragment, a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra- bodies, Tandabs etc.), when paired with an appropriate light chain. The antibody may be an IgGl, IgG2, IgG3, or IgG4 or a modified variant thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. The antibody may also be a chimeric antibody of the type described in WO86/01533 which comprises an antigen binding region and a non- immunoglobulin region.

One of skill in the art will recognize that the ABPs, such as antibodies, of the invention bind an epitope of 0X40. The epitope of an ABP is the region of its antigen to which the ABP binds. Two ABPs bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a lx, 5x, lOx, 20x or lOOx excess of one antibody inhibits binding of the other by at least 50%, 75%, 90% or even 99% as measured in a competitive binding assay compared to a control lacking the competing antibody (see, e.g., Junghans, et ah, Cancer Res. 50: 1495, 1990.

Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. In addition, the same epitope may include“overlapping epitopes”, e.g., if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

The strength of binding may be important in dosing and administration of an ABP or antibody of the invention. In one embodiment, the ABP of the invention binds to 0X40, preferably human 0X40, with high affinity. For example, when measured by surface plasmon resonance (for example with a BIACORE instrument) the ABP binds to 0X40, preferably human 0X40, with KD (equilibrium affinity or equilibrium dissociation constant) of 1 to 1000 nM, or 500 nM or less, or an affinity of 200 nM or less, or an affinity of 100 nM or less, or an affinity of 50 nM or less, or an affinity of 500 pM or less, or an affinity of 400 pM or less, or 300 pM or less. In a further aspect the ABP binds to 0X40, preferably human 0X40, when measured by surface plasmon resonance of between about 50 nM and about 200 nM, or between about 50 nM and about 150 nM. In one aspect of the present invention the ABP binds 0X40, preferably human 0X40, with an affinity of less than 100 nM.

In a further embodiment, binding is measured by BIACORE. Affinity is the strength of binding of one molecule, e.g., an ABP of the invention, to another, e.g., its target antigen, at a single binding site. The binding affinity of an ABP to its target may be determined by equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE SPR data analyzed with a kinetic model). For example, the BIACORE methods known in the art may be used to measure binding affinity and measure kinetic binding parameters.

Avidity is the sum total of the strength of binding of two molecules to one another at multiple sites, e.g., taking into account the valency of the interaction.

In an aspect, the equilibrium dissociation constant (K_D) of the ABP of the invention and 0X40, preferably human 0X40, interaction is 100 nM or less, 10 nM or less, 2 nM or less or 1 nM or less. Alternatively, the K_D may be between 5 and 10 nM; or between 1 and 2 nM. The K_D may be between 1 pM and 500 pM; or between 500 pM and 1 nM. A skilled person will appreciate that the smaller the K_D numerical value, the stronger the binding. The reciprocal of K_D (i.e., 1/Kn) is the equilibrium association constant (K_A) having units M ¹. A skilled person will appreciate that the larger the K_A numerical value, the stronger the binding.

The dissociation rate constant (ka) or“off-rate” describes the stability of the complex of an ABP on one hand and 0X40, preferably human 0X40 on the other hand, i.e., the fraction of complexes that decay per second. For example, a ka of 0.01 s ¹ equates to 1% of the complexes decaying per second. In one embodiment, the dissociation rate constant (ka) is lxlO^-3 s^-1 or less, lxlO ⁴ s^-1 or less, lxlO ⁵ s^-1 or less, or lxlO ⁶ s^-1 or less. The ka may be between lxlO ⁵ s ¹ and lxlO ⁴ s ¹; or between lxlO ⁴ s ¹ and lxlO ³ s ¹.

Competition between an ABP (e.g., 0X40 binding protein of the invention), and a reference antibody, e.g., for binding 0X40, an epitope of 0X40, or a fragment of the 0X40, may be determined by competition ELISA, FMAT (fluorometric microvolume assay technology) or BIACORE. In one aspect, the competition assay is carried out by BIACORE. There are several possible reasons for this competition: the two proteins may bind to the same or overlapping epitopes, there may be steric inhibition of binding, or binding of the first protein may induce a conformational change in the antigen that prevents or reduces binding of the second protein.

“Binding fragments” as used herein means a portion or fragment of the ABPs of the invention that include the antigen-binding site and are capable of binding 0X40 as defined herein, e.g., but not limited to capable of binding to the same epitope of the parent or full length antibody.

Functional fragments of the ABPs of the invention are contemplated herein.

Thus,“binding fragments” and“functional fragments” may be Fab and F(ab')2 fragments that lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl, et al., J. Nuc. Med. 24:316-325 (1983)). Also included are Fv fragments (Hochman, et al., Biochemistry 12: 1130-1135 (1973); Sharon, et al, Biochemistry 15: 1591-1594 (1976)). These various fragments are produced using conventional techniques such as protease cleavage or chemical cleavage (see, e.g., Rousseaux, et al., Meth. Enzymol., 121 :663-69 (1986)).

“Functional fragments”, as used herein, means a portion or fragment of the ABPs of the invention that include the antigen-binding site and are capable of binding the same target as the parent ABP, e.g., but not limited to, binding the same epitope, and that also retain one or more modulating or other functions described herein or known in the art.

As the ABPs of the present invention may comprise heavy chain variable regions and light chain variable regions of the invention which may be formatted into the structure of a natural antibody, a functional fragment is one that retains binding or one or more functions of the full length ABP as described herein. A binding fragment of an ABP of the invention may therefore comprise the VL or VH regions, a (Fab')2 fragment, a Fab fragment, a fragment of a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs etc.), when paired with an appropriate light chain.

The term,“CDR”, as used herein, refers to the complementarity determining region amino acid sequences of an antigen binding protein (ABP). These are the hypervariable regions of immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin.

It will be apparent to those skilled in the art that there are various numbering conventions for CDR sequences; Chothia (Chothia el al. (1989) Nature 342: 877-883), Rabat (Rabat et al. , Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath) and Contact (University College London). The minimum overlapping region using at least two of the Rabat, Chothia, AbM and contact methods can be determined to provide the“minimum binding unit”. The minimum binding unit may be a subportion of a CDR. The structure and protein folding of the antibody may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person. It is noted that some of the CDR definitions may vary depending on the individual publication used.

Unless otherwise stated and/or in absence of a specifically identified sequence, references herein to“CDR”,“CDRL1”,“CDRL2”,“CDRL3”,“CDRH1”,“CDRH2”,“CDRH3” refer to amino acid sequences numbered according to any of the known conventions; alternatively, the CDRs are referred to as“CDR1 ,”“CDR2,”“CDR3” of the variable light chain and“CDR1,”“CDR2,” and“CDR3” of the variable heavy chain. In some embodiments, the numbering convention is the Rabat convention.

The term,“CDR variant”, as used herein, refers to a CDR that has been modified by at least one, for example 1 , 2 or 3, amino acid substitution(s), deletion(s) or addition(s), wherein the modified antigen binding protein comprising the CDR variant substantially retains the biological characteristics of the antigen binding protein pre-modification. It will be appreciated that each CDR that can be modified may be modified alone or in combination with another CDR. In one aspect, the modification is a substitution, particularly a conservative substitution, for example as shown in Table 1. Table 1

For example, in a variant CDR, the amino acid residues of the minimum binding unit may remain the same, but the flanking residues that comprise the CDR as part of the Rabat or Chothia definition(s) may be substituted with a conservative amino acid residue.

Such antigen binding proteins comprising modified CDRs or minimum binding units as described above may be referred to herein as“functional CDR variants” or“functional binding unit variants”.

The antigen binding protein may be of any species, or modified to be suitable to administer to a cross species. For example, the CDRs from a mouse antibody may be humanized for administration to humans. In any embodiment, the antigen binding protein is optionally a humanized antibody.

A“humanized antibody” refers to a type of engineered antibody having its CDRs derived from a non-human donor immunoglobulin, the remaining immunoglobulin-derived parts of the molecule being derived from one (or more) human immunoglobulin(s). In addition, framework support residues may be altered to preserve binding affinity (see, e.g., Queen, et ah, Proc. Natl Acad Sci USA, 86: 10029-10032 (1989), Hodgson, et ah, Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody may be one selected from a conventional database, e.g., the RABAT database, Los Alamos database, and Swiss Protein database, by homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by a homology to the framework regions of the donor antibody (on an amino acid basis) may be suitable to provide a heavy chain constant region and/or a heavy chain variable framework region for insertion of the donor CDRs. A suitable acceptor antibody capable of donating light chain constant or variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains are not required to originate from the same acceptor antibody. The prior art describes several ways of producing such humanised antibodies - see, for example EP-A-0239400 and EP-A-054951.

In yet a further embodiment, the humanized antibody has a human antibody constant region that is an IgG. In another embodiment, the IgG is a sequence as disclosed in any of the above references or patent publications. In a particular embodiment, the humanized antibody has a human antibody constant region that is an IgGl .

For nucleotide and amino acid sequences, the term“identical" or“identity” indicates the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (/.<?., % identity = number of identical positions/total number of positions multiplied by 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

Percent identity between a query nucleic acid sequence and a subject nucleic acid sequence is the“Identities” value, expressed as a percentage, which is calculated by the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair-wise BLASTN alignment is performed. Such pair-wise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute’s website with the filter for low complexity regions turned off. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein. Percent identity between a query amino acid sequence and a subject amino acid sequence is the“Identities” value, expressed as a percentage, which is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed. Such pair-wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute’s website with the filter for low complexity regions turned off. Importantly, a query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein.

In any embodiment of the invention herein, the ABP may have any one or all CDRs, VH, VL, with 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90 percent identity to the sequence shown or referenced, e.g., as defined by a SEQ ID NO disclosed herein.

ABPs that bind human 0X40 (also known as 0X40 receptor) are provided herein (e.g., an 0X40 ABP, or 0X40 binding protein, or 0X40 antigen binding protein, or an anti human 0X40 receptor (hOX40R) antibody, sometimes referred to herein as an“anti- 0X40 ABP” or“an anti- 0X40 antibody” and/or other variations of the same). These antigen binding proteins are useful in the treatment or prevention of acute or chronic diseases or conditions whose pathology involves 0X40 signaling. In one aspect, an antigen binding protein, such as an isolated human antibody or functional fragment of such protein or antibody, that binds to human OX40R and is effective as a cancer treatment or treatment against disease is described, for example in combination with another compound such as a TLR4 modulator or TLR4 agonist. Any of the antigen binding proteins such as anti-OX40 antibodies disclosed herein may be used as a medicament. Any one or more of the antigen binding proteins such as anti-OX40 antibodies may be used in the methods or compositions to treat cancer, e.g., those disclosed herein.

The isolated antigen binding proteins, such as antibodies, as described herein bind to 0X40, and may bind to 0X40 encoded from the following genes: NCBI Accession Number NP_0033l7, Genpept Accession Number P23510, or genes having 90 percent homology or 90 percent identity thereto. The isolated antigen binding proteins provided herein may further bind to the 0X40 receptor having one of the following GenBank Accession Numbers: AAB39944, CAE 11757, or AAI05071.

Antigen binding proteins, such as antibodies, that bind and/or modulate 0X40 receptor are known in the art. Exemplary ABPs and antibodies of the invention are disclosed, for example in International Publication No. WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, and WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. (To the extent any definitions conflict, this instant application controls). In one embodiment, 0X40 antibodies of the present invention are disclosed in ETS Patent No. 9,163,085.

In one embodiment, the 0X40 binding protein is one disclosed in WO2012/027328 (PCT/US2011/048752), international filing date 23 August 2011. In another

embodiment, the antigen binding protein comprises the CDRs of an antibody disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011 , or CDRs with 90% identity to the disclosed CDR sequences. In a further embodiment the antigen binding protein comprises a VH, a VL, or both of an antibody disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011 , or a VH or a VL with 90% identity to the disclosed VH or VL sequences. In some

embodiments, the 0X40 binding protein is an agonist.

In another embodiment, the 0X40 binding protein is disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012. In another embodiment, the antigen binding protein comprises the CDRs of an antibody disclosed in

WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, or CDRs with 90% identity to the disclosed CDR sequences. In a further embodiment, the antigen binding protein comprises a VH, a VL, or both of an antibody disclosed in

WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012, or a VH or a VL with 90% identity to the disclosed VH or VL sequences.

In another embodiment, the 0X40 binding protein of the invention comprises one or more of the CDRs or VH or VL sequences, or sequences with 90% identity thereto, shown in the Figures herein. In one embodiment, the ABP of the invention comprises the CDRs of the 106-222 antibody, e.g., of Figures 6-7 herein, e.g. , CDRH1, CDRH2, and CDRH3 having the amino acid sequence as set forth in SEQ ID NOs 1 , 2, and 3, as disclosed in Figure 6, and e.g.,CDRLl, CDRL2, and CDRL3 having the sequences as set forth in SEQ ID NOs 7, 8, and 9 respectively as disclosed in Figure 7. In one embodiment, the ABP of the invention comprises the CDRs of the 106-222, Hul06 or Hul 06-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In a further embodiment, the 0X40 binding protein of the invention comprises the VH and VL regions of the 106-222 antibody as shown in Figures 6-7 herein, e.g., a VH having an amino acid sequence as set forth in SEQ ID NO:4 and a VL as in Figure 7 having an amino acid sequence as set forth in SEQ ID NO: 10. In another embodiment, the ABP of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO:5 in Figure 6 herein, and a VL having an amino acid sequence as set forth in SEQ ID NO: 11 in Figure 7 herein. In a further embodiment, the 0X40 binding protein of the invention comprises the VH and VL regions of the Hul 06-222 antibody or the 106-222 antibody or the Hul06 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In a further embodiment, the 0X40 binding protein of the invention is 106-222, Hul06-222 or Hul06, e.g., as disclosed in

WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In a further embodiment, the ABP of the invention comprises CDRs or VH or VL or antibody sequences with 90% identity to the sequences in this paragraph.

In another embodiment, the 0X40 binding protein of the invention comprises the CDRs of the 1 19-122 antibody, e.g., of Figures 10-11 herein, e.g., CDRH1, CDRH2, and CDRH3 having the amino acid sequence as set forth in SEQ ID NOs 13, 14, and 15 respectively. In another embodiment, the 0X40 binding protein of the invention comprises the CDRs of the 119-122 or Hul 19 or Hul 19-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In a further embodiment, the 0X40 binding protein of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 16 in Figure 10 herein, and a VL having the amino acid sequence as set forth in SEQ ID NO: 22 as shown in Figure 11 herein. In another embodiment, the 0X40 binding protein of the invention comprises a VH having an amino acid sequence as set forth in SEQ ID NO: 17 and a VL having the amino acid sequence as set forth in SEQ ID NO: 23. In a further embodiment, the 0X40 binding protein of the invention comprises the VH and VL regions of the 119-122 or Hul 19 or Hul 19-222 antibody as disclosed in WO2012/027328 (PCT/US201 1/048752), international filing date 23 August 2011. In a further embodiment, the ABP of the invention is 119-222 or Hul 19 or Hul 19-222 antibody, e.g., as disclosed in

In another embodiment, the 0X40 binding protein of the invention comprises the CDRs of the 1 19-43-1 antibody, e.g., as shown in Figures 14-15 herein. In another

embodiment, the 0X40 binding protein or antibody of the invention comprises the CDRs of the 1 19-43-1 antibody as disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012. In a further embodiment, the 0X40 binding protein of the invention comprises one of the VH and one of the VL regions of the 1 19-43-1 antibody as shown in Figures 14-17. In a further embodiment, the 0X40 binding protein of the invention comprises the VH and VL regions of the 119-43-1 antibody as disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012. In a further embodiment, the 0X40 binding protein of the invention is 119-43-1 or 119-43-1 chimeric as disclosed in Figures 14-17 herein. In a further embodiment, the ABP of the invention as disclosed in WO2013/028231 (PCT/US2012/024570), international filing date 9 Feb. 2012. In further embodiments, any one of the ABPs described in this paragraph are humanized. In further embodiments, any one of the any one of the ABPs described in this paragraph are engineered to make a humanized antibody. In a further embodiment, the ABP of the invention comprises CDRs or VH or VL or antibody sequences with 90% identity to the sequences in this paragraph.

In some embodiments, the 0X40 binding protein is an 0X40 agonist.

In another embodiment, any mouse or chimeric sequences of any 0X40 binding protein of the invention are engineered to make a humanized antibody. In one embodiment, the 0X40 binding protein of the invention comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1 ; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:2; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:3; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 7; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:8; and (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:9.

In another embodiment, the 0X40 binding protein of the invention comprises: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 14; (c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 15; (d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 19; (e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:20; and (f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:2l .

In another embodiment, the 0X40 binding protein of the invention comprises: a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: l or 13; a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:2 or 14; and/or a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:3 or 15, or a heavy chain variable region CDR having 90% identity thereto.

In yet another embodiment, the 0X40 binding protein of the invention comprises: a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:7 or 19; a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:8 or 20 and/or a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:9 or 21 , or a heavy chain variable region having 90 percent identity thereto.

In a further embodiment, the 0X40 binding protein of the invention comprises: a light chain variable region (“Vi”) comprising the amino acid sequence of SEQ ID NO: 10, 11, 22 or 23, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 10, 1 1, 22 or 23. In another embodiment, the 0X40 binding protein of the invention comprises a heavy chain variable region (“V_H”) comprising the amino acid sequence of SEQ ID NO:4, 5, 16 and 17, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO:4, 5, 16 and 17.

In another embodiment, the 0X40 binding protein of the invention comprises a variable heavy chain sequence of SEQ ID NO:5, or a sequence having 90 percent identity thereto, and a variable light chain sequence of SEQ ID NO: 1 1 , or a sequence having 90 percent identity thereto. In another embodiment, the 0X40 binding protein of the invention comprises a variable heavy chain sequence of SEQ ID NO: 17, or a sequence having 90 percent identity thereto, and a variable light chain sequence of SEQ ID NO:23 or a sequence having 90 percent identity thereto.

In another embodiment, the 0X40 binding protein of the invention comprises a variable light chain encoded by the nucleic acid sequence of SEQ ID NO: 12, or 24, or a nucleic acid sequence with at least 90 percent identity to the nucleotide sequences of SEQ ID NO: 12 or 24. In another embodiment, the 0X40 binding protein of the invention comprises a variable heavy chain encoded by a nucleic acid sequence of SEQ ID NO:6 or 18, or a nucleic acid sequence with at least 90 percent identity to nucleotide sequences of SEQ ID NO:6 or 18.

Also provided herein are monoclonal antibodies. In one embodiment, the monoclonal antibodies comprise a variable light chain comprising the amino acid sequence of SEQ ID NO: 10 or 22, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO: 10 or 22. Further provided are monoclonal antibodies comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:4 or 16, or an amino acid sequence with at least 90 percent identity to the amino acid sequences of SEQ ID NO:4 or 16.

In another embodiment, the 0X40 binding protein of the invention comprises a variable heavy chain sequence of SEQ ID NO:5 and a variable light chain sequence of SEQ ID NO: l l . In another embodiment, the 0X40 binding protein or antibody of the invention is an antibody that comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:48 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:49.

Heavy Chain of ANTIBODY 106-222:

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLKWMGWINTETGEPTYADDFK GRFVFSLDTSVSTAYLQISSLKAEDTAVYYCANPYYDYVSYYAMDYWGQGTTVTVSSASTKGPSV FPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(SEQ ID NO: 48)

Light Chain of ANTIBODY 106-222:

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYLYTGVPSRFSGS GSGTDFTFTISSLQPEDIATYYCQQHYSTPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO : 49 )

Heavy Chain Variable Region of ANTIBODY 106-222:

QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLKWMGWINTETGEPTYADDFK GRFVFSLDTSVSTAYLQISSLKAEDTAVYYCANPYYDYVSYYAMDYWGQGTTVTVSS (SEQ ID NO: 5)

Light Chain Variable Region of ANTIBODY 106-222:

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYLYTGVPSRFSGS GSGTDFTFTISSLQPEDIATYYCQQHYSTPRTFGQGTKLEIK (SEQ ID NO: 11)

CDR sequences of ANTIBODY 106-222:

HC CDRl : Asp Tyr Ser Met His (SEQ ID NO : 1 )

HC CDR2 : Trp lie Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp

Phe Lys Gly (SEQ ID NO: 2)

HC CDR3 : Pro Tyr Tyr Asp Tyr Val Ser Tyr Tyr Ala Met Asp Tyr

(SEQ ID NO : 3 )

LC CDRl : Lys Ala Ser Gin Asp Val Ser Thr Ala Val Ala (SEQ ID

NO: 7)

LC CDR2 : Ser Ala Ser Tyr Leu Tyr Thr (SEQ ID NO: 8)

LC CDR3 : Gin Gin His Tyr Ser Thr Pro Arg Thr (SEQ ID NO: 9)

TLR4 Agonists The combinations of the invention comprise TLR4 agonists, that is, molecules that agonize TLR4, for example, by binding and initiating conformational changes or signaling by engaging TLR4. TLR4 agonists bind to TLR4 and activate the receptor, triggering signaling to initiate a TLR4 signaling pathway response. In one embodiment, TLR4 modulators are aminoalkyl glucosaminide phosphate compounds (AGPs). TLR4 recognizes bacterial LPS (lipopolysaccharide) and when activated initiates an innate immune response. AGPs are a monosaccharide mimetic of the lipid A protein of bacterial LPS and have been developed with ether and ester linkages on the“acyl chains” of the compound. Processes for making these compounds are known and disclosed, for example, in WO 2006/016997, U.S. Patent Nos. 7,288,640 and 6,113,918, and WO 01/90129. Other AGPs and related processes are disclosed in U.S. Patent No. 7,129,219, U.S. Patent No. 6,525,028 and U.S. Patent No 6,911,434. AGPs with ether linkages on the acyl chains employed in the composition of the invention are known and disclosed in WO 2006/016997. The AGP compounds set forth and described according to Formula (III) at paragraphs [0019] through [0021] in WO 2006/016997 may be employed in the presently claimed methods and combinations.

AGP compounds employed in the present invention have the structure set forth in Formula 1 as follows:

wherein

m is 0 to 6 n is 0 to 4;

X is O or S, preferably O;

Y is O or NH;

Z is O or H;

each Ri, R₂, R3 is selected independently from the group consisting of a Cl -20 acyl and a Cl -20 alkyl;

R₄ is H or Me;

R5 is selected independently from the group consisting of -H, -OH, -(C1-C4) alkoxy, -PO3R8R9, -OPO3R8R9, -SO3R8, -OSO3R8, -NR8R9, -SRs, -CN, -N0₂, - CHO, -C0₂R₈, and -CONR8R9, wherein Rs and R9 are each independently selected from H and (C1-C4) alkyl; and

each Re and R₇ is independently H or P0₃H₂.

In Formula 1 the configuration of the 3' stereogenic centers to which the normal fatty acyl residues (that is, the secondary acyloxy or alkoxy residues, e.g., RiO, R₂0, and R₃0) are attached is R or S, preferably R (as designated by Cahn-Ingold-Prelog priority rules). Configuration of aglycon stereogenic centers to which R₄ and R5 are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the present invention.

The number of carbon atoms between heteroatom X and the aglycon nitrogen atom is determined by the variable“n”, which can be an integer from 0 to 4, or an integer from 0 to 2.

The chain length of normal fatty acids Ri, R₂, and R3 can be from about 6 to about 16 carbons, or from about 9 to about 14 carbons. The chain lengths can be the same or different. Some embodiments include chain lengths where Ri, R₂ and R3 are 6 or 10 or 12 or 14.

Formula 1 encompasses L/D-seryl, -threonyl, -cysteinyl ether and ester lipid AGPs, both agonists and antagonists and their homologs (n=l-4), as well as various carboxylic acid bioisosteres (i.e., R5 is an acidic group capable of salt formation; the phosphate can be either on 4- or 6- position of the glucosamine unit, preferably, is in the 4-position). In a one embodiment of the invention employing an AGP compound of Formula 1 , n is 0, R₅ is CO₂H, Re is PO₃H₂, and R₇ is H. This AGP compound is set forth as the structure in Formula la as follows:

wherein X is O or S; Y is O or NH; Z is O or H; each Ri, R₂, R₃ is selected independently from the group consisting of a Cl -20 acyl and a Cl -20 alkyl; and R₄ is H or methyl.

In Formula la the configuration of the 3' stereogenic centers to which the normal fatty acyl residues (that is, the secondary acyloxy or alkoxy residues, e.g., RiO, R2O, and R3O) are attached as R or S, preferably R (as designated by Cahn-Ingold-Prelog priority rules). Configuration of aglycon stereogenic centers to which R₄ and CO₂H are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the present invention.

Formula la encompasses L/D-seryl, -threonyl, -cysteinyl ether or ester lipid AGPs, both agonists and antagonists.

In both Formula 1 and Formula la, Z is O attached by a double bond or two hydrogen atoms which are each attached by a single bond. That is, the compound is ester-linked when Z=Y=0; amide-linked when Z =0 and Y=NH; and ether-linked when Z=H/H and Y=0.

CRX-601 and CRX-527 are compounds of Formula 1. Their structures are set forth as follows:

Additionally, another preferred embodiment employs CRX-547 having the structure shown. CRX-547

Still other embodiments include AGPs, such as CRX-602 or CRX-526 providing increased stability to AGPs having shorter secondary acyl or alkyl chains.

In a further embodiment of the invention, the TLR4 modulator is an agonist. In a further embodiment, the TLR4 modulator that is an agonist is selected from the group consisting of: CRX-601, CRX-547, and CRX-527.

AGP Buffers

In one embodiment of the present invention, the composition comprising a TLR4 modulator, such as an AGP, is buffered using a zwitterionoic buffer. In one embodiment of the invention, the zwitterionic buffer is an aminoalkanesulfonic acid or suitable salt. Examples of amninoalkanesulfonic buffers include, but are not limited, to HEPES, HEPPS/EPPS, MOPS, MOBS and PIPES. In one embodiment of the invention, the buffer is a pharmaceutically acceptable buffer, suitable for use in humans, such as in for use in a commercial injection product. In one embodiment of the invention, the buffer is HEPES. Dosing Regimens

In some aspects, the disclosure is drawn to a method of treatment of a subject with cancer, wherein an agonist 0X40 binding protein and a TLR4 agonist are administered to the subject, wherein the agonist 0X40 binding protein is administered at a dose of about 8 mg to about 24 mg and the TLR4 agonist is administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to a method of treatment of a subject with cancer, wherein an agonist 0X40 binding protein and a TLR4 agonist are administered to the subject, wherein the agonist 0X40 binding protein is administered at a dose of about 24 mg and the TLR4 agonist is administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to an agonist 0X40 binding protein and a TLR4 agonist for simultaneous or sequential use in treating cancer, wherein the agonist 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to an agonist 0X40 binding protein and a TLR4 agonist for simultaneous or sequential use in treating cancer, wherein the agonist 0X40 binding protein is to be administered at a dose of about 24 mg and the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to an agonist 0X40 binding protein for use in treating cancer, wherein the agonist 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and is to be administered simultaneously or sequentially with a TLR4 agonist at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to an agonist 0X40 binding protein for use in treating cancer, wherein the agonist 0X40 binding protein is to be administered at a dose of about 24 mg and is to be administered simultaneously or sequentially with a TLR4 agonist at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to a TLR4 agonist for use in treating cancer, wherein the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng and is to be administered simultaneously or sequentially with an agonist 0X40 binding protein at a dose of about 8 mg to about 24 mg.

In some aspects, the disclosure is drawn to a TLR4 agonist for use in treating cancer, wherein the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng and is to be administered simultaneously or sequentially with an agonist 0X40 binding protein at a dose of about 24 mg.

In some aspects, the disclosure is drawn to use of an agonist 0X40 binding protein in the manufacture of a medicament for treating cancer, wherein the 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and is to be administered simultaneously or sequentially with a TLR4 agonist at a dose of about 50 ng to about 250 ng-

In some aspects, the disclosure is drawn to use of an agonist 0X40 binding protein in the manufacture of a medicament for treating cancer, wherein the 0X40 binding protein is to be administered at a dose of about 24 mg and is to be administered simultaneously or sequentially with a TLR4 agonist at a dose of about 50 ng to about 250 ng.

In some aspects, the disclosure is drawn to use of a TLR4 agonist in the manufacture of a medicament for treating cancer, wherein the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng and is to be administered simultaneously or sequentially with an agonist 0X40 binding protein at a dose of about 8 mg to about 24 mg.

In some aspects, the disclosure is drawn to use of a TLR4 agonist in the manufacture of a medicament for treating cancer, wherein the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng and is to be administered simultaneously or sequentially with an agonist 0X40 binding protein at a dose of about 24 mg.

In some aspects, the disclosure is drawn to a pharmaceutical kit comprising about 8 mg to about 24 mg of an agonist 0X40 binding protein and about 50 ng to 250 ng of a TLR4 agonist.

In some aspects, the disclosure is drawn to a pharmaceutical kit comprising about 24 mg of an agonist 0X40 binding protein and about 50 ng to 250 ng of a TLR4 agonist. In some embodiments, the agonist 0X40 binding protein is administered intravenously ( e.g ., intravenous infusion).

In some embodiments, the TLR4 agonist is administered intravenously (e.g., intravenous injection).

In some embodiments, the TLR4 agonist is administered intratumorally (e.g., by intratumoral injection).

In some embodiments, the agonist 0X40 binding protein is administered intravenously (e.g., intravenous infusion) and the TLR4 agonist is administered intravenously (e.g., intravenous injection).

In some embodiments, the agonist 0X40 binding protein is administered at a dose of about 8 mg.

In some embodiments, the agonist 0X40 binding protein is administered at a dose of about 24 mg.

In some embodiments, the TLR4 agonist is administred in a dose in the range of about 5 ng to about 1000 ng.

In an embodiment, the TLR4 agonist is to be administered at a dose of about 50 ng to about 250 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 50 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 550 ng.

In some embodiments, the TLR4 agonist is administered at a dose of about 600 ng.

In some embodiments, the TLR4 agonist is selected from the group consisting of: CRX- 601; CRX-547; CRX-602; and, CRX-527.

In some embodiments, the TLR4 agonist is CRX-527. In some embodiments, the TLR4 agonist is CRX-601.

In some embodiments, the cancer comprises a solid tumor.

In some embodiments, the TLR4 agonist ( e.g ., CRX-601) and the agonist 0X40 binding protein (e.g. , an agonist 0X40 binding protein that comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:48 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:49) are both administered to the subject every three weeks.

In some embodiments, the TLR4 agonist (e.g., CRX-601) is administered for a two-week run in period (e.g., the TLR4 agonist is administered on day 1 and 8 of the run in period) prior to administering the agonist 0X40 binding protein (e.g. , an agonist 0X40 binding protein that comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:48 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:49).

In some embodiments, after the run in period, the TLR4 agonist (e.g., CRX-601) and the agonist 0X40 binding protein (e.g., an agonist 0X40 binding protein that comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:48 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:49) are both administered to the subject every three weeks.

In some embodiments, the TLR4 agonist (e.g., CRX-601) is administered for a two-week run in period (e.g., the TLR4 agonist is administered on day 1 and day 8 of the two-week run in period), and following the run in period, the TLR4 agonist and the agonist 0X40 binding protein (e.g. , an agonist 0X40 binding protein that comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:48 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:49) are both administered to the subject, e.g., every three weeks. Methods of Treatment

The combinations of the invention are believed to have utility in disorders wherein the engagement of 0X40 and/or TLR4, is beneficial.

The present invention thus also provides a combination of the invention, for use in therapy, particularly, in the treatment of disorders wherein the engagement of 0X40 and/or TLR4, is beneficial, particularly cancer.

In one embodiment, the present invention provides methods of treating cancer in a patient with the combination of a TLR4 agonist, such as CRX-601, with a humanized monoclonal 0X40 antibody, wherein the humanized 0X40 antibody is administered intravenously, and the TLR4 agonist is administered intratumorally, resulting in an abscopal effect in the tumor(s) in the patient.

As used herein, the term“abscopal effect”, means a phenomenon in which local treatment causes tumor regression at not only the treated site, but also at distant tumor sites. Postow, et ah, N Engl J Med 366 (10): 925-31 (2012).

A further aspect of the invention provides a method of treatment of a disorder wherein engagement of 0X40 and/or TLR4 is beneficial, comprising administering a combination of the invention.

A further aspect of the present invention provides the use of a combination of the invention in the manufacture of a medicament for the treatment of a disorder engagement of 0X40 and/or TLR4 is beneficial. In some embodiments, the disorder is cancer.

Suitably, the present invention provides the use of the combinations of the present invention for the treatment of cancer.

The cancer can comprise a solid cancer, e.g. , solid tumor.

Examples of cancers that are suitable for treatment with combination of the invention include, but are limited to, both primary and metastatic forms of head and neck, breast, lung, colon, ovary, and prostate cancers. Suitably the cancer is selected from: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,

medulloblastoma, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid, lymphoblastic T cell leukemia, Chronic myelogenous leukemia, Chronic lymphocytic leukemia, Hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, AML, Chronic neutrophilic leukemia, Acute lymphoblastic T cell leukemia, plasmacytoma, Immunoblastic large cell leukemia, Mantle cell leukemia, Multiple myeloma Megakaryoblastic leukemia, multiple myeloma, acute megakaryocytic leukemia, promyelocytic leukemia, Erythro leukemia, malignant lymphoma, hodgkins lymphoma, non-hodgkins lymphoma, lymphoblastic T cell lymphoma, Burkitt’s lymphoma, follicular lymphoma, neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulval cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, esophageal cancer, salivary gland cancer, hepatocellular cancer, gastric cancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST (gastrointestinal stromal tumor); and testicular cancer.

Additionally, examples of a cancer to be treated include Barret’s adenocarcinoma; biliary tract carcinomas; breast cancer; cervical cancer; cholangiocarcinoma; central nervous system tumors including primary CNS tumors such as glioblastomas, astrocytomas ( e.g ., glioblastoma multiforme) and ependymomas, and secondary CNS tumors (/.<?., metastases to the central nervous system of tumors originating outside of the central nervous system); colorectal cancer including large intestinal colon carcinoma; gastric cancer; carcinoma of the head and neck including squamous cell carcinoma of the head and neck; hematologic cancers including leukemias and lymphomas such as acute lymphoblastic leukemia, acute myelogenous leukemia (AML), myelodysplastic syndromes, chronic myelogenous leukemia, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, megakaryoblastic leukemia, multiple myeloma and erythroleukemia;

hepatocellular carcinoma; lung cancer including small cell lung cancer and non-small cell lung cancer; ovarian cancer; endometrial cancer; pancreatic cancer; pituitary adenoma; prostate cancer; renal cancer; sarcoma; skin cancers including melanomas; and thyroid cancers. Suitably, the present invention relates to a method for treating or lessening the severity of a cancer selected from: brain (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast, colon, head and neck, kidney, lung, liver, melanoma, ovarian, pancreatic, prostate, sarcoma and thyroid.

In one embodiment, the present invention relates to a method for treating or lessening the severity of a cancer selected from ovarian, breast, pancreatic and prostate.

In another embodiment, the present invention relates to a method for treating or lessening the severity of pre-cancerous syndromes in a mammal, including a human, wherein the pre-cancerous syndrome is selected from: cervical intraepithelial neoplasia, monoclonal gammapathy of unknown significance (MGUS), myelodysplastic syndrome, aplastic anemia, cervical lesions, skin nevi (pre-melanoma), prostatic intraepithelial (intraductal) neoplasia (PIN), Ductal Carcinoma in situ (DCIS), colon polyps and severe hepatitis or cirrhosis.

The combination of the invention may be used alone, or in combination with, one or more other therapeutic agents. The invention thus provides in a further aspect a further combination comprising a combination of the invention with a further therapeutic agent or agents, compositions and medicaments comprising the combination and use of the further combination, compositions and medicaments in therapy, in particular, in the treatment of diseases susceptible to engagement of 0X40 and/or TLR4.

In the embodiment, the combination of the invention may be employed with other therapeutic methods of cancer treatment. In particular, in anti-neoplastic therapy, combination therapy with other chemotherapeutic, hormonal, antibody agents as well as surgical and/or radiation treatments other than those mentioned above are envisaged. Combination therapies according to the present invention thus include the administration of an 0X40 binding protein of the invention and/or a TLR4 modulator as well as optional use of other therapeutic agents including other anti-neoplastic agents. Such combination of agents may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order, both close and remote in time. In one embodiment, the pharmaceutical combination includes an 0X40 binding protein of the invention and a TLR4 modulator, and optionally at least one additional anti-neoplastic agent.

In one embodiment, the further anti-cancer therapy is surgical and/or radiotherapy.

In one embodiment, the further anti-cancer therapy is at least one additional anti neoplastic agent.

Any anti-neoplastic agent that has activity versus a susceptible tumor being treated may be utilized in the combination. Typical anti-neoplastic agents useful include, but are not limited to, anti-microtubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such as anthracyclins, actinomycins and bleomycins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogues and anti-folate compounds; topoisomerase I inhibitors such as camptothecins; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; and cell cycle signaling inhibitors.

Anti-microtubule or anti-mitotic agents: Anti-microtubule or anti-mitotic agents are phase specific agents active against the microtubules of tumor cells during M or the mitosis phase of the cell cycle. Examples of anti-microtubule agents include, but are not limited to, diterpenoids and vinca alkaloids.

Diterpenoids, which are derived from natural sources, are phase specific anti -cancer agents that operate at the G₂/M phases of the cell cycle. It is believed that the

diterpenoids stabilize the b-tubulin subunit of the microtubules, by binding with this protein. Disassembly of the protein appears then to be inhibited with mitosis being arrested and cell death following. Examples of diterpenoids include, but are not limited to, paclitaxel and its analog, docetaxel.

Paclitaxel, 5P,20-epoxy-l,2a,4,7P,l0P,l3a-hexa-hydroxytax-l l-en-9-one 4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine; is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available as an injectable solution TAXOL. It is a member of the taxane family of terpenes. Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States (Markman, et ah, Yale Journal of Biology and Medicine , 64:583 (1991); McGuire, et ah, Ann. Intern, Med., 11 1 :273 (989), and for the treatment of breast cancer (Holmes, et al., J. Nat. Cancer Inst., 83: 1797 (1991)). Paclitaxel is a potential candidate for treatment of neoplasms in the skin (Einzig, et. al., Proc. Am. Soc. Clin. Oncol., 20:46 (2001) and head and neck carcinomas (Forastire, et. al., Sem. Oncol.,

20:56, (1990)). The compound also shows potential for the treatment of polycystic kidney disease (Woo, et al., Nature, 368:750 (1994)), lung cancer and malaria. Treatment of patients with paclitaxel results in bone marrow suppression (multiple cell lineages,

Ignoff, et. al, Cancer Chemotherapy Pocket Guide, 1998) related to the duration of dosing above a threshold concentration (50nM) (Kearns, et. al., Seminars in Oncology, 3(6) r.16-23 (1995)).

Docetaxel, (2R,3S)- N-carboxy-3-phcnylisoscrinc,N-/e/7-butyl ester, 13-ester with 5b-20- epoxy-l,2a,4,7P,lOP,l3a-hexahydroxytax-l l-en-9-one 4-acetate 2-benzoate, trihydrate; is commercially available as an injectable solution as TAXOTERE. Docetaxel is indicated for the treatment of breast cancer. Docetaxel is a semisynthetic derivative of paclitaxel q.v., prepared using a natural precursor, 10-deacetyl-baccatin III, extracted from the needle of the European Yew tree.

Vinca alkaloids are phase specific anti-neoplastic agents derived from the periwinkle plant. Vinca alkaloids act at the M phase (mitosis) of the cell cycle by binding specifically to tubulin. Consequently, the bound tubulin molecule is unable to polymerize into microtubules. Mitosis is believed to be arrested in metaphase with cell death following. Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, and vinorelbine.

Vinblastine, vincaleukoblastine sulfate, is commercially available as VELBAN as an injectable solution. Although, it has possible indication as a second line therapy of various solid tumors, it is primarily indicated in the treatment of testicular cancer and various lymphomas including Hodgkin’s Disease; and lymphocytic and histiocytic lymphomas. Myelosuppression is the dose-limiting side effect of vinblastine. Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commercially available as ONCOVIN as an injectable solution. Vincristine is indicated for the treatment of acute leukemias and has also found use in treatment regimens for Hodgkin’s and non-Hodgkin’s malignant lymphomas. Alopecia and neurologic effects are the most common side effect of vincristine and to a lesser extent myelosupression and gastrointestinal mucositis effects occur.

Liposomally formulated vincristine sulfate is commercially available as MARQUIBO. Liposomally formulated vincristine sulfate is indicated for the treatment of patients with Philadelphia chromosome-negative (Ph-) acute lymphoblastic leukemia (ALL).

Liposomally formulated vincristine sulfate is also useful for the treatment of acute leukemias generally and has also found use in treatment regimens for Hodgkin’s and non- Hodgkin’s malignant lymphomas.

Vinorelbine, 3 ',4'-di dehydro -4'-deoxy-C'-norvincaleukoblastine [R-(R*,R*)-2,3- dihydroxybutanedioate (l :2)(salt)], commercially available as an injectable solution of vinorelbine tartrate (NAVELBINE), is a semi-synthetic vinca alkaloid. Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, in the treatment of various solid tumors, such as non-small cell lung, advanced breast, and hormone refractory prostate cancers. Myelosuppression is the most common dose-limiting side effect of vinorelbine.

Platinum coordination complexes: Platinum coordination complexes are non-phase specific anti-cancer agents, which are interactive with DNA. The platinum complexes enter tumor cells, undergo, aquation and form intra- and interstrand cross-links with DNA causing adverse biological effects to the tumor. Examples of platinum coordination complexes include, but are not limited to, oxaliplatin, cisplatin and carboplatin.

Cisplatin, cis-diamminedichloroplatinum, is commercially available as PLATINOL as an injectable solution. Cisplatin is primarily indicated in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer. Carboplatin, platinum, diammine [l ,l-cyclobutane-dicarboxylate(2-)-0,0'], is commercially available as PARAP LATIN as an injectable solution. Carboplatin is primarily indicated in the first and second line treatment of advanced ovarian carcinoma.

Alkylating agents: Alkylating agents are non-phase anti-cancer specific agents and strong electrophiles. Typically, alkylating agents form covalent linkages, by alkylation, to DNA through nucleophilic moieties of the DNA molecule such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups. Such alkylation disrupts nucleic acid function leading to cell death. Examples of alkylating agents include, but are not limited to, nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; and triazenes such as dacarbazine.

Cyclophosphamide, 2-[bis(2-chloroethyl)amino]tetrahydro-2H-l,3,2-oxazaphosphorine 2-oxide monohydrate, is commercially available as an injectable solution or tablets as CYTOXAN. Cyclophosphamide is indicated as a single agent, or in combination with other chemotherapeutic agents, in the treatment of malignant lymphomas, multiple myeloma, and leukemias.

Melphalan, 4-[bis(2-chloroethyl)amino]-L-phenylalanine, is commercially available as an injectable solution or tablets as ALKERAN. Melphalan is indicated for the palliative treatment of multiple myeloma and non-resectable epithelial carcinoma of the ovary.

Bone marrow suppression is the most common dose-limiting side effect of melphalan.

Chlorambucil, 4-[bis(2-chloroethyl)amino]benzenebutanoic acid, is commercially available as LEUKERAN tablets. Chlorambucil is indicated for the palliative treatment of chronic lymphatic leukemia, and malignant lymphomas such as lymphosarcoma, giant follicular lymphoma, and Hodgkin’s disease.

Busulfan, 1 ,4-butanediol dimethanesulfonate, is commercially available as MYLERAN TABLETS. Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia. Carmustine, l ,3-[bis(2-chloroethyl)-l -nitrosourea, is commercially available as single vials of lyophilized material as BiCNU. Carmustine is indicated for the palliative treatment as a single agent or in combination with other agents for brain tumors, multiple myeloma, Hodgkin’s disease, and non-Hodgkin’s lymphomas.

Dacarbazine, 5-(3,3-dimethyl-l-triazeno)-imidazole-4-carboxamide, is commercially available as single vials of material as DTIC-Dome. Dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents for the second line treatment of Hodgkin’s Disease.

Antibiotic anti-neoplastics: Antibiotic anti-neoplastics are non-phase specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts ordinary function of the nucleic acids leading to cell death. Examples of antibiotic anti-neoplastic agents include, but are not limited to, actinomycins such as dactinomycin, anthrocyclins such as daunorubicin and doxorubicin; and bleomycins.

Dactinomycin, also known as Actinomycin D, is commercially available in injectable form as COSMEGEN. Dactinomycin is indicated for the treatment of Wilm’s tumor and rhabdomyosarcoma.

Daunorubicin, (8S-cis-)-8-acetyl-l0-[(3-amino-2,3,6-trideoxy-a-L-lyxo- hcxopyranosyl )oxy] -7,8,9, 10-tetrahydro-6,8 , 11 -trihydroxy- 1 -methoxy-5, 12

naphthacenedione hydrochloride, is commercially available as a liposomal injectable form as DAUNOXOME or as an injectable as CERUBIDINE. Daunorubicin is indicated for remission induction in the treatment of acute nonlymphocytic leukemia and advanced HIV associated Kaposi’s sarcoma.

Doxorubicin, (8S, lOS)-lO-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-8- glycoloyl, 7,8,9, lO-tetrahydro-6, 8,1 1 -trihydroxy- 1 -methoxy-5, 12 naphthacenedione hydrochloride, is commercially available as an injectable form as RUB EX or

ADRIAMYCIN RDF. Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblastic leukemia, but is also a useful component in the treatment of some solid tumors and lymphomas.

Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus, is commercially available as BLENOXANE. Bleomycin is indicated as a palliative treatment, as a single agent or in combination with other agents, of squamous cell carcinoma, lymphomas, and testicular carcinomas.

Topoisomerase II inhibitors: Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins .

Epipodophyllotoxins are phase specific anti-neoplastic agents derived from the mandrake plant. Epipodophyllotoxins typically affect cells in the S and G₂ phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA causing DNA strand breaks. The strand breaks accumulate and cell death follows. Examples of

epipodophyllotoxins include, but are not limited to, etoposide and teniposide.

Etoposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R )-ethylidene-P-D- glucopyranoside], is commercially available as an injectable solution or capsules as VePESID and is commonly known as VP- 16. Etoposide is indicated as a single agent, or in combination with, other chemotherapy agents in the treatment of testicular and non small cell lung cancers.

Teniposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R )-thenylidene-P-D- glucopyranoside], is commercially available as an injectable solution as VUMON and is commonly known as VM-26. Teniposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia in children.

Antimetabolite neoplastic agents: Antimetabolite neoplastic agents are phase specific anti-neoplastic agents that act at S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis and thereby limiting DNA synthesis. Consequently, S phase does not proceed and cell death follows. Examples of antimetabolite anti-neoplastic agents include, but are not limited to, fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, and gemcitabine.

5-fluorouracil, 5-fluoro-2,4- (lH,3H) pyrimidinedione, is commercially available as fluorouracil. Administration of 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into both RNA and DNA. The result typically is cell death. 5- fluorouracil is indicated as a single agent or in combination with other chemotherapy agents in the treatment of carcinomas of the breast, colon, rectum, stomach and pancreas. Other fluoropyrimidine analogs include 5-fluoro deoxyuridine (floxuridine) and 5- fluorodeoxyuridine monophosphate.

Cytarabine, 4-amino- l-P-D-arabinofuranosyl-2 (lH)-pyrimidinone, is commercially available as CYTOSAR-U and is commonly known as Ara-C. It is believed that cytarabine exhibits cell phase specificity at S-phase by inhibiting DNA chain elongation by terminal incorporation of cytarabine into the growing DNA chain. Cytarabine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other cytidine analogs include 5-azacytidine and 2', 2'- difluorodeoxycytidine (gemcitabine).

Mercaptopurine, l,7-dihydro-6H-purine-6-thione monohydrate, is commercially available as PURINETHOL. Mercaptopurine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism. Mercaptopurine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. A useful mercaptopurine analog is azathioprine.

Thioguanine, 2-amino- l,7-dihydro-6H-purine-6-thione, is commercially available as TABLOID. Thioguanine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism. Thioguanine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other purine analogs include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine. Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine monohydrochloride (b-isomer), is commercially available as GEMZAR. Gemcitabine exhibits cell phase specificity at S- phase and by blocking progression of cells through the Gl/S boundary. Gemcitabine is indicated in combination with cisplatin in the treatment of locally advanced non-small cell lung cancer and alone in the treatment of locally advanced pancreatic cancer.

Methotrexate, N-[4[[(2,4-diamino-6-pteridinyl) methyl] methylamino] benzoyl]-L- glutamic acid, is commercially available as methotrexate sodium. Methotrexate exhibits cell phase effects specifically at S-phase by inhibiting DNA synthesis, repair and/or replication through the inhibition of dyhydrofolic acid reductase which is required for synthesis of purine nucleotides and thymidylate. Methotrexate is indicated as a single agent or in combination with other chemotherapy agents in the treatment of

choriocarcinoma, meningeal leukemia, non-Hodgkin’s lymphoma, and carcinomas of the breast, head, neck, ovary and bladder.

Topoisomerase I inhibitors: Camptothecins, including, camptothecin and camptothecin derivatives are available or under development as Topoisomerase I inhibitors.

Camptothecins cytotoxic activity is believed to be related to its Topoisomerase I inhibitory activity. Examples of camptothecins include, but are not limited to, irinotecan, topotecan, and the various optical forms of 7-(4-methylpiperazino-methylene)-l0,l 1- ethylenedioxy-20-camptothecin described below.

Irinotecan HC1, (4S)-4,l l-diethyl-4-hydroxy-9-[(4-piperidinopiperidino) carbonyloxy]- 1 H-pyrano [3 ',4',6 ,7] indolizino [ 1 ,2-b]quinoline-3 , 14(4H, 12H)-dione hydrochloride, is commercially available as the injectable solution CAMPTOSAR. Irinotecan is a derivative of camptothecin which binds, along with its active metabolite SN-38, to the topoisomerase I - DNA complex. It is believed that cytotoxicity occurs as a result of irreparable double strand breaks caused by interaction of the topoisomerase I : DNA : irintecan or SN-38 ternary complex with replication enzymes. Irinotecan is indicated for treatment of metastatic cancer of the colon or rectum. Topotecan HC1, (S)- 10-[(dimethylamino)methyl]-4-ethyl-4, 9-dihydroxy- 1 H- pyrano[3',4',6,7]indolizino[l,2-b]quinoline-3,l4-(4H,l2H)-dione monohydrochloride, is commercially available as the injectable solution HYCAMTIN. Topotecan is a derivative of camptothecin which binds to the topoisomerase I - DNA complex and prevents re ligation of singles strand breaks caused by Topoisomerase I in response to torsional strain of the DNA molecule. Topotecan is indicated for second line treatment of metastatic carcinoma of the ovary and small cell lung cancer.

Hormones and hormonal analogues: Hormones and hormonal analogues are useful compounds for treating cancers in which there is a relationship between the hormone(s) and growth and/or lack of growth of the cancer. Examples of hormones and hormonal analogues useful in cancer treatment include, but are not limited to, adrenocorticosteroids such as prednisone and prednisolone which are useful in the treatment of malignant lymphoma and acute leukemia in children ; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrazole, vorazole, and exemestane useful in the treatment of adrenocortical carcinoma and hormone dependent breast carcinoma containing estrogen receptors; progestrins such as megestrol acetate useful in the treatment of hormone dependent breast cancer and endometrial carcinoma; estrogens, androgens, and anti-androgens such as flutamide, nilutamide, bicalutamide, cyproterone acetate and 5a- reductases such as finasteride and dutasteride, useful in the treatment of prostatic carcinoma and benign prostatic hypertrophy; anti-estrogens such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, as well as selective estrogen receptor modulators (SERMS) such those described in ET.S. Patent Nos. 5,681,835, 5,877,219, and 6,207,716, useful in the treatment of hormone dependent breast carcinoma and other susceptible cancers; and gonadotropin-releasing hormone (GnRH) and analogues thereof which stimulate the release of leutinizing hormone (LH) and/or follicle stimulating hormone (FSH) for the treatment prostatic carcinoma, for instance, LHRH agonists and antagonists such as goserelin acetate and luprolide.

Signal transduction pathway inhibitors: Signal transduction pathway inhibitors are those inhibitors, which block or inhibit a chemical process which evokes an intracellular change. As used herein this change is cell proliferation or differentiation. Signal transduction inhibitors useful in the present invention include, but are not limited to, inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, SH2/SH3 domain blockers, serine/threonine kinases, phosphotidyl inositol-3 kinases, myo-inositol signaling, and Ras oncogenes.

Several protein tyrosine kinases catalyse the phosphorylation of specific tyrosyl residues in various proteins involved in the regulation of cell growth. Such protein tyrosine kinases can be broadly classified as receptor or non-receptor kinases.

Receptor tyrosine kinases are transmembrane proteins having an extracellular ligand binding domain, a transmembrane domain, and a tyrosine kinase domain. Receptor tyrosine kinases are involved in the regulation of cell growth and are generally termed growth factor receptors. Inappropriate or uncontrolled activation of many of these kinases, i.e., aberrant kinase growth factor receptor activity, for example by over expression or mutation, has been shown to result in uncontrolled cell growth.

Accordingly, the aberrant activity of such kinases has been linked to malignant tissue growth. Consequently, inhibitors of such kinases could provide cancer treatment methods. Growth factor receptors include, for example, epidermal growth factor receptor (EGFr), platelet derived growth factor receptor (PDGFr), erbB2, erbB4, ret, vascular endothelial growth factor receptor (VEGFr), tyrosine kinase with immunoglobulin-like and epidermal growth factor identity domains (TIE-2), insulin growth factor -I (IGFI) receptor, macrophage colony stimulating factor (cfms), BTK, ckit, cmet, fibroblast growth factor (FGF) receptors, Trk receptors (TrkA, TrkB, and TrkC), ephrin (eph) receptors, and the RET protooncogene. Several inhibitors of growth receptors are under development and include ligand antagonists, antibodies, tyrosine kinase inhibitors and anti-sense oligonucleotides. Growth factor receptors and agents that inhibit growth factor receptor function are described, for instance, in Kath, John C., Exp. Opin. Ther. Patents (2000) 10(6): 803-818; Shawver, et al, DDT, Vol 2, No. 2 (February 1997); and Lofts, F. J., et al, “GROWTH FACTOR RECEPTORS AS TARGETS”, NEW MOLECULAR TARGETS FOR CANCER CHEMOTHERAPY (Workman, Paul and Kerr, David, CRC press 1994, London).

Tyrosine kinases, which are not growth factor receptor kinases are termed non-receptor tyrosine kinases. Non-receptor tyrosine kinases useful in the present invention, which are targets or potential targets of anti-cancer drugs, include cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (Focal adhesion kinase), Brutons tyrosine kinase, and Bcr-Abl. Such non-receptor kinases and agents which inhibit non-receptor tyrosine kinase function are described in Sinh, el al, Journal of Hematotherapy and Stem Cell Research, 8 (5): 465-80 (1999); and Bolen, et al., Annual review of Immunology, 15: 371-404 (1997).

SH2/SH3 domain blockers are agents that disrupt SH2 or SH3 domain binding in a variety of enzymes or adaptor proteins including, PI3-K p85 subunit, Src family kinases, adaptor molecules (She, Crk, Nek, Grb2) and Ras-GAP. SH2/SH3 domains as targets for anti-cancer drugs are discussed in Smithgall, T.E., Journal of Pharmacological and Toxicological Methods, 34(3) 125-32 (1995).

Inhibitors of Serine/Threonine Kinases including MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracellular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); and Protein kinase C family member blockers including blockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta). IkB kinase family (IKKa, IKKb), PKB family kinases, akt kinase family members, and TGF beta receptor kinases. Such Serine/Threonine kinases and inhibitors thereof are described in Yamamoto, et al., Journal of Biochemistry , 126 (5) 799-803 (1999) ; Brodt, et al., Biochemical Pharmacology, 60. 1101-1107 (2000); Massague, et al, Cancer Surveys, 27:41-64 (1996); Philip, et al., Cancer Treatment and Research, 78: 3-27 (1995), Lackey, et al., Bioorganic and Medicinal Chemistry Letters, (10) 223-226 (2000); U.S. Patent No. 6,268,391; and Martinez-Iacaci, et al, Int. J. Cancer, 88(1), 44- 52 (2000).

Inhibitors of Phosphotidyl inositol-3 Kinase family members including blockers of PI3- kinase, ATM, DNA-PK, and Ku are also useful in the present invention. Such kinases are discussed in Abraham, R.T. (1996), Current Opinion in Immunology. 8 (3) 412-8;

Canman, C.E., Lim, D.S. (1998), Oncogene 17 (25) 3301-3308; Jackson, S.P. (1997), International Journal of Biochemistry and Cell Biology. 29 (7):935-8; and Zhong, H., et al, Cancer Res., (2000) 60(6), 1541-1545.

Also useful in the present invention are myo-inositol signaling inhibitors, such as phospholipase C blockers and Myoinositol analogues. Such signal inhibitors are described in Powis, G., and Kozikowski A., (1994) NEW MOLECULAR TARGETS FOR CANCER CHEMOTHERAPY ED. (Paul Workman and David Kerr, CRC press 1994,

London).

Another group of signal transduction pathway inhibitor are inhibitors of Ras Oncogene. Such inhibitors include inhibitors of farnesyltransferase, geranyl-geranyl transferase, and CAAX proteases as well as anti-sense oligonucleotides, ribozymes and immunotherapy. Such inhibitors have been shown to block ras activation in cells containing wild-type mutant ras , thereby acting as antiproliferation agents. Ras oncogene inhibition is discussed in Scharovsky, et al. (2000), Journal of Biomedical Science. 7(4) 292-8;

Ashby, M.N. (1998), Current Opinion in Lipidology. 9 (2) 99 - 102; and BioChim. Biophys. Acta, (1989) 1423(3): 19-30.

As mentioned above, antibody antagonists to receptor kinase ligand binding may also serve as signal transduction inhibitors. This group of signal transduction pathway inhibitors includes the use of humanized antibodies to the extracellular ligand binding domain of receptor tyrosine kinases. For example, Imclone C225 EGFR specific antibody (see Green, el al., Monoclonal Antibody Therapy for Solid Tumors, Cancer Treat. Rev., (2000), 26(4), 269-286); Herceptin erbB2 antibody (see’’Tyrosine Kinase Signaling in Breast cancenerbB Family Receptor Tyrosine Kinases”, Breast Cancer Res., 2000, 2(3), 176-183); and 2CB VEGFR2 specific antibody (see Brekken, et al.,“Selective Inhibition of VEGFR2 Activity by a monoclonal Anti-VEGF antibody blocks tumor growth in mice”, Cancer Res. (2000) 60, 5117-5124).

Anti-angiogenic agents: Anti-angiogenic agents including non-receptorMEKngiogenesis inhibitors may also be useful. Anti-angiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the anti-vascular endothelial cell growth factor antibody bevacizumab [AVASTIN], and compounds that work by other mechanisms (for example linomide, inhibitors of integrin anb3 function, endostatin and angio statin);

Immuno therapeutic agents: Agents used in immunotherapeutic regimens may also be useful in combination with the compounds of formula (I). Immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines and approaches using anti- idiotypic antibodies

Proapoptotic agents: Agents used in proapoptotic regimens ( e.g ., bcl-2 antisense oligonucleotides) may also be used in the combination of the present invention.

Cell cycle signaling inhibitors: Cell cycle signaling inhibitors inhibit molecules involved in the control of the cell cycle. A family of protein kinases called cyclin dependent kinases (CDKs) and their interaction with a family of proteins termed cyclins controls progression through the eukaryotic cell cycle. The coordinate activation and inactivation of different cyclin/CDK complexes is necessary for normal progression through the cell cycle. Several inhibitors of cell cycle signaling are under development. For instance, examples of cyclin dependent kinases, including CDK2, CDK4, and CDK6 and inhibitors for the same are described in, for instance, Rosania, el ah, Exp. Opin. Ther. Patents (2000) 10(2):215-230.

In one embodiment, the combination of the present invention comprises an 0X40 binding protein or antibody and a TLR4 modulator and at least one anti-neoplastic agent selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine MEK angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, and cell cycle signaling inhibitors.

In one embodiment, the combination of the present invention comprises an 0X40 binding protein and a TLR4 modulator and at least one anti-neoplastic agent which is an anti microtubule agent selected from diterpenoids and vinca alkaloids.

In a further embodiment, the anti-neoplastic agent is a diterpenoid.

In a further embodiment, the anti-neoplastic agent is a vinca alkaloid. In one embodiment, the combination of the present invention comprises an 0X40 binding protein and a TLR4 modulator and at least one anti-neoplastic agent, which is a platinum coordination complex.

In a further embodiment, the anti-neoplastic agent is paclitaxel, carboplatin, or vinorelbine.

In one embodiment, the combination of the present invention comprises an 0X40 binding protein and a TLR4 modulator and at least one anti-neoplastic agent which is a signal transduction pathway inhibitor.

In a further embodiment, the signal transduction pathway inhibitor is an inhibitor of a growth factor receptor kinase, VEGFR2, TIE2, PDGFR, BTK, erbB2, EGFr, IGFR-l , TrkA, TrkB, TrkC, or c-fms.

In a further embodiment, the signal transduction pathway inhibitor is an inhibitor of a serine/threonine kinase rafk, akt, or PKC-zeta.

In a further embodiment, the signal transduction pathway inhibitor is an inhibitor of a non- receptor tyrosine kinase selected from the src family of kinases.

In a further embodiment, the signal transduction pathway inhibitor is an inhibitor of c-src.

In a further embodiment, the signal transduction pathway inhibitor is an inhibitor of Ras oncogene selected from inhibitors of farnesyl transferase and geranylgeranyl transferase.

In a further embodiment, the signal transduction pathway inhibitor is an inhibitor of a serine/threonine kinase selected from the group consisting of PI3K.

In a further embodiment, the signal transduction pathway inhibitor is a dual EGFr/erbB2 inhibitor, for example N- {3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (structure below):

In one embodiment, the combination of the present invention comprises a compound of formula I or a salt or solvate thereof and at least one anti-neoplastic agent which is a cell cycle signaling inhibitor. In further embodiment, cell cycle signaling inhibitor is an inhibitor of CDK2, CDK4, or CDK6.

In one embodiment the mammal in the methods and uses of the present invention is a human.

As indicated, therapeutically effective amounts of the combinations of the invention (an 0X40 binding protein or antibody and a TLR4 modulator) are administered to a human. Typically, the therapeutically effective amount of the administered agents of the present invention will depend upon a number of factors including, for example, the age and weight of the subject, the precise condition requiring treatment, the severity of the condition, the nature of the formulation, and the route of administration. Ultimately, the therapeutically effective amount will be at the discretion of the attendant physician.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, controls. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The following examples are intended for illustration only, and are not intended to limit the scope of the invention in any way. EXAMPLES

Example 1: Treatment of 0X86 Monotherapy in a CT-26 syngeneic mouse model for colon cancer

The CT26 mouse colon carcinoma (CT26.WT; ATCC #CRL-2638) cell line was obtained from ATCC. It is an N-nitroso-N-methylurethane-(NNMU) induced, undifferentiated colon carcinoma cell line known in the art. For example, it is described in: Wang M, et al., Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor-associated antigen, J. Immunol. 154: 4685-4692, 1995 (PubMed: 7722321). Rat IgGl was obtained from Bioxcell. 0X86 (Hybridoma 134) cells were obtained from the European Cell Culture collection and manufactured by Harlan; 0X86 is the name for a tool anti-OX40 monoclonal antibody used in rodents; it is a rodent antibody that binds rodent 0X40, e.g., mouse 0X40 (receptor).

0X86 and rat IgGl were diluted in diluted DPBS.

For preparation of tumor cells, a frozen (-l40°C) vial of CT-26 (mouse colon carcinoma cells), from ATCC (cat# CRL-2638, lot# 59227052) were thawed and cultured in basic RPMI (with 10% FBS) media over the following week.

CT-26 cells (passage 12) were harvested from the flask in complete medium. Cells were centrifuged and resuspended in RPMI (without FBS), this step is repeated 3 times. Cell density and viability were checked via trypan blue exclusion. Cells were then diluted to desired density (5xl0⁵ cells per mL) and kept on ice.

Escalating doses of 0X40 monoclonal antibody (mAh) 0X86 were evaluated for their efficacy in reducing tumor growth. Animals were weighed and inoculated on the right hind quarter with 0.5x10⁵ CT26 tumor cells per mouse on Day 0. A total of 130 mice were inoculated with tumor cells - assuming 30% failure rate (either too big or too small at time of start of study), the goal was to have h=10 for each group. After tumor cell inoculation, tumor growth and total body weight are measured 3 times a week for the duration of the study. Randomization occurred on day 10 or 11 when the average tumor volume was approximately 100 mm³. Beginning on the day of randomization, animals were dosed with 0X86 mAh or Rat IgGl isotype i.p. biweekly, for a total of 6 doses. Mice remained on study until tumors reach >2000 cu mm for two consecutive

measurements, they were removed from study for other reasons (/.<?., weight loss >20%, ulceration on tumor, etc.) or until the end of the study. After euthanization the tumors were removed and subject to dissociation for flow analysis and/or FFPE for IHC analysis.

Day 0: sc inoculation with tumor cells

Days 1, 4, 6, 8: Animals were weighed and checked for tumors and if present, tumors measured.

Randomization day (approx day 10): Animals were randomized and placed into cages representing appropriate groups

Dosing, biweekly through end of study: Animals were dosed ip with 0X86 or anti Rat IgGl, where the amounts shown above were on a per mouse basis.

Measurements, triweekly through end of study: Animals were weighed and tumors were measured

The mean tumor weights from about 10 animals were averaged. Error bars show SEM analysis. P values were calculated based on the following: P value tested the null hypothesis that the survival curves were identical in the overall populations. In other words, the null hypothesis is that the treatment did not change survival. Raw p- values adjusted for multiple comparisons via the Stepdown Bonferroni method

The above protocol was used to generate the results in Figure 1B, and results of the individual mice can be found in Figure 4. These figures demonstrate that mice inoculated with CT-26 cells and treated with rat IgGl developed tumors that grew unabated as expected, whereas dosing with 0X40 monoclonal antibody (mAh) 0X86 led to clear inhibition of tumor growth and increased survivability when compared to the rat IgGl control group.

Example 2: Results of CT-26 study with treatment with TLR4 (CRX-527)

The addition of TLR4 modulators such as CRX-527 to the above 0X40 monotherapy treatment protocol were used to study TLR4 monotherapy and the combination of anti- mOX40 immunotherapy with TLR4 modulators.

Day 0: sc inoculation with tumor cells

Days 1, 4, 6, 8: Animals were weighed and checked for tumors and measured.

Dosing, biweekly through end of study: Animals dosed ip with TLR compound CRX-527 at amounts shown above (per mouse), or vehicle

Measurements, triweekly through end of study: Animals weighed and tumors measured.

The above protocol was used to generate the results in Figure 1 A and Figures 2-6 at the dosages indicated. In almost every case, Balb/c mice that were inoculated with 0.5x10⁵ CT-26 colorectal tumor cells on the right hind quarter developed tumors that, when treated i.p. with vehicle (2% glycerol) only, and progressed as expected. TLR 4 agonists CRX-527 (Figures 2-5) and CRX-601 (Figure 6) inhibited tumor growth in a dose- dependent manner when compared to the vehicle treated animals. Dose dependence was also seen in the survivability of the model.

Example 3: Combination Treatment with 0X40 (Le., OX-86, an antibody raised against rodent 0X40 receptor) and CRX-527

The following treatment schedule was performed:

Day 0: SC inoculation with tumor cells

Days 1, 4, 6, 8: Animals checked for tumors and if present, tumors measured. Study enrollment day (approx day 10): Animals randomized and received treatment 1.

Biweekly post enrollment: starting with day of enrollment, mice received i.p. dose biweekly for a total 6 doses.

Triweekly through end of study: Animals weighed and tumors measured

When 0X86 treatment was combined with TLR4 modulator treatment (CRX-527), mice exhibited a higher reduction in tumor burden and survived longer than either treatment alone.

Example 4: Monotherapy and Combination Treatment with anti-mOX40R antibody and TLR4 targeting molecules of Formula I Mice were administered 0X40 antibody; a compound of Formula 1 (including a compound of Formula la, CRX-527, CRX-547, and CRX-601 (TLR4 agonists), or a combination of both. Each treatment has significant anti-tumor activity.

There are at least two significant findings. First, in mice, anti-OX40R or combination of anti-OX40 antibody and TLR4 agonist combination each delayed the growth of established CT-26 tumors relative to an untreated control group. Secondly, in mice significant anti-tumor effect was observed in TLR4 agonist and anti-OX40R antibody combinations as compared to monotherapy treatment.

Example 5: Combination Treatment with an OX40R ABS (Le., anti-mOX40 receptor antibody clone OX-86, an antibody raised against rodent 0X40 receptor) and CRX-601

Materials and Methods

In vivo anti-tumor efficacy studies

The in vivo anti -tumor efficacy of the TLR4 agonist (CRX601) was assessed in the murine CT-26 colon carcinoma syngeneic solid tumor model as a monotherapy and in combination with a rate anti-mouse 0X40 antibody clone 0X86. Seven to eight week old female Balb/c mice (B ALB/c AnNCrl, Charles River) were used in these studies. Murine CT-26 colon carcinoma cells (ATCC catalog number CRL-2638 lot# 59227052) were cultured in RPMI growth medium supplemented with 10% fetal bovine serum (FBS) in a humidified 37°C incubator with 5% CO 2. CT-26 cells cultured in logarithmic growth were harvested from tissue culture flasks and centrifuged for 5 minutes at 450xg at 4°C for ten minutes to pellet cells. The supernatant was discarded, and cells were washed in ice cold phosphate buffered saline (PBS) without calcium and magnesium and centrifuged again for 5 minutes at 450xg at 4°C for ten minutes to pellet cells. The cells were resuspended in sterile RPMI media without FBS and adjusted to a cell concentration of 500,000 cells/mL. 100 mΐ of the cell stock was implanted via subcutaneous injection into the right flank of each Balb/c mouse. After ten or eleven days when the average tumor size reached approximately 100 mm³, mice were randomized into study cohorts according to tumor size and the first treatment dose was given. The TLR4 agonist (CRX601) or vehicle was dosed via a systemic intravenous or direct intratumoral injection as indicated. The CRX-601 vehicle used for intravenous and intratumoral dosing was 0.5% where indicated. For CRX-601 liposomal intratumoral dosing, a DOPC/CHOL liposome prepared by GSK Lot #1783-157-B was used. The rat anti mouse 0X40 receptor antibody (clone 0X86) (expressed and purified in-house from the rat hybridoma Grits ID 50776, BP232 2013) or Rat IgGl isotype control antibody (BioXCell catalog # BE0088) was dosed via an intraperitoneal injection given twice per week for a total of six doses. Caliper measurements were taken three times per week to assess tumor growth, and mice with tumors <2,000 mm³ were maintained on study from 30 up to approximately 1 15 days. Mice with tumors >2,000 mm³ for 2 consecutive measurements or mice with tumors which formed open ulcers were removed from the study. Tumor volume was calculated using the formula (0.52) x (Length) x (Width²). In studies 6 and 7, tumor-free mice were re-challenged with CT-26 tumor cells as described above, on the opposite flank from the original inoculation site and tumor growth was monitored, as described above. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and Use Committee at GSK.

Immune phenotyping and cytokine analysis

Tumors, blood and tissues were harvested from CT-26 mice on day 0, day 1 and day 8 after first CRX-601 dosing. Mouse white blood cells and dissociated tumor single cells were stained freshly with surface or intracellular staining antibodies for multicolor flow cytometry analysis for immune phenotyping. Multiplex cytokine analysis was performed using mouse plasma samples from the same study.

Statistical Analysis

For studies 1-4, to determine significance of tumor growth inhibition, tumor volumes at 1 1 (study 1), 15 (studies 2 and 3), or 19 (study 4) days after first dose were compared between the different treatment groups. Prior to the analysis, tumor volumes were natural log transformed due to the inequality of variance in the different treatment groups.

ANOVA followed by pair-wise comparison was then carried out on the log transformed data. SAS 9.3 and R 3.0.2 analysis software was used. Kaplan-Meier (KM) method was carried out to estimate the survival probability of different treatment groups at a given time. The event for survival analysis was tumor volume of 2000 mm³ or tumor ulceration, whichever came first. The exact time to cut-off volume was estimated by fitting a linear line between log tumor volume and day of two observations, the first observation that exceed the cut-off volume and the one observation that immediately preceded the cut-off volume. The median time to endpoint and its corresponding 95% confidence interval was calculated. Whether or not KM survival curves were statistically different between any two groups was then tested by log-rank test. The raw p-value, as well as the false discovery rate (FDR) adjusted p-values, from the comparisons of days to events by survival analysis and the comparisons of log transformed tumor volume at indicated days between treatment groups was determined. The ones with FDR adjusted p-values < 0.05 were declared to be statistically significant.

For studies 6 and 7, to determine significance of tumor growth inhibition, tumor volumes at 12 days after first dose were compared between the different treatment groups.

Treatments were compared by standard ANOVA methods followed by FDR adjustment for multiplicity. Response is square root of volume, for homoscedasticity (equal variance) reasons. Kaplan-Meier (KM) method was carried out to estimate the survival probability of different treatment groups at a given time. For these survival analyses, “Death” means crossing the tumor volume cutoff (2000 mm³). “Survival” means proportion of mice not“Dead”, and“Survival time” means days until“Death”. If a mouse crossed the volume cutoff between two measurement days, then the day of“death” was estimated by linear interpolation. If a mouse crossed the volume cutoff more than once, the first crossing was used. Treatments were compared by the standard log-rank test for two treatments. The log-rank p-values were adjusted for multiplicity using the FDR (false discovery rate) method. Significance was defined as FDR < 0.05. All calculations and graphs were done using R software, version 3.2.3.

Results

Six studies (Studies 1 through 4 and Studies 6 through 7) were conducted to assess tumor size and survival time in mice treated with CRX601 and rat anti-mouse 0X40 Receptor antibody clone 0X86, both alone and in combination with each other. One additional study (Study 5 below) was conducted to assess cytokine release and T cell activation in mice treated with CRX601 and rat anti-mouse 0X40 Receptor antibody clone 0X86, both alone and in combination with each other.

Study 1

In order to determine CRX-601 monotherapy activity with intratumoral dosing, mice were inoculated with 5xl0⁴ CT-26 cells and randomized into groups of 10 listed below when tumor size reached approximately 100 mm³ as described in Materials and Methods.

Group 1 : Vehicle dosed intratumoral twice per week for 6 doses total

Group 2: CRX-601 0.1 pg/mouse dosed intratumoral twice per week for 6 doses total

Group 3: CRX-601 1 pg/mouse dosed intratumoral twice per week for 6 doses total

Group 4: CRX-601 10 pg/mouse dosed intratumoral twice per week for 6 doses total

Group 5: CRX-601 50 pg/mouse single dose

With intratumoral dosing, dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) was observed for the TLR4 agonist CRX-601 in the CT-26 syngeneic mouse tumor model. The 10 pg and 50 pg dosed mice showed statistically significant (*p-values < 0.05) tumor growth inhibition 11 days after the initial dose compared to vehicle. Results are shown in Figure 18.

Mice treated with the TLR4 agonist CRX-601 in this study also showed a statistically significant increase in survival time. The 50 pg dosed mice showed a statistically significant (*p-values < 0.05) increase in survival compared to vehicle by day 42 post CT26 tumor cell inoculation when the study was ended. On this day, only mice from the 50 pg and 10 pg CRX-601 groups remained on study. Three of the four mice in the 50 pg group were tumor-free, with the fourth mouse showing a tumor volume of 854.19 mm³. The single mouse remaining in the 10 pg group was tumor-free (see Figure 19).

Study 2

In order to determine CRX-601 monotherapy activity with intravenous dosing, mice were inoculated with 5x10⁴ CT-26 cells and randomized into groups of 10 below when tumor size reached approximately 100 mm³ as described in Materials and Methods. Group 1 : Vehicle dosed intravenous twice per week for 6 doses total

Group 2: CRX-601 1 pg/mouse dosed intravenous twice per week for 6 doses total Group 3: CRX-601 10 pg/mouse dosed intravenous twice per week for 6 doses total Group 4: CRX-601 100 pg/mouse single dose

With intravenous dosing, dose-dependent anti-tumor activity (as measured by tumor growth inhibition over time) was observed for the TLR4 agonist CRX-601 in this CT-26 syngeneic mouse tumor model. The 10 pg and 100 pg dosed mice showed statistically significant (*p-values < 0.05) tumor growth inhibition 15 days after the initial dose compared to vehicle (see Figure 20).

Mice treated with the TLR4 agonist CRX-601 in this CT-26 syngeneic mouse tumor model also showed statistically significant increase in survival compared with vehicle. The 100 pg dosed mice showed a statistically significant increase (*p-values < 0.05) in survival compared to vehicle when the study was ended on day 32 post CT-26 tumor cell inoculation. One of the three mice remaining in this group was tumor- free, while the other mice showed tumor volumes of 1500.49 and 962.61 mm³. The single mouse remaining in the 10 pg dose group had a tumor volume of 188.0 mm³. (See Figure 21)

Study 3

In order to determine CRX-601 activity alone and in combination with anti-OX40, mice were inoculated with 5xl0⁴ CT-26 cells and randomized into groups of 10 below when tumor size reached approximately 100 mm³ as described in Materials and Methods.

Group 1 : Vehicle dosed intravenous once per week for 3 doses total

Group 2: Rat IgGl 10 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 3: 0X86 25 pg/mouse dosed twice per week for 6 doses total

Group 4: CRX-601 10 pg/mouse dosed intravenous once per week for 3 doses total

Group 5: CRX-601 25 pg/mouse dosed intravenous once per week for 3 doses total Group 6: CRX-601 10 pg/mouse dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 7: CRX-601 25 pg/mouse dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Anti-tumor activity was assessed (as measured by tumor growth inhibition over time) for 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed intravenous twice per week for 6 doses total, 10 pg or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in this CT-26 syngeneic mouse model. The sub-optimal monotherapy CRX-601 doses of 10 pg/mouse or 25 pg/mouse dosed once per week did not show statistically significant tumor growth inhibition when dosed alone compared to vehicle, nor did the 0X86 25 pg/mouse dose compared to Rat IgGl . However, CRX601 dosed intravenous once per week at 10 pg or 25 pg/mouse for 3 doses total in combination with 25 pg/mouse 0X86 dosed twice per week for 6 doses total showed statistically significant (*p- values < 0.05) tumor growth inhibition 15 days after the initial dose compared to vehicle and Rat IgGl controls, and compared to CRX601 and 0X86 monotherapies (see Figure 22).

In this CT-26 syngeneic mouse model study, survival advantage was also determined for mice treated with 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX- 86), dosed intravenous twice per week for 6 doses total, 10 pg or 25 pg of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both. On day 106 post CT-26 tumor cell inoculation when the study was ended, CRX-601 10 pg and 25 pg/mouse dosed intravenous lx/week for 3 doses total in combination with 25 pg/mouse 0X86 dosed 2x/week for 6 doses total showed a statistically significant (^Up values < 0.05) increase in survival compared to both vehicle and Rat IgGl controls, and compared to 0X86 and CRX-601 monotherapies. The three remaining mice in the CRX- 601 25 pg/mouse + 0X86 group were tumor-free, and the one mouse in the CRX-601 10 pg/mouse + 0X86 group was tumor-free (see Figure 23).

Study 4

Study 3 was repeated with 25 pg/mouse of CRX-601 alone and in combination with anti- 0X40. Mice were inoculated with 5xl0⁴ CT-26 cells and randomized into groups of 10 below when tumor size reached approximately 100 mm³ as described in Materials and Methods.

Group 1 : Vehicle dosed intravenous once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 2: CRX-601 25 pg/mouse dosed intravenous once per week for 3 doses total

Group 3: Vehicle dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 4: CRX-601 25 pg/mouse dosed intravenous once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 5: CRX-601 25 pg/mouse dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Anti-tumor activity was observed (as measured by tumor volume over time) for

25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed intravenous twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. CRX601 dosed intravenous once per week at 25 pg/mouse for 3 doses total in combination with 25 pg/mouse 0X86 dosed twice per week for 6 doses total showed statistically significant (*p- values < 0.05) tumor growth inhibition compared to CRX601 and 0X86 monotherapies (see Figure 24).

Survival curves were measured for mice treated with 25 pg/mouse of a rat anti-mouse 0X40 receptor antibody (clone OX-86), dosed intravenous twice per week for 6 doses total, or 25 pg/mouse of TLR4 agonist CRX-601 dosed intravenous lx/week for 3 doses total, and the combination of both in a CT-26 syngeneic mouse model. CRX601 25 pg/mouse dosed intravenous lx/week for 3 doses total in combination with 25 pg/mouse 0X86 dosed 2x/week for 6 doses total showed a statistically significant (p-values < 0.05) increase in survival compared to monotherapies. This statistical analysis was conducted on day 64 post tumor cell inoculation when all remaining mice were tumor- free. These mice were monitored until study end on day 1 11. On this day, seven mice in Group 5 CRX-601 25 pg/mouse + 0X86 remained tumor-free, two mice in Group 3 CRX-601 25 pg/mouse + Rat IgGl remained tumor-free, and one mouse in Group 4 Vehicle + 0X86 remained tumor-free (see Figure 25).

Study 5

Results are the mean of five animals per cohort.

Leukocytes and immune-activation was assessed in mice treated with 10 pg of TLR4 agonist CRX-601, 25 pg of a rat anti-mouse 0X40 receptor antibody (clone OX-86), and the combination of both in a CT-26 syngeneic mouse model of colon cancer measured at 8 days post dosing. A significant increase of tumor-infiltrating leukocytes was observed in mice treated with CRX-601 and anti-OX86 in combination. A synergistic increase of expression of T cell activation marker CD25 on circulating CD4 T cells was observed in mice treated with CRX-601 and anti-OX86 in combination. A synergistic increase of T cell activation associated markers CTLA4, PD1 and ICOS on circulating CD4 T cells was observed in mice treated with CRX-601 and anti-OX86 in combination. Results are shown in Figure 26 A-C.

An increase of immune-activating cytokines TNF alpha and IL-l2p70 was observed in mice treated with 10 pg of TLR4 agonist CRX-601, a rat anti-mOX40R antibody (OX- 86), and the combination of both in a CT-26 syngeneic mouse model of colon cancer measured at 1 and 8 days post dosing. IL-l2p70 was only detectable at 8 days post dosing as shown in Figure 27B. Results are shown in Figures 27 A-B.

Study 6

To compare CRX-601 activity alone and in combination with anti-OX40 when CRX-601 was dosed either (IV) or intratumoral (IT) in a 0.5% glyceroF4% dextrose vehicle, mice were inoculated with 5xl0⁴ CT-26 cells and randomized into groups of 10 below when tumor size reached approximately 100 mm³ as described in Materials and Methods.

Group 2: CRX-601 25 pg/mouse dosed intravenous once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total Group 3: 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 4: CRX-601 25 pg/mouse dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 5: Vehicle dosed intratumoral once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 6: CRX-601 25 pg/mouse dosed intratumoral once per week for 3 doses total +

Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 7: CRX-601 25 pg/mouse dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal (i.p.) twice per week for 6 doses total

Anti-tumor activity was assessed (as measured by tumor growth inhibition over time) for treatment groups. The sub-optimal monotherapy CRX-601 dose of 25 pg/mouse did not show statistically significant tumor growth inhibition when dosed intravenous (Group 2) or intratumoral (Group 6) compared to corresponding control groups (Group 1 and Group 5 respectively). The monotherapy 0X86 25 pg/mouse dose did not show statistically significant tumor growth inhibition compared to control Groups 1 and 5 either. However, the CRX601 25 pg/mouse dose given intravenous in combination with the 0X86 25 pg/mouse i.p. dose (Group 4) showed statistically significant (*p- values < 0.05) tumor growth inhibition 12 days after the initial dose compared to control Group 1 and 0X86 monotherapy Group 3. The CRX601 25 pg/mouse dose given intratumoral in

combination with the 0X86 25 pg/mouse i.p. dose (Group 7) also showed statistically significant (*p-values < 0.05) tumor growth inhibition 12 days after the initial dose compared to control Group 5 and 0X86 monotherapy Group 3. The combination of CRX601 25 pg/mouse dosed intravenous (Group 4) or intratumoral (Group7) with 0X86 25 pg/mouse i.p. was not statistically significant compared to the CRX601 monotherapy Group 2 or Group 6 for tumor growth inhibition in this study (See Figures 28 and 29).

In this CT-26 syngeneic mouse model, study survival advantage was also determined. 68 days after the first dose, the combination of CRX601 25 pg/mouse dosed intravenous (Group 4) or intratumoral (Group 7) with 0X86 25 pg/mouse i.p. showed a statistically significant (*p-values < 0.05) increase in survival compared to its control Group 1 or Group 5 respectively. The intravenous dose of CRX-601 in combination with 0X86 i.p. (Group 4) resulted in 6 out of 10 mice tumor- free, and the intratumoral dose of CRX-601 in combination with 0X86 i.p. (Group 7) resulted in 3 out of 10 mice tumor- free. The monotherapy groups did not show a statistically significant increase in survival compared to control groups (see Figures 30 and 31). Naive control mice and fully regressed tumor- free mice on day 68 were re-challenged with CT26 tumor cells. CT26 tumors grew as expected in naive control mice, but were rejected with no tumor growth in the treatment group mice. This indicates a persistent anti-tumor memory immunity due to CRX-601 or CRX-601 in combination with 0X86 treatment (see Figure 32). The two mice in the 0X86 monotherapy Group 3 on day 68 had tumor volumes of 27.86 and 1576.27 mm³, and were not re-challenged.

Study 7

To compare CRX-601 activity alone and in combination with anti-OX40 when CRX-601 was dosed either intravenous (IV) using a 0.5% GlyceroF4% dextrose vehicle, or intratumoral (IT) using a DOPC/CHOL liposomal formulation, mice were inoculated with 5xl0⁴ CT-26 cells and randomized into groups of 10 below when tumor size reached approximately 100 mm³ as described in Materials and Methods

Group 1 : Vehicle (0.5% GlyceroP4% dextrose) dosed intravenous once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 2: CRX-601 25 pg/mouse (in 0.5% GlyceroF4% dextrose) dosed intravenous once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 3: Vehicle (0.5% GlyceroF4% dextrose) dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 4: CRX-601 25 pg/mouse (in 0.5% GlyceroF4% dextrose) dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total Group 5: Vehicle (DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 6: Vehicle (DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 7: CRX-601 25 pg/mouse (in DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 8: CRX-601 25 pg/mouse (in DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Anti-tumor activity was assessed (as measured by tumor growth inhibition over time) for treatment groups 12 days after the initial dose. The sub-optimal monotherapy CRX-601 dose of 25 pg/mouse showed statistically significant (*p- values < 0.05) tumor growth inhibition when dosed intravenous (Group 2) or intratumoral (Group 7, liposomal formulation) compared to corresponding control groups (Group 1 and Group 5 respectively). The monotherapy 0X86 25 pg/mouse IP dose Group 3 and Group 7 also showed statistically significant (*p- values < 0.05) tumor growth inhibition compared to control Groups 1 and 5. The CRX601 25 pg/mouse dose given intravenous in combination with the 0X86 25 pg/mouse i.p. dose (Group 4) showed statistically significant (*p-values < 0.05) tumor growth inhibition compared to control Group 1 and 0X86 monotherapy Group 3. The CRX601 25 pg/mouse dose given intratumoral with the DOPC/CHOL liposomal formulation in combination with the 0X86 25 pg/mouse i.p. dose (Group 8) also showed statistically significant (*p- values < 0.05) tumor growth inhibition compared to control Group 5. The combination of CRX601 25 pg/mouse dosed intravenous (IV) (Group 4) or intratumoral (IT) (Group 8) with 0X86 25 pg/mouse IP was not statistically significant compared to the CRX601 monotherapy Group 2 or Group 7 for tumor growth inhibition in this study on day 12 (See Figures 33 and 34).

In this CT-26 syngeneic mouse model study, survival advantage was also determined 80 days after the first dose. CRX601 dosed as a monotherapy IV (Group 2), or dosed IV in combination with 0X86 i.p. (Group 4) showed a statistically significant (*p-values < 0.05) increase in survival compared to control Group 1. Groups 2 and 4 had 5 out of 10 mice each showing full tumor regressions (see Figure 35). Both CRX601 dosed as a monotherapy intratumoral with the DOPC/CHOL liposome formulation (Group 7), and the 0X86 monotherapy with the liposomal intratumoral control (Group 6) showed a statistically significant (*p- values < 0.05) increase in survival compared to control Group 5. The intratumoral CRX601 DOPC/CHOL liposomal formulation dose in combination with 0X86 IP (Group 8) showed a statistically significant (*p-values < 0.05) increase in survival compared to control Group 5, as well as compared to the CRX601 intratumoral (Group 7) and 0X86 (Group 6) monotherapy control groups. 9 out of 10 mice were fully regressed and tumor- free in the intratumoral CRX601 DOPC/CHOL liposomal dose in combination with 0X86 i.p., compared to 3 and 2 mice in the intratumoral monotherapy control Groups 6 and 7. Thus, synergy was observed with the intratumoral CRX601 liposomal formulation dose in combination with 0X86 compared to the intratumoral control monotherapy Groups 6 and 7 (see Figure 36). Naive control mice and fully regressed tumor-free mice on day 80 were re-challenged with CT26 tumor cells. CT26 tumors grew as expected in naive control mice, but were rejected with no tumor growth in the treatment group mice. This result indicates a persistent anti-tumor memory is due to CRX-601 or CRX-601 in combination with 0X86 treatment (see Figure 37). This lack of tumor growth indicates a persistent anti -tumor memory due to CRX-601 or CRX-601 in combination with 0X86 treatment (see Figure 37).

Study 8

An abscopal effect is described as distant tumor regression after a local tumor treatment. In order to asses abscopal effects, mice were inoculated with 5x10⁴ CT-26 cells on the left flank, and 5xl0⁴ CT-26 cells on the right flank as described in Materials and Methods for single tumor inoculation. Thus, in this study, each mouse possessed two tumors, one on the right flank, and one on the left flank. Mice were randomized into groups of 10 as shown below when tumor size reached approximately 100 mm³ for the right flank, and left flank tumor size was similar. To determine abscopal effect of CRX-601 activity alone and in combination with anti-OX40, CRX-601 was dosed intratumoral (IT) in the left flank tumor only using a DOPC/CHOL liposomal formulation or a 0.5% glycerol/4% dextrose formulation. Tumor size was monitored for both the right and left flank tumors. In addition, CRX-601 was dosed intravenous (IV) using a 0.5% glycerol/4% dextrose vehicle, alone and in combination with anti-OX40 as a control for systemic activity (Group 7).

Group 1 : Vehicle (0.5% glycerol/4% dextrose) dosed intravenous once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 2: Vehicle (0.5% glycerol/4% dextrose) dosed intratumoral once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 3: CRX-601 25 pg/mouse (in 0.5% glycerol/4% dextrose) dosed intravenous once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 4: CRX-601 25 pg/mouse (in 0.5% glycerol/4% dextrose) dosed intratumoral once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 5: Vehicle (0.5% glycerol/4% dextrose) dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 6: Vehicle (0.5% glycerol/4% dextrose) dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 7: CRX-601 25 pg/mouse (in 0.5% glycerol/4% dextrose) dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 8: CRX-601 25 pg/mouse (in 0.5% glycerol/4% dextrose) dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total Group 9: Vehicle (DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 10: Vehicle (DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 11 : CRX-601 25 pg/mouse (in DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + Rat IgGl 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Group 12: CRX-601 25 pg/mouse (in DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total

Anti-tumor activity was assessed (as measured by tumor growth inhibition over time) for treatment groups. Mice were removed from study if either or both tumors reached 2,000 mm³. By study day 60 post first dose, all mice remaining on study were completely tumor free, and abscopal effect and survival advantage was determined. For the systemic dosing combination Group 7, CRX-601 25 pg/mouse (in 0.5% Glycerol/4% dextrose) dosed intravenous once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total, 7 out of 10 mice were tumor free for both right and left flank tumors (Figure 38). For the combination Group 8, CRX-601 25 pg/mouse (in 0.5% Glycerol/4% dextrose) dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total, 3 out of 10 mice showed full tumor regression for both tumors, even though only the left flank tumor received intratumoral injection (Figure 39). For the combination Group 12, CRX- 601 25 pg/mouse (in DOPC/CHOL Liposome) dosed intratumoral once per week for 3 doses total + 0X86 25 pg/mouse dosed intraperontoneal twice per week for 6 doses total, 5 out of 10 mice showed full tumor regression for both tumors, even though only the left flank tumor received intratumoral injection (Figure 40). Thus, CRX-601 formulations dosed intratumoral in combination with 0X86 dosed intraperontoneal demonstrated an abscopal effect (Groups 8 and 12). The local left flank tumor IT injection resulted in distant right flank tumor regression. There was no statistical difference in survival advantage between the three combination groups 7, 8, and 12. Group 7 demonstrated a statistically significant increase in survival compared to all vehicle and isotype controls, and also compared to all CRX-601 and 0X86 monotherapy groups (***p-values <

0.006). The Group 12 combination showed a statistically significant increase in survival compared to Group 10 Liposome Vehicle IT + 0X86 (**p-values = 0.006), although it was not statistically significant versus the Group 1 1 CRX-601 25 pg/mouse Liposome formulation + Rat IgGl (p-values = 0.119). The Group 8 combination showed a statistically significant increase in survival compared to Group 4 CRX-601 25 pg/mouse (in 0.5% Glycerol/4% dextrose) IT + Rat IgGl (*p- values = 0.013), although it was not statistically significant versus the Group 6 Vehicle (0.5% Glycerol/4% dextrose) IT + 0X86 (p-values = 0.5). Figure 41 shows the survival curves for all groups.

Example 6: 0X40 expression induced by CRX601 treatment with a range of concentrations (0.01 - 1000 ng/mL) on human CD4+ T cells (A), dendritic cells (B), and monocytes (C) at 24 hours in in vitro cell culture.

Experiment description:

In vitro human peripheral blood mononuclear cell (PBMC) assay was performed to assess the effect of CRX601 on 0X40 expression. Freshly isolated human PBMCs were checked for viability and were cultured in AIM-V serum free media at a density of two million cells per well in a 24- well non tissue culture treated plate. PBMCs were stimulated with a dose concentration (O.Ol pg/mL - 1,000 pg/mL, including a vehicle blank) of CRX-601 for 24 hours. By the end of incubation, cells were collected for flow cytometry assessment of 0X40 expression. The quick upregulation of 0X40 receptor expression by CRX601 on T cells, dendritic cells and monocytes demonstrated that CRX601 upregulates the target of anti-OX40 antibody, which may potentiate the therapeutic activity of anti-OX40 antibody and lead to the synergistic anti-tumor activity of TLR4+OX40 combination in vivo (Figure 42A, Figure 42B, and Figure 42C).

Example 7: A Phase I, Open-Label Study of CRX-601 in Combination with

Immunotherapies in Participants with Selected Advanced Solid Tumors 1. Synopsis

Protocol Title: A Phase I, Open-Label Study of CRX-601 in Combination with Immunotherapies in Participants with Selected Advanced Solid Tumors

Short Title: Study of combinations of CRX-601 and immunotherapies in participants with advanced solid tumors

Rationale: The combination of two or more immunotherapies holds promise in treating patients with cancer. One model of a“cancer-immune cycle” describes a series of feed forward steps by which the immune system recognizes and kills tumor cells, a cycle which is counterbalanced by tumor and host derived factors which suppress anti-tumor immune activation [Chen, 2013; Chen, 2017] These steps include:

Release of cancer cell antigens

Cancer antigen presentation

Priming and activation

Trafficking of T-cells to tumors

Infiltration of T-cells into tumors

Recognition of cancer cells by T-cells

Killing of cancer cells

Rational combination strategies, such as immunotherapies acting at different steps in the immune cycle, could produce meaningful and synergistic activity compared to monotherapies [Hoos, 2016] Combining a TLR4 agonist with a checkpoint modulator targets two complementary steps in the cancer-immunity cycle; TLR engagement results in the production of various inflammatory cytokines/chemokines such as tumor necrosis factor (TNF)a, interleukin (IL) 6, granulocyte colony-stimulating factor (G-CSF), and type I interferons (i.e., IFNa, IFNP) and enhanced uptake, processing, and presentation of antigens. Based on nonclinical data, the combinations of CRX-601 (a TLR4 agonist) and ANTIBODY 106-222 (an 0X40 agonist) is anticipated to have antitumor activity greater than either of the monotherapies.

Objectives and Endpoints:

Overall Design:

This is a Phase I, open-label, non-randomized, multicenter and multi-country study designed to evaluate the safety, tolerability, PK, pharmacodynamic, and preliminary clinical activity of CRX-601 administered in combination with other immunotherapies to participants with advanced solid tumors.

In Part 1 , the safety and tolerability of escalating doses of CRX-601 and a single dose level of a monoclonal antibody (mAh) combination partner (ANTIBODY 106-222) will be evaluated in separate cohorts of participants with advanced solid tumor cancers according to an Neuenschwander-Continual Reassessment Method (N-CRM) design to identify doses for evaluation in Part 2 [Neuenschwander, 2008] Part 1 will include a CRX-601 run-in period of 2 weeks (i.e., CRX-601 administration on Days 1 and 8) prior to administration of the combination partner beginning at Day 15. Approximately 5 dose levels of CRX-601 in combination with a single fixed dose level of the combination partner are planned to be evaluated in Part 1. Following protocol amendment, CRX-601 may also be further evaluated by additional routes of administration.

In Part 2, expansion cohorts of approximately 6 to 15 participants with squamous cell carcinoma of the head and neck (SCCHN) will be enrolled in the combination treatment arm to further evaluate the safety and activity of dose(s) identified in Part 1. The dose(s) of CRX-601 administered in combination with 24 mg ANTIBODY 106-222 will be determined based on data from Part 1 and may differ for the combination treatment. Following protocol amendment, additional expansion cohorts in other tumor types may be enrolled, based on emerging nonclinical and clinical data.

In addition, PK/Pharmacodynamic cohorts for the combination will be opened to enrollment during Part 1 to obtain additional PK and pharmacodynamic data, with an emphasis to obtain insight on the potential impact of the combination treatment on the immune cells and status of the tumor microenvironment, in conjunction with PK and pharmacodynamic markers obtained from blood. Tumor biopsies are required for enrollment to the PK/Pharmacodynamic cohorts, whereas biopsies are strongly encouraged but not mandatory for Part 1 dose escalation cohorts. For the combination, participants in the PK/Pharmacodynamic cohorts may be enrolled to any dose level which has already been completed and supported by adequate safety and tolerability from dose escalation for the combination. Up to a maximum of 45 participants may be enrolled into the PK/Pharmacodynamic cohorts with up to approximately 6 per dose level for the combination.

Treatment Groups and Duration:

Participants will receive the combination of CRX-601 with ANTIBODY 106-222. In Part 1 , escalating doses of CRX-601 will be evaluated as guided by the N-CRM approach. In Part 2, participants will receive a single dose level of CRX-601 as identified based on data from Part 1 , in combination with ANTIBODY 106-222.

The study includes a screening period, a treatment period, and a follow-up period.

Participants will be screened for eligibility beginning 4 weeks before the start of treatment. The duration of study treatment is expected to be up to 2 years. For participants that discontinue study treatment prior to a determination of progressive disease (PD), the follow-up period will include disease assessments every 12 weeks until documented PD occurs (PFS Follow Up [FU]). Following PD or for participants that discontinue study treatment for PD, participants will be contacted every 12 weeks to assess survival status (Survival FU [SFU]) for up to 2 years from the start of the study treatment.

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3. Introduction

3.1. Study Rationale

The model of a“cancer-immune cycle” describes a series of feed-forward steps by which the immune system recognizes and kills tumor cells, a cycle which is counterbalanced by tumor and host derived factors which suppress anti-tumor immune activation [Chen,

2013; Chen, 2017] The steps of tumor immune recognition and killing include release of cancer cell antigens, cancer antigen presentation, priming and activation, trafficking of T- cells to tumors, recognition of cancer cells by T-cells, and killing of cancer cells. Immune suppressive factors which may be operative in tumor microenvironment include checkpoint pathways ( e.g ., programmed death receptor- 1 [PD-l], cytotoxic T- lymphocyte-associated antigen 4 [CTLA-4]) and a range of immunosuppressive factors (e.g., IDO, TGF-b), as well as immune inhibitory cell populations including T regulatory (Treg) cells, myeloid derived suppressor cells, and immune suppressive macrophages (M2 -macrophages). Thus, cancer persistence and growth results from aberrant cell replication together with a relative imbalance in favor of immune suppressive factors as compared to anti -tumor immune activating responses.

The therapeutic benefit of blocking the immuno-inhibitory checkpoint pathways PD- 1 and CTLA-4 recently has been demonstrated across multiple tumor types, yielding durable responses in some patients. However, a majority of patients do not respond to monotherapy with checkpoint inhibitors, and strategies to increase their activity by combination approaches are being actively explored. It is also possible that

immunotherapies acting at different steps in the cycle and on different cells and pathways could have improved therapeutic indices over currently available monotherapies.

Furthermore, engaging novel pathways and combinations may provide therapeutic options for patients wherein the pre-existing host and tumor microenvironment factors do not favor response to PD-l or CTLA-4.

Toll-like receptors (TLRs) are a family of‘sensor’ proteins primarily expressed on certain immune and epithelial cells that function as activators of innate immunity in response to microbial-related molecules known as Pathogen- Associated Molecular Patterns (PAMPs). PAMPs include molecules such as nucleic acids, flagellar proteins, and lipopolysaccharide (LPS), the prototypical ligand for TLR4. Ligand-driven activation of TLRs results in the production of various inflammatory cytokines and chemokines such as tumor necrosis factor (TNF)a, IL-6, IL-8, IP- 10, G-CSF, interferons (IFNs), and enhanced uptake, processing, and presentation of antigens by antigen presenting cells. Further effects of TLR4 agonism observed in animal models of cancer include reduction of Treg cells and promotion of macrophage phenotypic switching from an immunosuppressive M2 phenotype to an immune active Ml phenotype. CRX-601 is a synthetic TLR4 agonist that is being developed by GlaxoSmithKline as an immunological adjuvant to be administered in combination with other immune system modulators for the treatment of cancers. CRX-601 is not being developed as a monotherapy given the lack of robust anti-tumor activity that has been reported for the drug class in participants with advanced malignancies [Guha, 2012; Weihrauch, 2015; Pashenkov, 2006] Therefore, the first-time in human (FTIH) study of CRX-601 was performed in healthy participants to evaluate preliminary safety, PK, pharmacodynamics, and to identify a pharmacologically active starting dose to initiate studies in cancer patients.

The adverse event (AE) profile in the CRX-601 FTIH study was characterized by cytokine-related effects such as flu-like symptoms and changes in temperature and heart rate (see below). One (1) study participant experienced an elevation in transaminases (see below). Overall, the clinical profile of CRX-601 as evaluated in healthy participants was consistent with that anticipated by the repeat dose GLP toxicology studies in rats and monkeys and with the profiles of other TLR agonists reported in both healthy participants and cancer patients [Kanzler, 2007; Astiz ME, Rackow EC, Still JG, Howell ST, Cato A, Von Eschen KB, et al. Pretreatment of normal humans with monophosphoryl lipid A induces tolerance to endotoxin: a prospective, double -blind, randomized, controlled trial. Crit Care Med. 1995 Jan;23(l):9-l7.

Bahador, 2007; Schmoll, 2014; Northfelt, 2014; Isambert, 2013] Data from the FTIH study was used to support the starting dose and dose rationale in the present study (see below).

Other attractive immunotherapy anticancer targets include costimulatory pathways that enhance T-cell function. 0X40 is one such costimulatory receptor expressed primarily on activated CD4+ and CD8+ T-cells. 0X40 agonists have been shown to increase antitumor immunity and improve tumor- free survival in non-clinical models, and 0X40 agonist monoclonal antibodies (mAbs) are currently being evaluated in Phase I clinical trials. ANTIBODY 106-222 is a humanized wild-type IgGl anti-OX40 agonistic mAh being developed for the treatment of advanced malignancies.

This study will assess the safety, PK, pharmacodynamics, and preliminary clinical activity of CRX-601 administered in combination with other immunotherapies to participants with advanced solid tumors. ANTIBODY 106-222 is well-suited for combination with CRX-601 based on mechanisms of action targeting complementary nodes of the cancer-immunity cycle and compelling antitumor activity observed in preclinical models. Subsequent combination partners and/or additional routes of administration may be evaluated (following protocol amendment/s) based on biologic rationale, nonclinical data, and/or emerging clinical data.

3.2. Brief Background 3.2.1. CRX-601 Background

An overview of CRX-601 is provided below.

CRX-601 was initially developed in the course of structure-activity studies on LPS (also known as‘endotoxin’), the naturally occurring ligand of TLR4. CRX-601 is a monosaccharide from the aminoalkyl glucosaminide 4-phosphate class of compounds intended for use as a vaccine adjuvant or an immune modulator. CRX-601 is an agonist of TLR4 that induces immunologic responses in vitro and in vivo. CRX-601 , as a single agent, stimulates cytokine production (in vitro and in vivo), changes in immune cell populations (in vivo) and generates fever response (in vivo).

3.2.1.1. CRX-601 Nonclinical Activity

CRX-601 has shown immunomodulatory activity in multiple in vitro and in vivo models. CRX-601 added to whole blood ex vivo induces cytokine production, and when administered to BALB/c mice, induces phenotypic trends in peripheral blood leukocytes including decreased regulatory T-cells (Tregs), increased T-cell activation, and expansion of myeloid cells and monocyte/macrophages. CRX-601 administered to CT-26 tumor bearing BALB/c mice resulted in an increase in survival compared to control groups.

The in vitro and in vivo pharmacology of CRX-601 is consistent with other TLR agonists [Kanzler, 2007] In vitro cytokine induction (IL-l p, IL-6, IP-10 and TNFa) by CRX-601 is similar to that of LPS. In rabbits, a species used for assessing endotoxin contamination of parenteral formulations due to their high sensitivity, CRX-601 produced a transient increase in body temperature similar to that which occurs following LPS administration. In the repeat dose intravenous toxicity studies in rats and monkeys, CRX-601 was associated with the expected pro-inflammatory actions of a TLR4 agonist. The primary systemic effect seen with weekly dosing of CRX-601 in rat (dosing up to 4 weeks) and monkey (dosing up to 6 weeks) studies was increased levels of specific cytokines; all other findings were transient and considered secondary to this primary response.

Adverse findings were only noted in rat and include microscopic changes in the heart valves and lymphocytic inflammatory cell infiltrates in the liver.

The no-observed-adverse-effect-level (NOAEL) is 15 pg/kg/dosc and 200 pg/kg/dose, in the rat and monkey, respectively. Based on the predicted human exposure at the highest planned clinical dose of 250 ng (predicted maximum concentration [C max] IS

approximately 0.036 ng/mL, and predicted area under the plasma concentration-time curve [AUC] is approximately 1.21 ng-h/mL), the margin to the NOAEL dose in rat is approximately 2061X for Cmax and 214X for AUC and in monkey is approximately 40,000X for C_max and 24,l32X for AUC.

3.2.1.2. CRX-601 Development Plan and Clinical Experience This protocol describes a study evaluating the combination of CRX-601 with other immunotherapies. The study will be the second evaluation of CRX-601 in humans and the first in participants with cancer.

CRX-601 is not planned for development as a monotherapy in cancer participants given that the TLR agonist drug class has not produced robust monotherapy antitumor activity in multiple prior clinical trials of participants with advanced malignancies. However, the safety, PK, and pharmacodynamics results from the FTIH study support the design and conduct of a clinical trial in cancer participants where the benefits of CRX-601 are more likely to be realized as an adjuvant in combination with other immune therapies with complementary modes of action. Should the combination(s) demonstrate robust anti tumor activity and a favorable safety profile, monotherapy study arms could be added by future amendment to explore the relative contributions of the study treatments.

Like CRX-601, other TLR agonists including the prototypical TLR4 agonist, LPS, have been evaluated in both healthy participants and cancer patients for experimental and therapeutic purposes. Administration causes dose-dependent increases in cytokines including TNFa, IL-6, and IL-8, which peak within 2 to 4 hours and return to normal within 24 hours. In cases where the same TLR agonist has been administered to both healthy participants and to cancer participants, the PK and pharmacodynamic profiles have been similar for the two populations [Riella, L.V., S. Dada, L. Chabtini, B. Smith,

L. Huang, P. Dakle, B. Mfarrej, F. D'Addio, L.-T. Adams, N. Kochupurakkal, A.

Vergani, P. Fiorina, A.L. Mellor, A.H. Sharpe, H. Yagita, and I. Guleria. 2013. B7h (ICOS-L) Maintains Tolerance at the Fetomaternal Interface. The American Journal of Pathology 182:2204-2213.

Schmidt, 2015; Dietsch, 2014] Clinical safety data for the drug class is characterized by a predictable tolerability profile of transient fever and flu-like symptoms ( e.g ., chills, nausea, malaise, etc.) attributable to cytokine production [Astiz ME, Rackow EC, Still JG, Howell ST, Cato A, Von Eschen KB, et al. Pretreatment of normal humans with monophosphoryl lipid A induces tolerance to endotoxin: a prospective, double-blind, randomized, controlled trial. Crit Care Med. 1995 Jan;23(l):9-l7.

Bahador, 2007] A review of studies comprising thousands of healthy participants that have been administered LPS notes that long-term toxicities have not been described [Astiz ME, Rackow EC, Still JG, Howell ST, Cato A, Von Eschen KB, et al.

Pretreatment of normal humans with monophosphoryl lipid A induces tolerance to endotoxin: a prospective, double -blind, randomized, controlled trial. Crit Care Med. 1995 Jan;23(l):9-l7.

Bahador, 2007] Although elderly participants have greater decreases in blood pressure as compared to young participants administered 2 ng/kg doses of LPS [Kanzler H, Barrat FJ, Hessel EM, Coffman RL. Therapeutic targeting of innate immunity with Toll-like receptor agonists and antagonists. Nat Med. 2007 May;l3(5):552-9. Review.

Krabbe, 2001], the difference in sensitivity has not prevented administration of even higher doses (5 ng/kg) to cancer participants [Engelhardt, 1991] At these doses, peak TNFa and IL-6 levels can exceed 5000 pg/ml, and further dose escalation has been limited by rising transaminase levels [Engelhardt, 1991] In other trials of TLR4 agonists in participants with advanced malignancies, cytokine-associated SAEs such as bronchospasm and hypotension have been reported [Vosika, 1984; de Bono, 2000] Overall, the cytokine-associated adverse events associated with TLR agonists overlaps minimally with the profiles of checkpoint modulators. Thus, the safety profile of TLR agonists is well suited for administration in combination with other immune therapies such as checkpoint modulators.

The FTIH study of CRX-601 was a randomized, double-blind (sponsor unblinded), placebo-controlled, ascending dose and parallel group study in healthy participants. In Part 1 , single IV bolus doses of placebo or CRX-601 at 7, 21, 60, or 100 ng were administered (n=6 CRX-601, n=2 placebo per dose level, except the 60 ng cohort which was repeated, i.e., n=l2 CRX-601 , n=4 placebo). Dose escalation was stopped, per protocol, following the 100 ng cohort, in which 3 of 6 participants experienced AEs of moderate intensity. In Part 2, participants were to receive repeat doses of CRX-601. However, Part 2 was not started following an elevation in transaminases on study day 30 for 1 participant in cohort 4 (60 ng) of Part 1 (see details herein).

Preliminary PK assessments of CRX-601 were performed based on available data. No quantifiable concentrations were observed at the 7 ng dose (assay lower limit of quantification [LLOQ] = 2 pg/mL), and insufficient PK concentrations were above the LLOQ at the 21 ng dose to enable calculation of AUC. Median peak concentrations (C_max) at doses 21 (n=6), 60 (n=l2) and 100 ng (n=6) doses were 3.90, 10.02 and 23.26 pg/mL, respectively. The C_max values from these three single doses were evaluated for dose-proportionality using the power model loge (PK parameter) = a + b * loge (dose) where“a” is the intercept and“b” is the slope and was fitted by restricted maximum likelihood using SAS Proc Mixed. An estimate of the slope with corresponding 90% confidence interval (Cl) was obtained from the power model to assess the degree of dose- proportionality, wherein a slope equal to 1.0 is indicative of dose-proportionality. The 90% confidence interval for the slope was (0.84, 1.20) and (0.96, 1.24) with inclusion and without inclusion, respectively, of a participant from the 100 ng dose cohort who showed an approximately 3-fold lower C_max compared to other participants from this cohort.

These intervals were contained within the interval (0.8, 1.25) indicating that slope is around unity implying that dose-proportionality is observed for C_max within this dose range. Half-life could not be reliably estimated for lower dose groups due to limited data above the assay LLOQ. The median half-life calculated from the 100 ng dose is ~72 h, which is in agreement with expectations based on extrapolation of preclinical data. The safety profile of CRX-601 in the FTIH study, included AEs consistent with cytokine production and was generally qualitatively similar to profiles of other TLR agonists. Based on preliminary, unblinded safety data, as of 7 days after completing dosing in the 100 ng cohort, the most common clinical findings were influenza- like illness (10 participants), body temperature increased (4 participants), abdominal pain, back pain, dizziness, headache, oropharyngeal pain, presyncope, (2 participants). No other AEs were observed in more than 1 participant. The frequency of safety observations increased with dose as described below.

• Following administration of 7 or 21 ng CRX-601 (6 participants in each group), the reported AEs and changes in vital signs resembled the placebo group.

• Following administration of 60 ng CRX-601 (12 participants), mild influenza-like symptoms or headache were reported for 7 participants. One (1) participant had moderate abdominal cramps considered possibly related to study treatment. One (1) participant experienced a 58 bpm increase in heart rate to a maximum of

125 bpm. One (1) participant experienced a 12-fold increase in alanine aminotransferase (ALT) (as described herein).

• Following administration of 100 ng CRX-601 (6 participants), influenza-like symptoms were reported for 5 participants. Three (3) participants experienced moderate AEs considered at least possibly related to study treatment by the investigator, including orthostatic presyncope, muscle tremor, back pain, nausea, dizziness, influenza-like symptoms. Because 3 of 6 participants experienced AEs of moderate intensity dose escalation was stopped, per protocol.

3.2.I.2.I. Transaminase elevation

One participant received a single 60 ng IV dose of CRX-601 and experienced a 12-fold increase in ALT. The time course of the increase was notable in that the participant had a slightly elevated ALT immediately prior to dosing (53 U/L; upper limit of normal [ULN] = 50 U/L), and the value remained of low grade but trended upward to 89.6 U/L on day 7 post-dose and 122.5 U/L on day 21. On day 32, the ALT increased to 563.4 U/L, and it peaked on day 34 (626.7 U/L) before declining. Beginning with day 35, total bilirubin was elevated (29.9 pmol/L; ULN = 21 pmol/L). Day 35 aspartate aminotransferase (AST), indirect bilirubin, and direct bilirubin were 254.5 U/L (ULN = 50 U/L), 25.8 pmol/L (ULN = 17.6 pmol/L), and 4.1 pmol/L (ULN = 3.4 pmol/L), respectively. The participant experienced no other AEs on the trial and only had mild increases in body temperature (0.6°C) and heart rate (14 bpm) relative to predose values. A thorough evaluation by a hepatologist including comprehensive laboratory studies and liver ultrasound provided no additional insight into the elevations in hepatic laboratory values. Of note, the participant had multiple elevations in transaminases and total bilirubin before or during other clinical trials at the investigative site, although all were of low grade (maximum ALT <4-fold ULN; maximum total bilirubin < 1.5-fold ULN). The elevation in transaminases and total bilirubin was considered possibly related to CRX-601 by the investigator. In addition, the sponsor, in consultation with external hepatolo gists, considered an undefined, underlying, low-grade hepatic pathology to possibly have contributed to the elevation in transaminases, given the participant’s history. A potential role for CRX-601 as contributing to the observed increases cannot be ruled out based on the available information.

Transaminases were routinely measured in all participants in the study, and no other participant experienced an increase.

3.2.2. ANTIBODY 106-222 Background

An overview of ANTIBODY 106-222 is provided below. Detailed information concerning the biology, pharmacology, PK, and safety characteristics can be found in the IB.

ANTIBODY 106-222 is a humanized wild-type IgGl anti-OX40 agonistic mAh.

ANTIBODY 106-222 demonstrated several mechanisms of action in vitro including promoting effector CD4+ T-cell proliferation, inhibiting the induction of IL- 10-producing CD4+ Type 1 regulatory (Trl) cells and blocking the suppressive function of natural Tregs (nTregs), and binding to the fragment crystallizable region (Fc) receptor (FcR), which is anticipated to augment 0X40 signaling via cross-linking of the antibody via the Fc domain on FcR positive cells. Importantly, it has been shown that 0X40 activation gives a costimulatory signal to T-cells, dependent on a T-cell receptor (TCR)

engagement, suggesting that ANTIBODY 106-222 is not a super agonist in the models tested which is supported by available clinical experience.

3.2.2.1. ANTIBODY 106-222 Nonclinical Activity

ANTIBODY 106-222 (and mouse surrogate antibodies) have shown activity in multiple in vitro and in vivo models. ANTIBODY 106-222 demonstrated several mechanisms of action in vitro, including promoting effector CD4+ T-cell proliferation, inhibiting the induction of IL- 10 producing CD4+ Trl cells and blocking the suppressive function of nTregs, and binding to FcR, which is anticipated to augment 0X40 signaling via cross- linking of the antibody via the Fc domain on FcR positive cells. A surrogate mAh to murine 0X40 (0X86) was administered to female BALB/c mice bearing CT26 mouse colon carcinoma tumors, and produced an increase in survival compared to control groups. Together, these data provide rationale for ANTIBODY 106-222 to be used as an immunotherapy for the treatment of cancer.

The cynomolgus monkey was demonstrated to be an appropriate toxicology species due to human comparability with 0X40 protein sequence identity, ANTIBODY 106-222 binding and activity. ANTIBODY 106-222 was well tolerated in monkey toxicology studies following weekly IV dosing for up to 4 weeks at doses up to 100 mg/kg/week with no adverse test article-related findings noted. Anti -ANTIBODY 106-222 anti drug antibodies (AD As) were detected in most monkeys given <10 mg/kg; however, the ability to determine toxicity in the terminal necropsy animals was not compromised by AD As due to the fact that robust target engagement was observed. No infusion reactions were observed in monkeys, including those with ADA.

3.2.2.2. ANTIBODY 106-222 Clinical Experience

Eighty-two (82) participants with advanced solid tumors have been treated in the ongoing FTIH study 201212 as of 13 August 2017. Of the 82 participants, 45 were treated with ANTIBODY 106-222 monotherapy across 6 dose levels of (0.003 0.01, 0.3, 1, 3, and 10 mg/kg), and 39 participants were treated with the combination of ANTIBODY 106-222 and pembrolizumab 200 mg, at ANTIBODY 106-222 doses of 0.003 0.01, 0.3, 1 mg/kg. Two participants crossed over from monotherapy to combination therapy. To date, ANTIBODY 106-222 was well tolerated in humans with no indication of dose-related increases in AEs based on current data.

In the ANTIBODY 106-222 monotherapy study arm, no dose-limiting toxicities or treatment-related Grade 3, Grade 4, or Grade 5 toxicities were reported. A single event led to treatment discontinuation, Grade 3 aphasia due to Grade 5 stroke. The most common AEs were fatigue (1 1, 24%), back pain (9, 20%), diarrhea (9, 20%), nausea (8, 18%), asthenia (8, 18%), vomiting (8, 18%), anemia, (7, 16%), headache (5, 11%), dyspnoea (5, 11%), myalgia (4, 11%), pain in extremity (5, 11%), and pyrexia (5, 1 1%). The most common treatment related AEs included diarrhea (5, 11%) and fatigue (5,

1 1%).

In the ANTIBODY 106-222 combination study arm, no treatment related Grade 4 or Grade 5 toxicities were reported. Two patients reported Grade 3 treatment-related AEs. One patient with head and neck cancer treated with 1 mg/kg ANTIBODY 106-222 and 200 mg pembrolizumab reported treatment related Grade 3 AEs of asthenia and infusion reaction, both attributed to study treatment and occurring over one month apart from another. A second patient with triple-negative breast cancer (TNBC) treated with 3 mg/kg ANTIBODY 106-222 reported Grade 3 lymphopenia attributed to study treatment. One dose limiting toxicity of Grade 2 pleural effusion was reported in a patient with TNBC. The only AE leading to treatment discontinuation was Grade 3 fatigue. The most common AEs were pleural effusion (9, 23%), fatigue (6, 15%), decreased appetite, (5, 13%), pyrexia (5, 13%), arthralgia (4, 10%), and dyspnoea (4, 10%). The most common treatment related AEs included fatigue (5, 13%) and nausea (3, 8%).

Of note, there was no evidence for an acute cytokine release syndrome (CRS) associated with ANTIBODY 106-222 administration at any dose tested, despite having a measured receptor occupancy >80% in peripheral blood in the immediately post-dose timeframe for all tested doses (see Figure 45 for additional information on ANTIBODY 106-222 receptor occupancy). This is consistent with the mechanistic understanding of 0X40 providing a costimulatory signal to T-cells dependent on a TCR engagement and not functioning as a super agonist. Overall, the safety profile of ANTIBODY 106-222 does not appear to have overlapping toxicity with TLR agonists.

3.2.3. CRX-601 Combination Background

3.2.3.1. CRX-601 In Vivo Studies

Efficacy Studies

Based on the potentially complementary mechanisms of action of ANTIBODY 106-222 and CRX-601, the combination was evaluated in B ALB/c mice implanted with syngeneic CT-26 tumor xenografts. Four groups of 10 BALB/c mice with intact immune systems were implanted with CT-26 tumors. The mice received one of the following treatments: placebo, CRX-601 (TLR4 agonist), 0X86 agonist (mouse surrogate 0X40 agonist antibody), or the combination of CRX-601 and 0X86 agonist. While the monotherapies had modest effects on tumor growth (Figure 43), the combination treatment of CRX-601 and 0X86 produced greater activity and durable responses.

The reduction in tumor volume translated to significant improvement in the survival of animals (Figure 44). Approximately 70% of animals that received the combination of CRX-601 and 0X86 survived more than 100 days. By contrast, 10-20% of the animals that received either monotherapy survived 100 days.

These preclinical efficacy results for CRX-601 have been reproducible across a range of studies ( e.g ., testing of different doses, dose frequencies, and routes of administration), and together with in vitro and in vivo pharmacologic data, provide rationale for evaluating the combination of CRX-601 with ANTIBODY 106-222 in a clinical trial for the treatment of cancer.

Safety Studies

In a Safety PK/Pharmacodynamic study, groups of 3 cynomolgus monkeys were administered CRX-601 (2 pg/kg) monotherapy or CRX-601 in combination with ANTIBODY 106-222 (10 mg/kg). Changes in IL-6, IL-8, IL-10, MCP-l , and IP-10 were observed, with peak cytokine values at 2 hours post-dose of test articles. Minimal changes from baseline were observed for TNFa, IFNy, IL-2, and I L- 1 b . The ratios (combinatiommonotherapy) of median values (from lowest to highest) at 2 hours post dose were 0.59, 0.77, 1.4, 1.6, and 2.5 for MCP-l, IL-8, IP-10, IL-6, and lL-lO, respectively. Overall, cytokine values were variable and the distributions were generally overlapping when comparing the monotherapy and combination arms. There were no changes in hematology or clinical pathology parameters.

A review of the nonclinical toxicology findings with both CRX-601 and ANTIBODY 106-222 given as single agents indicate that additional combination toxicology studies in monkeys would not likely provide additional relevant clinical risk assessment data. 3.2.3.2. CRX-601 In Vitro Studies

The potential of CRX-601 alone and in combination with ANTIBODY 106-222 to induce cytokine release in vitro was evaluated in PBMCs isolated from 10 healthy human donors (5 males and 5 females) under conditions of no anti-CD3 stimulation (resting) and with anti-CD3 stimulation (pre-stimulation), which was previously shown to upregulate 0X40 surface expression on T-cells. Cytokines (IL-2, IL-6, IL-10, IFNy, and TNFa) were measured following 24 hours incubation with CRX-601 (10, 100 or 1000 pg/mL) and ANTIBODY 106-222 (0. 01, 0.1 , 1 or 10 pg/mL). Anti-CD28 and human IgGl were included as positive and negative controls, respectively. Positive cytokine induction by CRX-601 and ANTIBODY 106-222 combination was defined as >3-fold increase above that of CRX-601 alone. No effects on cytokine responses in resting or pre-stimulated PBMCs were detected at 10 pg/mL CRX-601 in combination with any concentration of ANTIBODY 106-222. For concentrations >100 pg/mL CRX-601, addition of 1 pg/mL ANTIBODY 106-222 to resting PBMCs produced minimal to mild increases (approx. 2-4 fold) in IL-6, IL-10, and/or TNFa in 2/10 donors, and in 1/10 donors in pre-stimulated PBMCs incubated with 1000 pg/mL CRX-601 in combination with >1 pg/mL

ANTIBODY 106-222. There was high donor-to-donor variability in cytokine responses, and individual donors had inconsistent concentration-response curves amongst the various incubation conditions. These results suggest that the potential for enhanced cytokine release when these agents are administered in combination in patients appears to be low, but cannot be eliminated due to the high amount of data variability.

3.3. Benefit/Risk Assessment

The following section outlines the risk assessment and mitigation strategy for this protocol.

3.3.2. Overall Benefit: Risk Conclusion

This is an open-label, dose escalation study and the first study of the combination of CRX- 601 with ANTIBODY 106-222 conducted in humans; this study will enroll participants with advanced solid tumors. There is biologic rationale to study the combination for the treatment of cancer based on complementary modes of action on the immune system, and antitumor activity of the combination that exceeds activity of the monotherapies in preclinical models. However, it is unknown if the combination will have clinical activity for patients with cancer.

Based on nonclinical in vivo and ex vivo combination evaluations and clinical experience to date, and the conservative starting dose of CRX-601, the safety profiles of the combination is not expected to exceed that of the monotherapies. In contrast to experience to date with ANTIBODY 106-222, it is expected that increasing the dose of CRX-601 past a certain threshold will be associated with DLTs given the TLR4 agonist mechanism of action and experience with other TLR agonists including LPS.

Consistent with other Phase 1 trials for the treatment of cancer, a target DLT frequency has been set as 16-33%, and a Bayesian adaptive dose escalation design for CRX-601 is employed to efficiently determine the dose(s) associated with this DLT frequency. In addition, it is possible that infrequent events unrelated to CRX-601 dose, such as increases in hepatic laboratory values, might be observed. This risk will, in part, be mitigated by a run-in period for CRX-601 prior to the initiation of combination study treatment. The run- in provides an evaluation of monotherapy CRX-601 safety and tolerability in participants with cancer and prevents the administration of combination therapy to participants that experience, with CRX-601 alone, a DLT, unacceptable tolerability, or an increase in ALT to 1 5x ULN and 1 5x baseline. Overall, the benefit: risk is typical of a Phase I study of participants with advanced cancer.

. Objectives and Endpoints

5. Study Design

5.1. Overall Design

This is a Phase I, open-label, non-randomized, multicentre, multi-country study designed to evaluate the safety, tolerability, PK, pharmacodynamic, and preliminary clinical activity of CRX-601 administered in combination with other immunotherapies to participants with advanced solid tumors.

The study will be conducted in two parts. Part 1 is a treatment arm based on the CRX-601 combination partner. The treatment arm may have up to 5 dose escalation cohorts to investigate the safety and tolerability of escalating doses of CRX-60lwith a single dose level of the combination partner. CRX-601 combination partner is:

ANTIBODY 106-222: 24 mg (Part la, Part 2a)

Part 1 will include a CRX-601 run-in period of 2 weeks (i.e., CRX-601 administration on days 1 and 8) prior to administration of the combinations beginning on day 15 (Week 3). Following protocol amendment, CRX-601 may also be evaluated by additional routes of administration. Safety data will be evaluated according to a Neuenschwander-Continual Reassessment Method (N-CRM) design [Neuenschwander, 2008] to help identify a dose for investigation in Part 2.

Part 2a is also a treatment arm for the expansion cohorts. Expansion cohorts of

approximately 6 to 15 participants with SCCHN will be enrolled to the combination to further evaluate safety and activity of the dose regimen(s) identified in Part la. The dose(s) of CRX-601 administered with 24 mg ANTIBODY 106-222 will be determined based on data from Part la. Following protocol amendment, additional expansion cohorts in other tumor types may be enrolled based on emerging nonclinical and/or clinical data.

For the combination of CRX-601 in Part la, PK/Pharmacodynamic cohorts will be opened at cleared dose levels for that combination (i.e. the most recent investigated dose level that supported dose escalation) to explore the potential relationships between dose, biological effects in the tumor microenvironment, and tumor response. A particular emphasis in the PK/Pharmacodynamic cohort is placed on evaluating the possible effects of the

combination on the immune cells and immune status within the tumor microenvironment. Thus, to be eligible for the PK/Pharmacodynamic cohort, participants must consent to mandatory fresh biopsy collection at baseline and on treatment (see above, So A). An additional radiographic disease assessment (see above, SoA) will support exploratory investigation of tumor growth kinetics in this cohort. Note that while consent to fresh tumor biopsy is not required for participation in the dose escalation cohorts in Part la, it is strongly encouraged. Up to 6 participants per dose level may be enrolled into the

PK/Pharmacodynamic cohort for each combination. The study includes a screening period, a treatment period, and a follow-up period.

Participants will be screened for eligibility beginning 4 weeks before the start of treatment. The duration of study treatment will be up to 2 years. For participants that discontinue study treatment prior to a determination of PD, the follow-up period includes disease assessments every 12 weeks until documented PD. Following PD or for participants that discontinue study treatment for PD, participants will be contacted every 12 weeks to assess survival status for 2 years from the start of the study.

Participants with confirmed PR or CR will be followed for response duration and may be eligible (outside of Canada) for continued study treatment at the time of

relapse/progression. The decision whether a participant will receive additional treatment will be discussed and agreed upon by the treating investigator and the Sponsor/Medical Monitor on a case-by-case basis.

Following protocol amendment(s), additional participants may be enrolled to evaluate additional routes of study treatment administration (e.g., intratumoral administration), additional agents to be used in combination with CRX-601, or additional indications, based on emerging nonclinical and/or clinical data.

5.1.1. Part 1: Dose Escalation of CRX-601 administered in combination with

ANTIBODY 106-222

In Part 1 , dose escalation will be performed to identify combination dose levels comprising CRX-601 with 24 mg ANTIBODY 106-222 (Part la. One (1) dose level of ANTIBODY 106-222 with up to 5 dose levels of CRX-601 are planned for evaluation, pending emerging safety and tolerability information as dose escalation proceeds.

Part 1 will include a run-in period of 2 weeks in which CRX-601 is administered once- weekly [i.e., administration on day 1 (Week 1) and day 8 (Week 2)] prior to initiation of combination treatment with ANTIBODY 106-222 beginning on day 15 (Week 3). During the run-in period, participants that experience a DLT, unacceptable toxicity, or an increase in ALT (1 5x ULN and 1 5x baseline) and not attributable to another cause will be discontinued from the study and will not receive CRX-601 in combination (as described herein).

Guidance for the management of toxicity, including dose modification algorithms, is provided below.

The starting schedule for CRX-601 will be at every l-week intervals (Ql W) from Week 1 through Week 12 including the 2-week monotherapy run in period (Week 1 and Week 2) (see SoA Table 1). Subsequently, CRX-601 will be administered at every 3-week intervals (Q3W) to coincide with ANTIBODY 106-222 dosing. Thus, beginning with Week 12 for Part 1 and Week 13 for Part 2, both CRX-601 and combination partners will be

administered on the same study day at a frequency of Q3W. Cohorts will be opened beginning with 50 ng CRX-601 administered in combination with either 24 mg ANTIBODY 106-222. Three (3) or more participants will be enrolled in each cohort. The total number of participants enrolled into each cohort and dose assignments will be guided by safety information (as described herein) from participants receiving the study treatment combinations according to N-CRM modelling (as described herein,

[Neuenschwander, 2008]).

Sequential cohorts will be enrolled and dose escalation (or de-escalation) will proceed guided by an N-CRM design. Dose escalation for each cohort will proceed independently of the other cohorts. The first 3 participants at each dose level will receive study treatment at least 3 days apart (e.g., if the first participant in a cohort were dosed on Monday, the earliest the next participant could be dosed is Thursday). Once the 6-week DLT evaluation period has been completed (as described herein), N-CRM analysis will be performed to guide the dose level to which the next 3 participants will be assigned based on DLT frequency (as described herein). The number of participants allocated to any cohort is an estimate; participants may also be allocated to PK/Pharmacodynamic cohorts at a previous dose level that supported dose escalation.

Dose levels -1 are available for ANTIBODY 106-222 (8 mg +50 ng CRX-601) if the target toxicity level is exceeded in Cohort 1 and a dose reduction is needed below planned doses.

5.1.1.1. Description of the Continual Reassessment Method

The N-CRM model-based design is a Bayesian adaptive dose escalation scheme that assumes a 2-parameter logistic model for the toxicity rate as a function of dose. It is a modified version of the original Continual Reassessment Method proposed by [O’ Quigley, 1990] The N-CRM method is fully adaptive and makes use of all DLT information, therefore is expected to locate the target dose level efficiently. In this case, the model will be applied to the dose escalation decision for CRX-601, which will be performed independently for each combination.

Dose escalation decisions will be held after participants within any given cohort have been observed for at least 6 weeks after starting the study treatment (as described herein). At the time of each dose escalation decision, the Fixed and Adaptive Clinical Trial Simulator (FACTS [Tessella, Abington, United Kingdom]) will be used to obtain the posterior probabilities for the DLT rate. The N-CRM estimates for each potential dose will provide the posterior probabilities that the DLT rate lies in each of four toxicity ranges:

The recommended dose for dose escalation, based on the N-CRM model, will be the dose with the highest posterior probability of lying in the target toxicity interval with the additional requirement that the sum of the posterior probabilities of the DLT rate lying in the excessive toxicity or unacceptable toxicity range is less than 25%. An updated estimate of the toxicity curve will be provided at the time of each dose escalation meeting. Note that de-escalation as well as escalation is possible using this method. Dose escalation will continue until conditions for either scenario (i) or (ii) are met:

i) Six participants have been treated at the current target dose

AND

For the current dose level, the posterior probabilities of the DLT rate lying within the excessive toxicity interval or within the unacceptable toxicity interval sum to less than 25%

AND

For the next higher dose, the posterior probabilities of the DLT rate lying within the excessive toxicity interval or within the unacceptable toxicity interval sum to greater than 25%. ii) No doses are usable (i.e., for all doses, the posterior probabilities of the DLT rate lying within the excessive toxicity interval or within the unacceptable toxicity interval sum to more than 25%)

AND

At least 2 DLTs have been observed.

Dose recommendations based on the N-CRM analysis will be used as guidance. To ensure safety of participants, additional participants may be enrolled at a current dose level at the discretion of the study investigators and sponsor, even though a higher dose is

recommended by N-CRM analysis.

5.1.1.2. Logistic Model for N-CRM

A two-parameter logistic model will be used for N-CRM analysis for dose level selection during the dose escalation phase. This model will estimate the probability of observing a DLT at each dose level in the study as DLT information becomes available.

The logistic model that used for describing the dose-toxicity relationship is:

where p_d is the probability of DLT at dose d, and d_m is a reference dose, and a and b are Bayesian priors. 5.1.2. PK/Pharmacodynamic Cohort(s)

Characterizing the effects of treatment on the tumor microenvironment is essential to the understanding the mechanism of action of CRX-601 and its combination partners at the site of action. Thus, for each combination of CRX-601 in Part 1, PK/Pharmacodynamic cohorts will be opened to characterize the biological effects in the tumor microenvironment and explore the potential relationships between dose and tumor response.

PK/Pharmacodynamic cohorts, with up to 6 participants per dose level, will be opened for CRX-601 dose levels previously cleared for dose escalation.

Pre- and on-treatment tumor biopsies are required for enrollment to this cohort. PK, pharmacodynamic markers, and safety samples will be drawn according to description herein to obtain additional PK and pharmacodynamic data. Participants in the

PK/Pharmacodynamic cohort may have the dose escalated to a higher completed dose level (not exceeding the target toxicity level) after Week 9 once the necessary

PK/Pharmacodynamic procedures and tissue biopsies have been completed. See herein for further instructions on intra-participant dose escalation.

5.1.3. Part 2: Expansion Cohort

Part 2 of the study will further characterize the safety and tolerability of CRX-601 administered in combination with ANTIBODY 106-222 (Part 2a) in participants with recurrent, locally advanced, or metastatic SCCHN as determined by safety and tolerability results from the respective cohorts in Part 1. Part 2 will also characterize antitumor activity, PK, and pharmacodynamics effects, including effects measured from tumor biopsy. Part 2 may be opened for a given combination before Part 1 has been completed provided a tolerable dose level within or below the target toxicity range has been identified for that combination. The dose of CRX-601 to be administered in the expansion cohort will be based on all available data and may have a DLT frequency within or below the target toxicity range.

Between approximately 6 and 15 participants with SCCHN will be enrolled in Part 2a expansion cohort. No more than 6 participants will be enrolled in an expansion cohort without meeting eligibility requirements for mandatory biopsy requirement.

Interim analysis for futility will be performed on an on-going basis for Part 2a and cohort(s) may be stopped if interim analysis reveals futility. Actual decisions will depend on the totality of the data. Futility criteria are described in greater detail in herein.

5.1.4. Tumor Types Enrolled During Parts 1 and 2

In Part 1, participants with advanced solid tumors will be enrolled. In Part 2, only participants with SCCHN will be enrolled.

5.1.5. Intra-Participant Dose Escalation Following the selection of a recommended combination dose for Part 2 (as described herein), participants in respective cohorts in Part 1 may be considered for escalation to the Part 2 dose level. Intra-participant dose escalation will be considered on a case-by-case basis provided the participant has completed at least 6 weeks of study treatment without the occurrence of a SAE or >Grade 2 drug-related toxicity. Approval by the Sponsor is required for intra-participant dose escalation.

5.1.6. Study Treatment

5.2. Scientific Rationale for Study Design

The combination of CRX-601 with ANTIBODY 106-222 was selected based on complementary mechanisms of action and robust antitumor activity in preclinical models.

Eligibility criteria require that participants have progressed after standard therapies or are otherwise unsuitable for standard therapies, and the criteria are intended to minimize the risk of adverse reactions to treatment with immunotherapies.

In Part 1, dose escalation of CRX-601, with a fixed dose of the combination partners, will be performed using an N-CRM model to optimize the allocation of participants to dose levels with a 16-33% DLT frequency. The DLT criteria are based on typical oncology rules with additional modifications for toxicities expected for the study treatments.

In Part 1, a 2-week run-in period for CRX-601 precedes the administration of the combination therapy. The run-in provides an evaluation of monotherapy CRX-601 safety and tolerability in participants with cancer and prevents the administration of combination therapy to participants that experience, with CRX-601 alone, a DLT, unacceptable tolerability, or an increase in ALT to l.5x ULN and l.5x baseline.

In Part 2, expansion cohorts will be opened to evaluate safety and tolerability of the combinations as well as preliminary activity in participants with SCCHN (as described herein). SCCHN was chosen for further study based on observations of responses to other immunotherapies and recognition of the considerable unmet need for this indication.

Additionally, since TLR agonists are being developed by different routes of administration, including intratumoral injection, SCCHN is a possible indication for future exploration of alternative approaches to dosing.

5.3. Dose Justification 5.3.1. Overview

CRX-601 and ANTIBODY 106-222 have been previously administered as monotherapies.

The selection of starting combination doses has taken into consideration all available data, including the safety, tolerability, and pharmacology data of monotherapy CRX-601, and monotherapy ANTIBODY 106-222, observed in the respective FTIH studies, together with pharmacology and safety data from animal models and human ex vivo (peripheral blood mononuclear cell [PBMC]) assays, conducted under monotherapy and combination conditions.

5.3.1.1. Starting dose for TLR4 agonist CRX-601

The starting dose of CRX-601 is 50 ng administered once-weekly IV. Previously CRX- 601 was administered at doses up to 100 ng IV to healthy participants in the FTIH Study. Based on data from the FTIH study, the starting dose in the current study (50 ng) is expected to produce low level pharmacological effects consistent with TLR4 agonism based on data from the FTIH study (as described herein).

Because robust TLR receptor saturation assays are not available, target engagement by CRX-601 in the FTIH study was monitored using representative inflammatory cytokine biomarkers. Based on review of available preliminary data (dose levels up to 100 ng), post-dose elevations of cytokines following administration of CRX-601 in the FTIH study were of a low magnitude compared to historical clinical studies of TLR agonists administered to cancer patients. For example, the peak levels of inflammatory cytokines at 2h, such as TNFa (median: 12 pg/mL; min: 6 pg/mL; max: 23 pg/mL) and IL-6 (median: 132 pg/mL; min: 81 pg/mL; max: 184 pg/mL pg/mL, respectively), associated with administration of 100 ng CRX-601 are below levels reported in previous studies of TLR agonists in cancer patients (>1000 pg/mL) [Chow, 2017; Engelhardt, 1991] These differences are likely not a function of differences in study populations, given that prior comparisons of TLR agonists in healthy participants and cancer participants have shown similar cytokine responses between populations [Riella, L.V., S. Dada, L. Chabtini, B. Smith, L. Huang, P. Dakle, B. Mfarrej, F. DAddio, L.-T. Adams, N. Kochupurakkal, A. Vergani, P. Fiorina, A.L. Mellor, A.H. Sharpe, H. Yagita, and I. Guleria. 2013. B7h (ICOS- L) Maintains Tolerance at the Fetomatemal Interface. The American Journal of Pathology 182:2204-2213.

Schmidt, 2015; Dietsch, 2014] Even acknowledging possible differences in

pharmacodynamic assay performance, the greater than 10-100 fold margin between cytokine concentrations associated with 100 ng doses of CRX-601 versus concentrations reported in other studies of cancer patients provides reassurance that a significant margin separates the starting dose of CRX-601 and maximum tolerated dose of other TLR agonists. Consistent with the mechanism of action of CRX-601, body temperature and heart rate increased with dose in an earlier study. Mean maximum change with 95% Cl in body temperature in the participants that received placebo was 0.4 ± 0.2°C. For CRX-601 dose levels, 7 ng, 21 ng, 60 ng, and 100 ng, mean maximum change with 95% Cl in body temperature was 0.5 ± 0.3, 0.6 ± 0.4, 0.8+.5, and 1.3 ± 0.3 °C, respectively. Mean maximum change with 95% Cl in heart rate in the participants that received placebo was 6 ± 5 beats per minute. For CRX-601 dose levels, 7 ng, 21 ng, 60 ng, and 100 ng, mean maximum change with 95% Cl in heart rate was 8 + 9, 10 + 18, 18 + 15, 21 ± 5 beats per minute, respectively. Thus, a 50 ng starting dose is expected to be associated with modest changes in body temperature and heart rate.

Based on preliminary, unblinded safety data (as described herein), the most common clinical findings were influenza-like symptoms and increased body temperature.

Predominantly mild AEs were reported when doses up to 60 ng were administered. Three (3) participants experienced moderate AEs following administration of 100 ng CRX-601.

In addition to the aforementioned dose-related AEs, 1 out of the 12 participants that received a 60 ng dose of CRX-601 experienced a 12-fold ULN increase in ALT, 5 -fold ULN increase in AST, and 1 4-fold ULN increase in total bilirubin on day 35. As only 1 event was observed and the dose administered was below the maximum administered, 100 ng, the data are too limited to relate elevations in hepatic laboratories to dose. Because of the possible risk of infrequent or idiosyncratic transaminase elevations, a 2-week CRX-601 run-in period will be performed in Part 1 , including monitoring and study treatment discontinuation criteria for ALT elevations, before CRX-601 and combination partners are administered.

In summary, at the CRX-601 starting dose of 50 ng, minimal pharmacodynamic effects and dose-related cytokine-associated clinical effects are expected. The risk of infrequent events of uncertain relationship to CRX-601 dose will be mitigated by a 2-week monotherapy run- in period.

5.3.1.2. Dose for OX-40 agonist ANTIBODY 106-222

The starting dose of ANTIBODY 106-222 is 24 mg (~0.3 mg/kg) IV administered every 3 weeks (Q3W). No DLTs were observed over a range from 0.003 mg/kg (-0.24 mg) to 10 mg/kg (-800 mg) in cancer patients receiving ANTIBODY 106-222 monotherapy in an earlier study. Thus, the starting dose of 24 mg is 1/33 of the top dose of ANTIBODY 1 Oh- 222 that was evaluated and produced no DLTs in an earlier study.

0X40 receptor occupancy (RO) in the central circulation is expected to be near maximal over the whole 3-week dosing interval with 24 mg ANTIBODY 106-222 based on measured RO in an earlier study (see the 0.3 mg/kg dose level in Figure 45). Efficacy responses to 0X40 agonism have been observed at widely varying dose levels, including 0.3 mg/kg.

Based on the totality of available data, including receptor occupancy, efficacy and safety, a 24 mg dose level was selected for ANTIBODY 106-222. Based on emerging safety, exposure and/or pharmacodynamic data, the dose for ANTIBODY 106-222 may be adjusted lower, to 8 mg.

5.3.1.3. Combination considerations

At the 50 ng starting dose of CRX-601, only minimal clinical and pharmacodynamic effects are expected. Therefore, CRX-601 is not expected to significantly alter the safety and tolerability profile of ANTIBODY 106-222.

Meanwhile, ANTIBODY 106-222 did not enhance cytokine induction by CRX-601 in vitro. Based on the nonclinical data, ANTIBODY 106-222 are not expected to

significantly alter the safety and tolerability profile of CRX-601.

In cynomolgus monkeys in vivo, ANTIBODY 106-222 did not enhance cytokine induction by CRX-601. Overall, when CRX-601 was evaluated alone or in combination with ANTIBODY 106-222, cytokine values were variable and the distributions were generally overlapping.

Overall, the clinical and nonclinical data for CRX-601 and ANTIBODY 106-222 administered as monotherapies and in combination support the starting combination of low doses of CRX-601 with ANTIBODY 106-222.

5.3.2. Dose Escalation and Top Dose

Based on available clinical data, the tolerability of CRX-601 approximates that of LPS. Therefore, the top dose of CRX-601 for study participants with cancer is expected to be similar to doses of LPS studied in similar populations, namely 2 to 4 ng/kg (i.e., 160 to 320 ng). The top dose of CRX-601 will not exceed approximately 250 ng, which would represent a less than 3-fold escalation beyond the 100 ng dose which has been studied in the FTIH healthy volunteer study. The dose escalation step size of 50 ng increments results in a dose escalation scheme with progressively more conservative relative increases ( e.g .,

50 ng to 100 ng = 100% increase; 100 to 150 ng = 50% increase; 150 ng to 200 ng = 33% increase; 200 ng to 250 ng = 25% increase).

6. Study Population

Prospective approval of protocol deviations to recruitment and enrollment criteria, also known as protocol waivers or exemptions, is not permitted.

Specific information regarding warnings, precautions, contraindications, AEs, and other pertinent information on the GSK investigational product or other study treatment that may impact participant eligibility is provided in the IBs/IB supplements. Deviations from inclusion and exclusion criteria are not allowed because they can potentially jeopardize the scientific integrity of the study, regulatory acceptability, or participant safety. Therefore, adherence to the criteria as specified in the protocol is essential.

6.1. Inclusion Criteria

Participants are eligible to be included in the study only if all of the following criteria apply:

Age

1. Participant must be >18 years of at the time of signing the informed consent.

Type of Participant and Disease Characteristics

2. Histological documentation of advanced solid tumor.

3. Archival tumor tissue obtained at any time from the initial diagnosis to study entry. Although a fresh biopsy obtained during screening is preferred, archival tumor specimen is acceptable if it is not feasible to obtain a fresh biopsy.

Note: Participants enrolled in a PK/Pharmacodynamic Cohort must provide a fresh biopsy of a tumour lesion not previously irradiated during the screening period and must agree to provide at least one additional on-treatment biopsy.

4. Disease that has progressed after standard therapies or for which standard

therapy is otherwise unsuitable (e.g., intolerance).

5. Measurable disease, i.e., presenting with at least 1 measurable lesion per

Response Evaluation Criteria in Solid Tumors (RECIST version 1.1). See D Appendix 9 for definition of a measurable lesion.

6. Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0-1.

7. Life expectancy of at least 12 weeks.

8. Adequate organ function (see Table 5):

9. In France, a participant will be eligible for inclusion in this study only if either affiliated to or a beneficiary of a social security category.

Table 5 Organ Function

Sex

10. Male or female

a. Female participants:

A female participant is eligible to participate if she is not pregnant, not breastfeeding, and at least 1 of the following conditions applies:

i. Not a woman of childbearing potential (WOCBP)

OR

ii. A WOCBP who agrees to follow the contraceptive guidance during the treatment period and for at least 120 days after the last dose of study treatment.

Informed Consent

11. Capable of giving signed informed consent a which includes compliance with the requirements and restrictions listed in the ICF and in this protocol. Additional Inclusion Criteria for Patients in Part 2a (ANTIBODY 106-222 expansion)

12. Histological or cytological documentation of SCCHN (oral cavity, oropharynx, hypopharynx, or larynx) that is recurrent, locally advanced, or metastatic and is not amenable to curative treatment options, surgery or definitive chemoradiation therapy.

13. Received or ineligible for platinum-based therapy and PD-1/PD-L1 therapy.

14. Received no more than 3 prior lines of systemic therapy for metastatic disease.

6.2. Exclusion Criteria

Participants are excluded from the study if any of the following criteria apply:

Medical Conditions

1. Malignancy other than disease under study with the exception of those from which the participant has been disease-free for more than 2 years and not expected to affect the safety of the participant or the endpoints of the trial.

2. Symptomatic central nervous system (CNS) metastases or asymptomatic CNS metastases that have required steroids within 2 weeks prior to first dose of study treatment.

3. Active autoimmune disease that has required systemic disease modifying or immunosuppressive treatment within the last 2 years.

Note: Replacement therapy ( e.g ., thyroxine or physiologic corticosteroid replacement therapy for adrenal or pituitary insufficiency, etc.) is permitted.

4. Concurrent medical condition requiring the use of systemic immunosuppressive treatment within 28 days before the first dose of study treatment.

5. Known human immunodeficiency virus infection.

6. Current unstable liver or biliary disease per investigator assessment defined by the presence of ascites, encephalopathy, coagulopathy, hypoalbuminaemia, oesophageal or gastric varices, persistent jaundice, or cirrhosis.

NOTE: Stable chronic liver disease (including Gilbert’s syndrome or

asymptomatic gallstones) or hepatobiliary involvement of malignancy is acceptable if participant otherwise meets entry criteria.

7. Presence of Hepatitis B surface antigen (HBsAg) at screening or within 3

months prior to first dose of study treatment

8. Positive Hepatitis C test result at screening or within 3 months prior to first dose of study treatment.

NOTE: Participants with positive Hepatitis C antibody due to prior resolved disease can be enrolled, only if a confirmatory negative Hepatitis C RNA test is obtained. Participants with negative Hepatitis C antibody test are not required to also undergo Hepatitis C RNA testing

9. QTcF >450 msec or QTcF >480 msec for participants with bundle branch block The QTcF is the QT interval corrected for heart rate according to Fridericia’s formula, machine-read or manually over-read.

10. Recent history (within the past 6 months) of acute diverticulitis, inflammatory bowel disease, intra-abdominal abscess, or gastrointestinal obstruction.

11. Recent history of allergen desensitization therapy within 4 weeks of starting study treatment.

12. History of severe hypersensitivity to mAbs.

13. History or evidence of cardiovascular (CV) risk including any of the following:

• Recent (within the past 6 months) history of serious uncontrolled cardiac

arrhythmia or clinically significant ECG abnormalities including second degree (Type II) or third degree atrioventricular block.

• Cardiomyopathy, myocardial infarction, acute coronary syndromes (including unstable angina pectoris), coronary angioplasty, stenting, or bypass grafting within the past 6 months before enrollment.

• Congestive heart failure (Class II, III, or IV) as defined by the New York Heart Association functional classification system [NYHA, 1994]

• Recent (within the past 6 months) history of symptomatic pericarditis.

14. History of idiopathic pulmonary fibrosis, pneumonitis, interstitial lung disease, or organizing pneumonia, or evidence of active, non-infectious pneumonitis. Note: post-radiation changes in the lung related to prior radiotherapy and/or asymptomatic radiation-induced pneumonitis not requiring treatment may be permitted if agreed by the investigator and Sponsor.

15. Recent history (within 6 months) of uncontrolled symptomatic ascites or pleural effusions.

16. Any serious and/or unstable pre-existing medical, psychiatric disorder, or other condition that could interfere with the participant’s safety, obtaining informed consent, or compliance to the study procedures.

17. Is or has an immediate family member (e.g., spouse, parent/legal guardian, sibling or child) who is investigational site or sponsor staff directly involved with this trial, unless prospective Institutional Review Board (IRB) approval (by chair or designee) is given allowing exception to this criterion for a specific participant.

Prior/Concomitant Therapy

18. Prior treatment with the following agents :

• Tumor necrosis factor receptor (TNFR) agonists, including 0X40, CD27, CD137 (4-1BB), CD357 (glucocorticoid-induced TNFR family-related gene) at any time.

• Prior systemic or intratumoral therapy with TLR agonist.

• Anticancer therapy or investigational therapy within 30 days or 5 half-lives of the drug, whichever is shorter. • Prior radiation therapy: permissible if at least 1 non-irradiated measurable lesion is available for assessment according to RECIST version 1.1 or if a solitary measurable lesion was irradiated, objective progression is documented. A wash out of at least 14 days before start of study treatment for radiation of any intended use to the extremities for bone metastases and 28 days for radiation to the chest, brain, or visceral organs is required.

19. Prior allogeneic or autologous bone marrow transplantation or another solid organ transplantation.

20. Toxicity from previous treatment including:

• Toxicity Grade >3 related to prior immunotherapy and that lead to study

treatment discontinuation.

• Toxicity related to prior treatment has not resolved to Grade <1 (except

alopecia, or endocrinopathy managed with replacement therapy).

21. Received transfusion of blood products (including platelets or red blood cells) or administration of colony stimulating factors (including G-CSF, granulocyte- macrophage colony-stimulating factor, and recombinant erythropoietin) within 2 weeks before the first dose of study treatment.

Other Exclusions

22. Major surgery <4 weeks before the first dose of study treatment. Participants must have also fully recovered from any surgery (major or minor) and/or its complications before initiating study treatment.

23. Known drug or alcohol abuse.

24. Receipt of any live vaccine within 4 weeks.

6.3. Lifestyle Restrictions

6.3.1. Meals and Dietary Restrictions

No dietary restrictions are required. Note that participants should be well-hydrated before receiving study treatment (see below)

6.3.2. Caffeine, Alcohol, and Tobacco

Participants who use products containing caffeine, alcohol, or tobacco are not required to change their habits of using these products during the study treatment.

6.3.3. Activity

Participants may experience orthostatic dizziness following administration of CRX-601. Precautions should be taken to avoid falls after rising from a lying or seated position for several hours after administration of study treatment. In addition, participants will abstain from strenuous exercise for 8 hours before each blood collection for clinical laboratory tests. Participants may participate in light recreational activities during studies (e.g., watching television, reading). 6.4. Screen Failures

Screen failures are defined as participants who consent to participate in the clinical study but are not subsequently entered in the study. A minimal set of screen failure information is required to ensure transparent reporting of screen failure participants to meet the

Consolidated Standards of Reporting Trials publishing requirements and to respond to queries from regulatory authorities. Minimal information includes demography, screen failure details, eligibility criteria, and any SAEs.

Individuals who do not meet the criteria for participation in this study (screen failure) may be rescreened once. This includes retesting specific vital sign measurements, laboratory assessments, etc. that may not have met eligibility criteria.

7. Treatments

Study treatment is defined as any investigational treatment(s), marketed product(s), placebo, or medical device(s) intended to be administered to a study participant according to the study protocol. The term‘study treatment’ is used throughout the protocol to describe any combination of products received by the participant as per the protocol design.

7.1. Treatments Administered

Participants receiving study treatment should be well-hydrated (TLR agonists have rarely been associated with severe bradycardia or asystole in clinical trials, attributed to poor hydration and/or history of syncope) [van Eijk, 2004] Oral hydration should be encouraged in the days prior to study treatment and/or IV fluids ( e.g ., 1 L or as clinically indicated) administered before CRX-601. Participants with a history of syncope and/or uncertain compliance with hydration recommendations should receive additional pre-dose and/or post-dose fluids at the discretion of the investigator.

Following administration of CRX-601, assessments must be performed as noted in the SoA. Cytokine-related AEs including changes in vital signs commonly begin within several hours of administration of CRX-601. Participants must be monitored for 6 hours after administration of the first dose of CRX-601 or longer as clinically indicated. Similarly, participants must be monitored for 6 hours after administration of the first 2 study treatments of CRX-601 and combination partners. Participants that tolerate CRX-601 without adverse changes in heart rate or blood pressure may have the duration of observation with subsequent study treatment reduced to 2 hours, provided the dose and schedule has not been changed. Guidelines for monitoring cytokine-related AEs are summarized herein.

CRX-601 and mAh combination partner ANTIBODY 106-222 will be administered to participants at each study site under medical supervision of an investigator or designee. ANTIBODY 106-222 will be administered first, and CRX-601 will be administered at least 1 hour after the completion of the mAh infusion. The date and time of administration will be recorded in the source documents and reported in the eCRF.

If a participant experiences an infusion reaction with the administration of the mAh combination partner, associated AEs should resolve before CRX-601 is administered. If AEs associated with the mAh are slow to resolve, it is acceptable to administer CRX-601 on the following day. Should further delay be required, the participant will be discontinued from study treatment. Any participant who experiences an infusion reaction attributable to the mAh may receive CRX-601 on the following day for all subsequent study treatments.

The specific time of study treatment administration (e.g., time of the week for first administration; time of the day for each administration) should take into consideration PK sampling time points and study visit procedures. See herein for dosing timepoints and visit windows and section below for additional details regarding dosing delays.

The Study Reference Manual (SRM) contains specific instructions for the preparation of CRX-601 and ANTIBODY 106-222.

Table 6 Investigational Product Dosage/ Administration

a. Infusions may be prolonged in the event of an infusion reaction. If multiple participants experience clinically significant infusion reactions, the infusion rate may be slowed for all future administrations of study treatment(s) for all participants. Should this global change in infusion rate be required, it will be communicated to the sites in writing. 7.2. Dose Modification

Safety management guidelines, including dose modification algorithms, are provided below. Please note, in cases where the investigator is directed to permanently discontinue study treatment, these instructions are mandatory as described herein.

An overview of the dose modification guidelines is presented in Table 7.

All AEs are to be graded according to NCI-CTCAE, version 4.0 (http://ctep.cancer.gov). All dose modifications and the reason(s) for the dose modification must be documented in the eCRF.

The major classes of toxicity described herein include“cytokine-related AEs and infusion reactions” and“immune-related AEs”. Even though both cytokine production and immune activity play roles in both categories of events, the nomenclature is intended to describe distinct classes of AEs, as described below.

In case a dose reduction is necessary, the dose level of CRX-601, the mAh or both may be changed as determined by the investigator and sponsor. Participants may not discontinue only 1 study treatment. If either study treatment is deemed intolerable and requires discontinuation despite optimal management, as described below, the participant must be discontinued from both study treatments. CRX-601 may be restarted at the next lower dose level, and/or the mAh (ANTIBODY 106-222) at the next lower dose level described above.

Table 7 General Dose Modification and Management Guidelines for Drug- Related Non-Hematologic Adverse Events Not Otherwise Specified

7.3. Efficacy Assessments

· Lesion assessment method and timing, evaluation of disease, disease progression and response criteria will be conducted according to Response Evaluation Criteria in Solid Tumors (RECIST 1.1) [Dillingh M, van Poelgeest E, Malone K, Kemper E, Stroes E, Moerland M, et al. Characterization of inflammation and immune cell modulation induced by low-dose LPS administration to healthy volunteers. Journal of

Inflammation. 2014; 11(1).

• Eisenhauer, 2009] as outlined below.

• Disease assessment modalities may include imaging ( e.g ., computed tomography [CT] scan, magnetic resonance imaging [MRI], bone scan, plain radiography) and physical examination (as indicated for palpable/superficial lesions). Scans will be collected centrally during the study and may be reviewed or analyzed by an independent central reviewer. Details will be provided in the SRM.

• The baseline disease assessment will be completed up to 28 days prior to the first dose of study treatment. See the Schedule of Activities Tables (above) for the schedule of assessments of anti-cancer activity subsequent to the baseline disease assessment.

• Assessments must be performed on a calendar schedule and should not be affected by dose interruptions/delays.

• For post-baseline assessments, a window of [+7 days] is permitted to allow for flexible scheduling. If the last radiographic assessment was 6 weeks or more prior to the participant’s withdrawal from study treatment and PD has not been documented, a disease assessment should be obtained at the time of withdrawal from study treatment.

• To ensure comparability between the baseline and subsequent assessments, the same method of assessment and the same technique will be used when assessing response.

7.3.1. Evaluation of Anti-Cancer Activity

• RECIST version 1.1 guidelines will be used to determine the overall tumor burden at baseline, select target and non-target lesions, and in the disease assessments throughout the duration of the study [Dillingh M, van Poelgeest E, Malone K, Kemper E, Stroes E, Moerland M, et al. Characterization of inflammation and immune cell modulation induced by low-dose LPS administration to healthy volunteers. Journal of Inflammation. 2014; 11(1).

• Eisenhauer, 2009] as further outlined in D Appendix 9 of this protocol. irRECIST assessments will be evaluated as well. Treatment decisions according to irRECIST are encouraged, including confirmatory disease assessments at least 4 weeks after the date disease progression was declared. Similarly, new lesions should be measured, as feasible, and may be incorporated into assessments of tumor burden according to irRECIST guidelines.

• Lymph nodes that have a short axis of <10 mm are considered non-patho logical and should not be recorded or followed.

• Pathological lymph nodes with <15 mm and but >10 mm short axis are considered non-measurable.

• Pathological lymph nodes with >15 mm short axis are considered measurable and can be selected as target lesions, however lymph nodes should not be selected as target lesions when other suitable target lesions are available.

• Measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs, should be identified as target lesions, and recorded and measured at baseline. These lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repeated measurements (either by imaging techniques or clinically).

Note: Cystic lesions thought to represent cystic metastases should not be selected as target lesions when other suitable target lesions are available. Note: Measurable lesions that have been previously irradiated and have not been shown to be progressing following irradiation should not be considered as target lesions.

• Lytic bone lesions or mixed lytic-blastic lesions, with identifiable soft tissue

components, that can be evaluated by CT or MRI can be considered measurable.

Bone scans, fluorodeoxyglucose (FDG)-positron-emission tomography (PET) scans or X-rays are not considered adequate imaging techniques to measure bone lesions.

• All other lesions (or sites of disease) should be identified as non-target and should also be recorded at baseline. Non-target lesions will be grouped by organ. Measurements of these lesions are not required, but the presence or absence of each should be noted throughout follow-up.

• The following are required at baseline (up to 28 days before first dose, see herein): CT scan with contrast of the chest, abdomen, and pelvis is required. For participants with SCCHN, a scan of the head and neck area is required. Other areas should be evaluated as indicated by the participant’s underlying disease prior to screening, including clinical disease assessment for palpable/visible lesions. Although CT scan is preferred, MRI may be used as an alternative method of baseline disease assessment, especially for those participants where a CT scan is contraindicated due to allergy to contrast, provided that the method used to document baseline status is used consistently throughout study treatment to facilitate direct comparison. At each post baseline assessment, evaluations of the sites of disease identified by these scans are required. Refer to RECIST version 1.1 guidelines for use of FDG-PET/CT [Dillingh M, van Poelgeest E, Malone K, Kemper E, Stroes E, Moerland M, et al. Characterization of inflammation and immune cell modulation induced by low-dose LPS administration to healthy volunteers. Journal of Inflammation. 2014; 11(1).

• Eisenhauer, 2009]

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9. Appendices

9.1. Appendix 1 Abbreviations and Trademarks

Abbreviations

9.2. D Appendix 9 Guidelines for Assessment of Disease, Disease Progression and Response Criteria - adapted from RECIST version 1.1

Assessment Guidelines

Please note the following:

• The same diagnostic method, including use of contrast when applicable, must be used throughout the study to evaluate a lesion. Contrast agents must be used in accordance with the Image Acquisition Guidelines.

• All measurements should be taken and recorded in millimeters (mm), using a ruler or calipers.

• Ultrasound is not a suitable modality of disease assessment. If new lesions are

identified by ultrasound, confirmation by CT or MRI is required.

• Fluorodeoxyglucose (FDG)-PET is generally not suitable for ongoing assessments of disease. However, FDG-PET can be useful in confirming new sites of disease where a positive FDG-PET scans correlates with the new site of disease present on CT/MRI or when a baseline FDG-PET was previously negative for the site of the new lesion. FDG-PET may also be used in lieu of a standard bone scan providing coverage allows interrogation of all likely sites of bone disease and FDG-PET is performed at all assessments.

• If PET/CT is performed then the CT component can only be used for standard

response assessments if performed to diagnostic quality, which includes the required anatomical coverage and prescribed use of contrast. The method of assessment should be noted as CT on the CRF.

Clinical Examination: Clinically detected lesions will only be considered measurable when they are superficial (e.g., skin nodules). In the case of skin lesions, documentation by color photography, including a ruler/calipers to measure the size of the lesion, is required [Dillingh M, van Poelgeest E, Malone K, Kemper E, Stroes E, Moerland M, et al.

Characterization of inflammation and immune cell modulation induced by low-dose LPS administration to healthy volunteers. Journal of Inflammation. 2014; 11(1).

Eisenhauer, 2009]

CT and MRI: Contrast enhanced CT with 5mm contiguous slices is recommended.

Minimum size of a measurable baseline lesion should be twice the slice thickness, with a minimum lesion size of 10 mm when the slice thickness is 5 mm. MRI is acceptable, but when used, the technical specification of the scanning sequences should be optimized for the evaluation of the type and site of disease and lesions must be measured in the same anatomic plane by use of the same imaging examinations. Whenever possible the same scanner should be used [Dillingh M, van Poelgeest E, Malone K, Kemper E, Stroes E, Moerland M, et al. Characterization of inflammation and immune cell modulation induced by low-dose LPS administration to healthy volunteers. Journal of Inflammation.

2014;! 1(1). Eisenhauer, 2009]

X-ray: In general, X-ray should not be used for target lesion measurements owing to poor lesion definition. Lesions on chest X-ray may be considered measurable if they are clearly defined and surrounded by aerated lung; however, chest CT is preferred over chest X-ray [Dillingh M, van Poelgeest E, Malone K, Kemper E, Stroes E, Moerland M, et al.

Eisenhauer, 2009]

Guidelines for Evaluation of Disease

Measurable and Non-Measurable Definitions

Measurable lesion:

A non-nodal lesion that can be accurately measured in at least 1 dimension (longest dimension) of

• >10 mm with MRI or CT when the scan slice thickness is no greater than 5mm. If the slice thickness is greater than 5mm, the minimum size of a measurable lesion must be at least double the slice thickness ( e.g ., if the slice thickness is 10 mm, a measurable lesion must be >20 mm).

• >10 mm caliper/ruler measurement by clinical exam or medical photography.

• >20 mm by chest x-ray.

Additionally, lymph nodes can be considered pathologically enlarged and measurable if

• >l5mm in the short axis when assessed by CT or MRI (slice thickness recommended to be no more than 5mm). At baseline and follow-up, only the short axis will be measured [Dillingh M, van Poelgeest E, Malone K, Kemper E, Stroes E, Moerland M, et al. Characterization of inflammation and immune cell modulation induced by low- dose LPS administration to healthy volunteers. Journal of Inflammation. 2014; 11(1).

• Eisenhauer, 2009]

Non-measurable lesion:

All other lesions including lesions too small to be considered measurable (longest diameter <10 mm or pathological lymph nodes with > 10 mm and <15 mm short axis) as well as truly non-measurable lesions, which include: leptomeningeal disease, ascites, pleural or pericardial effusions, inflammatory breast disease, lymphangitic involvement of the skin or lung, abdominal masses/abdominal organomegaly identified by physical exam that is not measurable by reproducible imaging techniques [Dillingh M, van Poelgeest E, Malone K, Kemper E, Stroes E, Moerland M, et al. Characterization of inflammation and immune cell modulation induced by low-dose LPS administration to healthy volunteers. Journal of Inflammation. 2014; 11(1).

Eisenhauer, 2009]

Measurable disease: The presence of at least 1 measurable lesion. Palpable lesions that are not measurable by radiologic or photographic evaluations may not be utilized as the only measurable lesion.

Non-Measurable only disease: The presence of only non-measurable lesions. Note: non- measurable only disease is not allowed per protocol.

Immune-Related RECIST Response Criteria

Table 8 Evaluation of Target Lesions

a. Measurable according to RECIST version 1.1.

b. Treatment decisions may be based upon the immune-related RECIST guidelines.

Antitumor response based on total measurable tumor burden

For Modified RECIST based on RECIST version 1.1 and irRECIST [Wolchok, 2009; Nishino, 2013], the initial target (‘index”) and measurable new lesions are taken into account. At the baseline tumor assessment, the sum of the diameters in the plane of measurement of all target lesions (maximum of 5 lesions in total and a maximum of 2 lesions per organ representative of all involved organs) is calculated.

Note: If pathological lymph nodes are included in the sum of diameters, the short axis of the lymph node(s) is added into the sum. The short axis is the longest perpendicular diameter to the longest diameter of a lymph node or nodal mass. At each subsequent tumor assessment, the sum of diameters of the baseline target lesions and of new, measurable nodal and non-nodal lesions (>10 mm), up to 2 new lesions per organ are added together to provide the total tumor burden:

Tumor Burden = Sum of diameterstarget lesions + sum of diametersnew, measurable lesions

Time-point response assessment using the Immune-Related RECIST criteria

Percentage changes in tumor burden per assessment time point describe the size and growth kinetics of both conventional and new, measurable lesions as they appear. At each tumor assessment, the response in index and new, measurable lesions is defined based on the change in tumor burden (after ruling out irPD). Decreases in tumor burden must be assessed relative to baseline measurements (i.e., the sum of diameters of all target lesions at screening).

Response Criteria

Evaluation of target lesions

Definitions for assessment of response for target lesion(s) are as follows:

• CR: Disappearance of all target lesions. Any pathological lymph nodes must be

<l0mm in the short axis.

• PR: At least a 30% decrease in the sum of the diameters of target lesions, taking as a reference, the baseline sum of the diameters ( e.g ., percent change from baseline).

• SD: Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD.

• PD: At least a 20% increase in the sum of the diameters of target lesions, taking as a reference, the smallest sum of diameters recorded since the treatment started (e.g., percent change from nadir, where nadir is defined as the smallest sum of diameters recorded since treatment start). In addition, the sum must have an absolute increase from nadir of 5 mm.

• Not Applicable (NA): No target lesions at baseline.

• Not Evaluable (NE): Cannot be classified by 1 of the 5 preceding definitions.

Note:

• If lymph nodes are documented as target lesions the short axis is added into the sum of the diameters (e.g., sum of diameters is the sum of the longest diameters for non-nodal lesions and the short axis for nodal lesions). When lymph nodes decrease to non- patho logical size (short axis <l0mm) they should still have a measurement reported in order not to overstate progression.

• If at a given assessment time point all target lesions identified at baseline are not

assessed, sum of the diameters cannot be calculated for purposes of assessing CR, PR, or SD, or for use as the nadir for future assessments. However, the sum of the diameters of the assessed lesions and the percent change from nadir should be calculated to ensure that progression has not been documented. If an assessment of PD cannot be made, the response assessment should be NE.

• All lesions (nodal and non-nodal) should have their measurements recorded even when very small ( e.g ., 2 mm). If lesions are present but too small to measure, 5 mm should be recorded and should contribute to the sum of the diameters, unless it is likely that the lesion has disappeared in which case 0 mm should be reported.

• If a lesion disappears and reappears at a subsequent time point it should continue to be measured. The response at the time when the lesion reappears will depend upon the status of the other lesions. For example, if the disease had reached a CR status then PD would be documented at the time of reappearance. However, if the response status was PR or SD, the diameter of the reappearing lesion should be added to the remaining diameters and response determined based on percent change from baseline and percent change from nadir.

Evaluation of non-target lesions

Definitions for assessment of response for non-target lesions are as follows:

• Complete Response: The disappearance of all non-target lesions. All lymph nodes identified as a site of disease at baseline must be non-patho logical (e.g., <10 mm short axis).

• Non-CR/Non-PD: The persistence of 1 or more non-target lesion(s) or lymph nodes identified as a site of disease at baseline > 10 mm short axis.

• Progressive Disease: Unequivocal progression of existing non-target lesions.

• Not Applicable (NA): No non-target lesions at baseline.

• Not Evaluable (NE): Cannot be classified by 1 of the 4 preceding definitions.

Note:

• In the presence of measurable disease, progression on the basis of solely non-target disease requires substantial worsening such that even in the presence of SD or PR in target disease, the overall tumor burden has increased sufficiently to merit

discontinuation of therapy.

• Sites of non-target lesions, which are not assessed at a particular timepoint based on the assessment schedule, should be excluded from the response determination (e.g., non-target response does not have to be "Not Evaluable").

New lesions

New malignancies denoting disease progression must be unequivocal. Lesions identified in follow-up in an anatomical location not scanned at baseline are considered new lesions.

Any equivocal new lesions should continue to be followed. Treatment can continue at the discretion of the investigator until the next scheduled assessment. If at the next assessment the new lesion is considered to be unequivocal, progression should be documented. Evaluation of overall response

Table 9 presents the overall response at an individual time point for all possible

combinations of tumor responses in target and non-target lesions with or without the appearance of new lesions for participants with measurable disease at baseline.

Table 9 Evaluation of Overall Response for Participants with Measurable

Disease at Baseline

CR=complete response, PR = partial response, SD=stable disease, PD=progressive disease, NA= Not applicable, and NE=Not Evaluable

Note:

• Participants with a global deterioration of health status requiring discontinuation of treatment without objective evidence of disease progression at that time should be classified as having "symptomatic deterioration." Objective response status is determined by evaluations of disease burden. Every effort should be made to document the objective progression even after discontinuation of treatment.

• In some circumstances, it may be difficult to distinguish residual disease from

normal tissue. When the evaluation of CR depends on this determination, it is recommended that the residual lesion be investigated (fine needle aspirate/biopsy) to confirm the CR.

Evaluation of best overall response

The best overall response is the best response recorded from the start of the treatment until disease progression/recurrence and will be determined programmatically by GSK based on the investigator’s assessment of response at each time point.

• To be assigned a status of SD, follow-up disease assessment must have met the SD criteria at least once after the first dose at a minimum interval of the first scheduled tumor evaluation.

• If the minimum time for SD is not met, best response will depend on the subsequent assessments. For example, if an assessment of PD follows the assessment of SD and SD does not meet the minimum time requirement the best response will be PD. Alternatively, participants lost to follow-up after an SD assessment not meeting the minimum time criteria will be considered not evaluable.

Confirmation Criteria:

To be assigned a status of PR or CR, a confirmatory disease assessment should be performed no less than 4 weeks (28 days) after the criteria for response are first met.

Claims

We claim:

1. A method of treatment of a human subject with cancer, the method comprising:

administering to the human subject an agonist 0X40 binding protein and a TLR4 agonist, wherein the agonist 0X40 binding protein is administered at a dose of about 8 mg to about 24 mg and the TLR4 agonist is administered at a dose of about 5 ng to about 1000 ng, optionally about 50 ng to about 250 ng.

2. An agonist 0X40 binding protein and a TLR4 agonist for simultaneous or sequential use in treating cancer, wherein the agonist 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and the TLR4 agonist is to be administered at a dose of about 5 ng to about 1000 ng, optionally about 50 ng to about 250 ng.

3. An agonist 0X40 binding protein for use in treating cancer, wherein the agonist 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and is to be administered simultaneously or sequentially with a TLR4 agonist at a dose of about 5 ng to about 1000 ng, optionally about 50 ng to about 250 ng.

4. A TLR4 agonist for use in treating cancer, wherein the TLR4 agonist is to be administered at a dose of about 5 ng to about 1000 ng, optionally about 50 ng to about 250 ng, and is to be administered simultaneously or sequentially with an agonist 0X40 binding protein at a dose of about 8 mg to about 24 mg.

5. Use of an agonist 0X40 binding protein in the manufacture of a medicament for treating cancer, wherein the 0X40 binding protein is to be administered at a dose of about 8 mg to about 24 mg and is to be administered simultaneously or sequentially with a TLR4 agonist at a dose of about 5 ng to about 1000 ng, optionally about 50 ng to about 250 ng.

6. Use of a TLR4 agonist in the manufacture of a medicament for treating cancer, wherein the TLR4 agonist is to be administered at a dose of about 5 ng to about 1000 ng, optionally about 50 ng to about 250 ng, and is to be administered simultaneously or sequentially with an agonist 0X40 binding protein at a dose of about 8 mg to about 24 mg.

7. A pharmaceutical kit comprising about 8 mg to about 24 mg of an agonist 0X40 binding protein and about 5 ng to 1000 ng, optionally about 50 ng to about 250 ng, of a TLR4 agonist.

8. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the agonist 0X40 binding protein is administered intravenously.

9. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the TLR4 agonist is administered by a route selected from the group consisting of intravenously, subcutaneously, and intratumorally.

10. The method, binding protein, agonist, use, or kit of any of claims 1-8, wherein the agonist 0X40 binding protein is administered intravenously and the TLR4 agonist is administered intravenously.

11. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the agonist 0X40 binding protein is administered at a dose of about 8 mg to about 24 mg.

12. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the agonist 0X40 binding protein comprises (a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:l; (b) a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2; (c) a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; (d) a light chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7; (e) a light chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:8; and (f) a light chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:9.

13. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the agonist 0X40 binding protein comprises a heavy chain variable region comprising the amino acid sequence as set forth in SEQ ID NO:5; and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 11.

14. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the agonist 0X40 binding protein is a humanized monoclonal antibody.

15. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the agonist 0X40 binding protein comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:48 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:49.

16. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the TLR4 agonist is selected from the group consisting of: CRX-601; CRX-547; CRX-602; and CRX-527.

17. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the cancer comprises squamous cell carcinoma of head and neck (SCCHN).

18. The method, binding protein, agonist, use, or kit of any of the preceding claims, wherein the TLR4 agonist and the agonist 0X40 binding protein are both administered to the subject every three weeks.

19. A method for treating metastatic solid tumors, the method comprising: (i) systemically administering CRX-601 in a dose from about 5 ng to about 1000 ng, optionally about 50 ng to about 250 ng; and, (ii) systemically administering a therapeutic amount of an agonist 0X40 antibody.