WO2021018941A1

WO2021018941A1 - Methods of treating cancer

Info

Publication number: WO2021018941A1
Application number: PCT/EP2020/071351
Authority: WO
Inventors: Patrick MAYES; Sapna YADAVILLI; Ping-Chiao TSAI
Original assignee: Glaxosmithkline Intellectual Property Development Limited
Priority date: 2019-07-31
Filing date: 2020-07-29
Publication date: 2021-02-04

Abstract

This invention relates to methods of treating cancer in a subject in need thereof, e.g., in a human in need thereof, comprising determining the level of soluble ICOS (sICOS) in a sample from the human, and administering to the human an effective amount of an agent directed to human ICOS if the level of soluble ICOS is increased relative to a reference level, thereby treating the cancer in the human. Also provided are kits for the treatment of cancer comprising a means for determining the level of soluble ICOS (sICOS) in a sample from a human.

Description

METHODS OF TREATING CANCER

FIELD OF THE INVENTION

The present invention relates generally to immunotherapy in the treatment of human disease. More specifically, the present invention relates to the use of soluble ICOS (sICOS) levels in serum to identify patients more likely to respond to anti-ICOS antibodies in the treatment of cancer.

BACKGROUND TO 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.

The expanding development and use of targeted therapies for cancer treatment reflects an increasing understanding of key oncogenic pathways, and how the targeted perturbation of these pathways corresponds to clinical response. Difficulties in predicting efficacy to targeted therapies is likely a consequence of the limited global knowledge of causal mechanisms for pathway deregulation (e.g. activating mutations, amplifications). Pre-clinical translational research studies for oncology therapies focuses on determining what tumor type and genotypes are most likely to benefit from treatment. Treating selected patient populations may help maximize the potential of a therapy. Pre- clinical cellular response profiling of tumor models has become a cornerstone in development of novel cancer therapeutics.

Immunomodulators such as anti-ICOS antibodies that are useful in treating cancer have been reported in US20160215059, which is incorporated by reference herein. It is desirable to identify patients that are more likely to respond to these agents.

SUMMARY OF THE INVENTION

In one aspect of the invention, there is provided a method of treating cancer in a human in need thereof, the method comprising determining the level of soluble ICOS (sICOS) in a sample from the human and administering to the human an effective amount of an agent directed to human ICOS if the level of the sICOS is increased relative to a reference level, thereby treating the cancer in the human.

In another aspect of the invention, there is provided an agent directed to human ICOS for use in the treatment of cancer in a human classified as a responder, wherein the responder is characterized by the presence of an increased level of soluble ICOS (sICOS) in a sample from the human relative to a reference level. In a further aspect of the invention, there is provided a kit for the treatment of cancer, the kit comprising a means for determining the level of soluble ICOS (sICOS) in a sample from a human.

In another aspect of the invention, there is provided a pharmaceutical composition is provided, comprising a comprising an agent directed to human ICOS, for use in treating cancer in a human wherein at least a first sample from the human is determined to have an increased level of soluble ICOS (sICOS) relative to a reference level.

In yet another aspect of the invention, there is provided a use of an agent directed to human ICOS in the manufacture of a medicament for the treatment of cancer in a human wherein one or more samples from the human is determined to have an increased level of soluble ICOS (sICOS) relative to a reference level.

DESCRIPTION OF DRAWINGS/FIGURES

FIG. 1 is a diagram showing the sequences of ICOS isoforms. The sequences of the ICOS isoforms were obtained from UniProt and NCBI.

FIG. 2 is a schematic showing MSD Platform to detect total sICOS.

FIG. 3 is a box plot showing sICOS levels in healthy donors vs. donors of different cancer types / autoimmune diseases.

FIG. 4 is set of plots and schematic showing the "2-Steps" competition assay.

FIG. 5 is set of plots and schematic showing the "1-Step" competition assay.

FIG. 6 is a plot showing correlation of sICOS level vs. response to "CD3+bICOS" activation (IFN-y increase from "Baseline").

FIG. 7 is a plot showing correlation of sICOS level vs. response to "CD3+bICOS" activation (IFN-y increase from "Baseline").

DETAILED DESCRIPTION OF THE INVENTION

DEFINITIONS

As used herein "ICOS" means any Inducible T-cell costimulator protein. Pseudonyms for ICOS (Inducible T-cell COStimulator) include AILIM; CD278; CVIDl, JTT-1 or JTT-2, MGC39850, or 8F4. ICOS is a CD28-superfamily costimulatory molecule that is expressed on activated T cells. The protein encoded by this gene belongs to the CD28 and CTLA-4 cell-surface receptor family. It forms homodimers and plays an important role in cell-cell signaling, immune responses, and regulation of cell proliferation. The amino acid sequence of human ICOS (isoform 2) (Accession No. : UniProtKB - Q9Y6W8-2) is shown below as SEQ ID NO: 11.

MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTV SI KSLKFCHSQLSN N SVSFFLYN LDHSH ANYYFCN LSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFW VCILGCILICWLTKKM (SEQ ID NO: 11)

The amino acid sequence of human ICOS (isoform 1) (Accession No. : UniProtKB - Q9Y6W8-1) is shown below as SEQ ID NO: 12.

MKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQ FKMQLLKGGQ ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYN LD HSHANYYFCN LSIFDPPPFK VTLTGGYLHI YESQLCCQLK FWLPIGCAAF VWCILGCIL ICWLTKKKYS SSVHDPNGEY MFMRAVNTAK KSRLTDVTL

(SEQ ID NO: 12)

Activation of ICOS occurs through binding by ICOS-L (B7RP-1/B7-H2). Neither B7-1 nor B7-2 (ligands for CD28 and CTLA4) bind or activate ICOS. However, ICOS-L has been shown to bind weakly to both CD28 and CTLA-4 (Yao S et al., "B7-H2 is a costimulatory ligand for CD28 in human", Immunity, 34(5); 729-40 (2011)). Expression of ICOS appears to be restricted to T cells. ICOS expression levels vary between different T cell subsets and on T cell activation status. ICOS expression has been shown on resting TH17, T follicular helper (TFH) and regulatory T (Treg) cells; however, unlike CD28; it is not highly expressed on naive TH1 and TH2 effector T cell populations (Paulos CM et al., "The inducible costimulator (ICOS) is critical for the development of human Thl7 cells", Sci Transl Med, 2(55); 55ra78 (2010)). ICOS expression is highly induced on CD4+ and CD8+ effector T cells following activation through TCR engagement (Wakamatsu E, et al., "Convergent and divergent effects of costimulatory molecules in conventional and regulatory CD4+ T cells", Proc Natal Acad Sci USA, 110(3); 1023-8 (2013)). Co-stimulatory signalling through ICOS receptor only occurs in T cells receiving a concurrent TCR activation signal (Sharpe AH and Freeman GJ. "The B7-CD28 Superfamily", Nat. Rev Immunol, 2(2); 116-26 (2002)). In activated antigen specific T cells, ICOS regulates the production of both TH1 and TH2 cytokines including IFN-y, TNF-a, IL-10, IL-4, IL-13 and others. ICOS also stimulates effector T cell proliferation, albeit to a lesser extent than CD28 (Sharpe AH and Freeman GJ. "The B7-CD28 Superfamily", Nat. Rev Immunol, 2(2); 116-26 (2002)).

As used herein, "soluble ICOS" or "sICOS" refers to ICOS secreted or shed by cells.

Levels of sICOS in a sample, for example, a serum sample, can be measured by a number of methods well known in the art and as shown in the Examples. For example, sICOS levels in a sample can be measured using enzyme-linked immunosorbent assays (ELISA), Western blot assays, or mass spectrometry. Measurement of soluble ICOS levels using ELISA is described in, for example, Hasegawa et al., Rheumatology 2013;52:242-251 and Yanaba et al., Arch Dermatol Res 2013, 305: 17- 23. Mass spectrometry and ELISA based assays include, but are not limited to, platforms by Meso Scale Discovery (MSD), SINGULEX and MYRIAD RBM.

By "agent directed to ICOS" is meant any chemical compound or biological molecule capable of binding to ICOS. In some embodiments, the agent directed to ICOS is an ICOS binding protein. In some other embodiments, the agent directed to ICOS is an ICOS agonist. In some embodiments, the ICOS binding protein is an agonist ICOS binding protein.

The term "ICOS binding protein" as used herein refers to a protein that binds to ICOS, including an antibody or an antigen binding fragment thereof, or engineered molecules that function in similar ways to antibodies that are capable of binding to ICOS. In one embodiment, the antibody is a monoclonal antibody. In some instances, the ICOS is human ICOS. The term "ICOS binding protein" can be used interchangeably with "ICOS binding agent", "ICOS antigen binding protein" or "ICOS antigen binding agent". Thus, as is understood in the art, anti-ICOS antibodies and/or ICOS antigen binding proteins would be considered ICOS binding proteins. This definition does not include the natural cognate ligand or receptor. References to ICOS binding proteins, in particular anti-ICOS antibodies, includes antigen binding portions or fragments thereof. As used herein "antigen binding portion" of an ICOS binding protein would include any portion of the ICOS binding protein capable of binding to ICOS, including but not limited to, an antigen binding antibody fragment.

In one embodiment, the ICOS binding proteins of the present invention comprise any one or a combination of the following CDRs:

CDRH1: DYAMH (SEQ ID NO: l)

CDRH2: LISIYSDHTNYNQKFQG (SEQ ID NO:2)

CDRH3: NNYGNYGWYFDV (SEQ ID NO:3)

CDRL1: SASSSVSYMH (SEQ ID NO:4)

CDRL2: DTSKLAS (SEQ ID NO: 5)

CDRL3: FQGSGYPYT (SEQ ID NO:6)

In one embodiment, the ICOS binding protein comprises a heavy chain variable region CDR1 ("CDRH1") comprising an amino acid sequence with one or two amino acid variation(s) ("CDR variant") to the amino acid sequence set forth in SEQ ID NO: l.

In one embodiment, the ICOS binding protein comprises a heavy chain variable region CDR2 ("CDRH2") comprising an amino acid sequence with five or fewer, such as four or fewer, three or fewer, two or fewer, or one amino acid variation(s) ("CDR variant") to the amino acid sequence set forth in SEQ ID NO:2. In a further embodiment, the CDRH2 comprises an amino acid sequence with one or two amino acid variation(s) to the amino acid sequence set forth in SEQ ID NO:2.

In one embodiment, the ICOS binding protein comprises a heavy chain variable region CDR3 ("CDRH3") comprising an amino acid sequence with one or two amino acid variation(s) ("CDR variant") to the amino acid sequence set forth in SEQ ID NO:3.

In one embodiment, the ICOS binding protein comprises a light chain variable region CDR1 ("CDRL1") comprising an amino acid sequence with three or fewer, such as one or two amino acid variation(s) ("CDR variant") to the amino acid sequence set forth in SEQ ID NO:4.

In one embodiment, the ICOS binding protein comprises a light chain variable region CDR2 ("CDRL2") comprising an amino acid sequence with one or two amino acid variation(s) ("CDR variant") to the amino acid sequence set forth in SEQ ID NO: 5.

In one embodiment, the ICOS binding protein comprises a light chain variable region CDR3 ("CDRL3") comprising an amino acid sequence with three or fewer, such as one or two amino acid variation(s) ("CDR variant") to the amino acid sequence set forth in SEQ ID NO:6.

In one embodiment, the ICOS binding protein comprises a CDRH1 comprising an amino acid sequence with up to one amino acid variation to the amino acid sequence set forth in SEQ ID NO: l; a CDRH2 comprising an amino acid sequence with up to five amino acid variations to the amino acid sequence set forth in SEQ ID NO:2; a CDRH3 comprising an amino acid sequence with up to one amino acid variation to the amino acid sequence set forth in SEQ ID NO:3; a CDRL1 comprising an amino acid sequence with up to three amino acid variations to the amino acid sequence set forth in SEQ ID NO:4; a CDRL2 comprising an amino acid sequence with up to one amino acid variation to the amino acid sequence set forth in SEQ ID NO: 5; and/or a CDRL3 comprising an amino acid sequence with up to three amino acid variations to the amino acid sequence set forth in SEQ ID NO:6.

In one embodiment of the present invention the ICOS binding protein comprises CDRH1 (SEQ ID NO: 1), CDRH2 (SEQ ID NO:2), and CDRH3 (SEQ ID NO:3) in the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7. ICOS binding proteins of the present invention comprising the humanized heavy chain variable region set forth in SEQ ID NO:7 are designated as "H2." In some embodiments, the anti-ICOS antibodies of the present invention comprise a heavy chain variable region having at least 90% sequence identity to SEQ ID NO:7. Suitably, the ICOS binding proteins of the present invention may comprise a heavy chain variable region having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7.

Humanized heavy chain (VH) variable region (H2): OVOLVOSGAEVKKPGSSVKVSCKASGYTFTDYAMHWVROAPGOGLEWMGLISIYSDHTNYNOKFOGRVTITA DKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGOGTTVTVSS (SEQ ID NO:7; underlined amino acid residues correspond to the positions of CDRs).

In one embodiment, the ICOS binding protein comprises a heavy chain variable region ("VH") comprising an amino acid sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the VH comprises an amino acid sequence with at least one amino acid variation to the amino acid sequence set forth in SEQ ID NO:7, such as between 1 and 5, such as between 1 and 3, in particular up to 2 amino acid variations to the amino acid sequence set forth in SEQ ID NO:7.

In one embodiment of the present invention the ICOS binding protein comprises CDRL1 (SEQ ID NO:4), CDRL2 (SEQ ID NO:5), and CDRL3 (SEQ ID NO:6) in the light chain variable region having the amino acid sequence set forth in SEQ ID NO:8. ICOS binding proteins of the present invention comprising the humanized light chain variable region set forth in SEQ ID NO: 8 are designated as "L5." Thus, an ICOS binding protein of the present invention comprising the heavy chain variable region of SEQ ID NO:7 and the light chain variable region of SEQ ID NO:8 can be designated as H2L5 herein.

In some embodiments, the ICOS binding proteins of the present invention comprise a light chain variable region having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:8. Suitably, the ICOS binding proteins of the present invention may comprise a light chain variable region having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 8.

Humanized light chain (VL) variable region (L5):

EIVLTOSPATLSLSPGERATLSCSASSSVSYMHWYOOKPGOAPRLLIYDTSKLASGIPARFSGSGSGTDYTLTISS LEPEDFAVYYCFOGSGYPYTFGOGTKLEIK (SEQ ID NO:8; underlined amino acid residues correspond to the positions of CDRs).

In one embodiment, the ICOS binding protein comprises a light chain variable region ("VL") comprising an amino acid sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In one embodiment, the VL comprises an amino acid sequence with at least one amino acid variation to the amino acid sequence set forth in SEQ ID NO: 8, such as between 1 and 5, such as between 1 and 3, in particular up to 2 amino acid variations to the amino acid sequence set forth in SEQ ID NO:8.

In one embodiment, the ICOS binding protein comprises a VH domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 7 and/or a VL domain comprising an amino acid sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:8 wherein said ICOS binding protein specifically binds to human ICOS. In one embodiment, the ICOS binding protein comprises a VH with the amino acid sequence set forth in SEQ ID NO:7; and a VL with the amino acid sequence set forth in SEQ ID NO:8.

In one embodiment, the ICOS binding protein comprises a VH comprising an amino acid sequence of SEQ ID NO:7 and a VL comprising an amino acid sequence of SEQ ID NO:8

In one embodiment, the ICOS binding protein comprises a VH comprising an amino acid sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and a VL comprising an amino acid sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

In one embodiment, the ICOS binding protein is a humanized monoclonal antibody comprising a heavy chain (HC) amino acid sequence having at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9.

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYAMHVWRQAPGQGLEWMGLISIYSDHTNYNQKFQGRVTITA

DKSTSTAYMELSSLRSEDTAVYYCGRNNYGNYGWYFDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAAL

GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES

KYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREE

QFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV

KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL

SLGK (SEQ ID NO:9)

In one embodiment, the HC comprises an amino acid sequence with at least one amino acid variation to the amino acid sequence set forth in SEQ ID NO:9, such as between 1 and 10, such as between 1 and 7, in particular up to 6 amino acid variations to the amino acid sequence set forth in SEQ ID NO:9. In a further embodiment, the HC comprises one, two, three, four, five, six or seven amino acid variations to the amino acid sequence set forth in SEQ ID NO:9.

In one embodiment, the ICOS binding protein is a humanized monoclonal antibody comprising a light chain (LC) amino acid sequence having at least 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10.

EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDYTLTISS LEPEDFAVYYCFQGSGYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10) In one embodiment, the LC comprises an amino acid sequence with at least one amino acid variation to the amino acid sequence set forth in SEQ ID NO: 10, such as between 1 and 10, such as between 1 and 5, in particular up to 3 amino acid variations to the amino acid sequence set forth in SEQ ID NO: 10. In a further embodiment, the LC comprises one, two or three amino acid variations to the amino acid sequence set forth in SEQ ID NO: 10.

In one embodiment, the ICOS binding protein comprises a HC comprising an amino acid sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; and a LC comprising an amino acid sequence with at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. Therefore, the antibody is an antibody with a heavy chain at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO:9 and/or with a light chain at least about 90% identical to the light chain amino acid sequence of SEQ ID NO: 10.

In one embodiment, the ICOS binding protein comprises a heavy chain amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:9 and/or a light chain amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO: 10.

In one embodiment, the ICOS binding protein comprises a heavy chain sequence of SEQ ID NO:9 and a light chain sequence of SEQ ID NO: 10.

In one embodiment there is provided an ICOS binding protein comprising a heavy chain constant region such that has reduced ADCC and/or complement activation or effector functionality as compared to IgGl heavy chain constant region. In one such embodiment the heavy chain constant region may comprise a naturally disabled constant region of IgG2 or IgG4 isotype or a mutated IgGl constant region.

In one embodiment, the ICOS binding protein comprises an IgG4 Fc region comprising the amino acid substitutions S228P and L235E or functional equivalents thereof. In one embodiment, the ICOS binding protein comprises an IgG4 Fc region comprising amino acid subsitutions S229P and L236E. Such embodiments may have the designation IgG4PE. Thus, an ICOS binding protein having the heavy chain variable region H2 and the light chain variable region L5 and an IgG4PE Fc region will be designated as H2L5 IgG4PE or synonymously as H2L5 hIgG4PE.

Antibodies to ICOS and methods of using in the treatment of disease are described, for instance, in W02012131004, US20110243929, and US20160215059. US20160215059 is incorporated by reference herein. CDRs for murine antibodies to human ICOS having agonist activity are shown in PCT/EP2012/055735 (W02012131004). Antibodies to ICOS are also disclosed in WO2008137915, W02010056804, EP1374902, EP1374901, and EP1125585. Agonist antibodies to ICOS or ICOS binding proteins are disclosed in W02012/13004, WO2014033327, WO2016120789, US20160215059, and US20160304610. Exemplary antibodies in US20160304610 include 37A10S713. Sequences of

37A10S713 are reproduced below as SEQ ID NOS: 13-20. 37A10S713 VH CDRl: GFTFSDYWMD (SEQ ID NO: 13)

37A10S713 VH CDR2: NIDEDGSITEYSPFVKG (SEQ ID NO: 14)

37A10S713 VH CDR3: WGRFGFDS (SEQ ID NO: 15)

37A10S713 VL CDRl: KSSQSLLSGSFNYLT (SEQ ID NO: 16)

37A10S713 VL CDR2: YASTRHT (SEQ ID NO: 17)

37A10S713 VL CDR3: HHHYNAPPT (SEQ ID NO: 18)

37A10S713 heavy chain variable region:

EVOLVESGGLVOPGGSLRLSCAASGFTFSDYWMDWVROAPGKGLVWVSNIDEDGSITEYSPFVKGRFTISRDN AKNTLYLOM NSLRAEDTAVYYCTRWG RFG FDSWGOGTLVTVSS (SEQ ID NO: 19; underlined amino acid residues correspond to the positions of CDRs)

37A10S713 light chain variable region:

DIVMTOSPDSLAVSLGERATINCKSSOSLLSGSFNYLTWYOOKPGOPPKLLIFYASTRHTGVPDRFSGSGSGTDF TLTISSLOAEDVAVYYCHHHYNAPPTFGPGTKVDIK (SEQ ID NO:20; underlined amino acid residues correspond to the positions of CDRs)

In an embodiment, the ICOS binding protein is vopratelimab. In one embodiment, the ICOS binding protein is JTX-2011.

Exemplary antibodies in US2018/0289790 include ICOS.33 IgGlf S267E. Sequences of ICOS.33 IgGlf S267E are reproduced below as SEQ ID NOS:21-22:

ICOS.33 IgGlf S267E heavy chain variable domain:

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMHVWRQAPGKGLEVWGVIDTKSFNYATYYSDLVKGRFTISR DDSKNTLYLQMNSLKTEDTAVYYCTATIAVPYYFDYWGQGTLVTVSS (SEQ ID NO: 21)

ICOS.33 IgGlf S267E light chain variable domain:

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLSWYQQKPGKAPKLLIYYTNLLAEGVPSRFSGSGSGTDFTFTI SSLQPEDIATYYCQQYYNYRTFGPGTKVDIK (SEQ ID NO: 22)

In one embodiment, the ICOS binding protein is BMS-986226. Exemplary antibodies in WO2018/029474 include STIM003. Sequences of STIM003 are reproduced below as SEQ ID NOS: 23-24.

STIM003 heavy chain variable domain:

EVQLVESGGGWRPGGSLRLSCVASGVTFDDYGMSVWRQAPGKGLEWVSGINWNGGDTDYSDSVKGRFTISR DNAKNSLYLQMNSLRAEDTALYYCARDFYGSGSYYHVPFDYWGQGILVTVSS (SEQ ID NO: 23)

STIM003 light chain variable domain:

EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRLLIYGASSRATGIPDRFSGDGSGTDFTLSI SRLEPEDFAVYYCHQYDMSPFTFGPGTKVDIK (SEQ ID NO: 24)

In one embodiment, the ICOS binding protein is KY1044.

Exemplary antibodies in W02018/045110 include XENP23104. Sequences of the ICOS binding Fab side ([ICOS]_H0.66_L0) of XENP23104 are reproduced below as SEQ ID NOS: 25-60.

XENP23104 [ICOS]_H0.66_L0 heavy chain variable domain:

OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYYMHWVROAPGOGLEWMGWINPHSGETIYAOKFOGRVTMT RDTSISTAYMELSSLRSEDTAVYYCARTYYYDTSGYYHDAFDVWGOGTMVTVSS (SEQ ID NO: 25; underlined amino acid residues correspond to the positions of CDRs).

XENP23104 [ICOS]_H0.66_L0 VH CDRl: GYYMH (SEQ ID NO: 26)

XENP23104 [ICOS]_H0.66_L0 VH CDR2: WINPHSGETIYAQKFQG (SEQ ID NO: 27)

XENP23104 [ICOS]_H0.66_L0 VH CDR3: TYYYDTSGYYHDAFDV (SEQ ID NO: 28)

XENP23104 [ICOS]_H0.66_L0 light chain variable domain:

DIOMTOSPSSVSASVGDRVTITCRASOGISRLLAWYOOKPGKAPKLLIYVASSLOSGVPSRFSGSGSGTDFTLTI SSLOPEDFATYYCOOANSFPWTFGOGTKVEIK (SEQ ID NO: 29; underlined amino acid residues correspond to the positions of CDRs).

XENP23104 [ICOS]_H0.66_L0 VL CDRl: RASQGISRLLA (SEQ ID NO: 30)

XENP23104 [ICOS]_H0.66_L0 VL CDR2: VASSLQS (SEQ ID NO: 31)

XENP23104 [ICOS]_H0.66_L0 VL CDR3: QQANSFPWT (SEQ ID NO: 32) As used herein "ICOS-L" and "ICOS Ligand" are used interchangeably and refer to the membrane bound natural ligand of human ICOS. ICOS ligand is a protein that in humans is encoded by the ICOSLG gene. ICOSLG has also been designated as CD275 (cluster of differentiation 275). Pseudonyms for ICOS-L include B7RP-1 and B7-H2.

"Antigen binding protein" (ABP) means a protein that binds an antigen, including antibodies or engineered molecules that function in similar ways to antibodies. Such alternative antibody formats include triabody, tetrabody, miniantibody, and a minibody. 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. The terms "ABP", "antigen binding protein", "binding protein", "antigen binding agent" and "binding agent" are used interchangeably herein. For example, disclosed herein are ICOS binding proteins, namely ICOS antibodies.

"Antigen binding site" refers to a site on an antigen binding protein that is capable of specifically binding to an antigen, this may be a single variable domain, or it may be paired VH/VL domains as can be found on a standard antibody. Single-chain Fv (scFv) domains can also provide antigen-binding sites.

The term "antibody" is used herein in the broadest sense to refer to molecules comprising an immunoglobulin-like domain (for example IgG, IgM, IgA, IgD or IgE) and includes monoclonal, recombinant, polyclonal, chimeric, human, humanized, multispecific antibodies, including bispecific antibodies, and heteroconjugate antibodies; a single variable domain ( e.g . VH, VHH, VL, domain antibody (DAB)), antigen binding antibody fragments, Fab, F(ab 2, Fv, disulphide linked Fv, single chain Fv, disulphide-linked scFv, diabodies, TANDABS, etc. and modified versions of any of the foregoing (for a summary of alternative "antibody" formats see, e.g. Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No. 9, 1126-1136).

A "chimeric antibody" refers to a type of engineered antibody that contains a naturally- occurring variable region (light chain and heavy chains) derived from a donor antibody in association with light and heavy chain constant regions derived from an acceptor 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 al. Proc. Natl Acad Sci USA, 86: 10029- 10032 (1989), Hodgson et al. Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody may be one selected from a conventional database, e.g. the KABAT 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 humanized antibodies - see, for example, EP-A-0239400 and EP-A-054951.

The term "fully human antibody" includes antibodies having variable and constant regions (if present) derived from human germline immunoglobulin sequences. The human sequence antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g. mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Fully human antibodies comprise amino acid sequences encoded only by polynucleotides that are ultimately of human origin or amino acid sequences that are identical to such sequences. As meant herein, antibodies encoded by human immunoglobulin-encoding DNA inserted into a mouse genome produced in a transgenic mouse are fully human antibodies since they are encoded by DNA that is ultimately of human origin. In this situation, human immunoglobulin-encoding DNA can be rearranged (to encode an antibody) within the mouse, and somatic mutations may also occur. Antibodies encoded by originally human DNA that has undergone such changes in a mouse are fully human antibodies as meant herein. The use of such transgenic mice makes it possible to select fully human antibodies against a human antigen. As is understood in the art, fully human antibodies can be made using phage display technology wherein a human DNA library is inserted in phage for generation of antibodies comprising human germline DNA sequence.

The term, full, whole or intact antibody, used interchangeably herein, refers to a heterotetra meric glycoprotein with an approximate molecular weight of 150,000 daltons. An intact antibody is composed of two identical heavy chains (HCs) and two identical light chains (LCs) linked by covalent disulphide bonds. This H2L2 structure folds to form three functional domains comprising two antigen-binding fragments, known as 'Fab' fragments, and a 'Fc' crystal Usable fragment. The Fab fragment is composed of the variable domain at the amino-terminus, variable heavy (VH) or variable light (VL), and the constant domain at the carboxyl terminus, CHI (heavy) and CL (light). The Fc fragment is composed of two domains formed by dimerization of paired CH2 and CH3 regions. The Fc may elicit effector functions by binding to receptors on immune cells or by binding Clq, the first component of the classical complement pathway. The five classes of antibodies IgM, IgA, IgG, IgE and IgD are defined by distinct heavy chain amino acid sequences which are called m, a, g, e and d respectively, each heavy chain can pair with either a K or l light chain. The majority of antibodies in the serum belong to the IgG class, there are four isotypes of human IgG, IgGl, IgG2, IgG3 and IgG4, the sequences of which differ mainly in their hinge region.

Fully human antibodies can be obtained using a variety of methods, for example using yeast- based libraries or transgenic animals ( e.g . mice) which are capable of producing repertoires of human antibodies. Yeast presenting human antibodies on their surface which bind to an antigen of interest can be selected using FACS (Fluorescence-Activated Cell Sorting) based methods or by capture on beads using labelled antigens. Transgenic animals that have been modified to express human immunoglobulin genes can be immunised with an antigen of interest and antigen-specific human antibodies isolated using B-cell sorting techniques. Human antibodies produced using these techniques can then be characterised for desired properties such as affinity, developability and selectivity.

Alternative antibody formats include alternative scaffolds in which the one or more CDRs of the antigen binding protein 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.

The term "domain" refers to a folded polypeptide structure that retains its tertiary structure independent of the rest of the polypeptide. Generally domains are responsible for discrete functional properties of polypeptides and in many cases may be added, removed or transferred to other polypeptides without loss of function of the remainder of the protein and/or of the domain.

The term "single variable domain" refers to a folded polypeptide domain comprising sequences characteristic of antibody variable domains. It therefore includes complete antibody variable domains such as VH, VHH and VL and modified antibody variable domains, for example, in which one or more loops have been replaced by sequences which are not characteristic of antibody variable domains, or antibody variable domains which have been truncated or comprise N- or C-terminal extensions, as well as folded fragments of variable domains which retain at least the binding activity and specificity of the full-length domain. A single variable domain is capable of binding an antigen or epitope independently of a different variable region or domain. A "domain antibody" or "DAB" may be considered the same as a "single variable domain". A single variable domain may be a human single variable domain, but also includes single variable domains from other species such as rodent, nurse shark and Camelid VHH DABS. Camelid VHH are immunoglobulin single variable domain polypeptides that are derived from species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies naturally devoid of light chains. Such VHH domains may be humanized according to standard techniques available in the art, and such domains are considered to be "single variable domains". As used herein VH includes camelid VHH domains.

The terms "VH" and "VL" are used herein to refer to the heavy chain variable region and light chain variable region respectively of an antigen binding protein. "CDRs" are defined as the complementarity determining region amino acid sequences of an antigen binding protein. 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. Thus, "CDRs" as used herein refers to all three heavy chain CDRs, all three light chain CDRs, all heavy and light chain CDRs, or at least two CDRs.

Throughout this specification, amino acid residues in variable domain sequences and variable domain regions within full length antigen binding sequences, e.g. within an antibody heavy chain sequence or antibody light chain sequence, are numbered according to the Kabat numbering convention. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" used in the Examples follow the Kabat numbering convention. For further information, see Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1991).

It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full length antibody sequences. There are also alternative numbering conventions for CDR sequences, for example those set out in Chothia et al. (1989) Nature 342: 877-883. The structure and protein folding of the antigen binding protein may mean that other residues are considered part of the CDR sequence and would be understood to be so by a skilled person.

Other numbering conventions for CDR sequences available to a skilled person include "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlapping region using at least two of the Kabat, Chothia, AbM and contact methods can be determined to provide the "minimum binding unit". The minimum binding unit may be a sub-portion of a CDR.

CDRs or minimum binding units may be modified by at least one amino acid substitution, deletion or addition, wherein the variant antigen binding protein substantially retains the biological characteristics of the unmodified protein, such as an antibody comprising SEQ ID NO:7 and SEQ ID NO:8.

CDRs or minimum binding units may be modified by at least one amino acid substitution, deletion or addition, wherein the variant antigen binding protein substantially retains the biological characteristics of the unmodified protein, such as an antibody comprising SEQ ID NO:7 and SEQ ID NO:8. It will be appreciated that each of CDR HI, H2, H3, LI, L2, L3 may be modified alone or in combination with any other CDR, in any permutation or combination. In one embodiment, a CDR is modified by the substitution, deletion or addition of up to 3 amino acids, for example 1 or 2 amino acids, for example 1 amino acid. Typically, the modification is a substitution, particularly a conservative substitution (referred herein also as a direct equivalent), for example as shown in Table 1 below. Table 1

"Percent identity" between a query amino acid sequence and a subject amino acid sequence is the "Identities" value, expressed as a percentage, that is calculated using a suitable algorithm or software, such as BLASTP, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle or EMBOSS infoalign, over the entire length of the query sequence after a pair-wise global sequence alignment has been performed using a suitable algorithm/software such as BLASTP, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene. Importantly, a query amino acid sequence may bthe se described by an amino acid sequence identified in one or more claims herein.

The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of amino acid or nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. Such alterations include at least one amino acid deletion, substitution (including conservative and non-conservative substitution), or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the query sequence or anywhere between those terminal positions, interspersed either individually among the amino acids or nucleotides in the query sequence or in one or more contiguous groups within the query sequence.

The % identity may be determined across the entire length of the query sequence, including the CDRs. Alternatively, the % identity may exclude one or more or all of the CDRs, for example all of the CDRs are 100% identical to the subject sequence and the % identity variation is in the remaining portion of the query sequence, e.g. the framework sequence, so that the CDR sequences are fixed and intact.

The variant sequence substantially retains the biological characteristics of the unmodified protein, such as an agonist for ICOS.

An antigen binding fragment may be provided by means of arrangement of one or more CDRs on non-antibody protein scaffolds. "Protein Scaffold" as used herein includes, but is not limited to, an immunoglobulin (Ig) scaffold, for example an IgG scaffold, which may be a four chain or two chain antibody, or which may comprise only the Fc region of an antibody, or which may comprise one or more constant regions from an antibody, which constant regions may be of human or primate origin, or which may be an artificial chimera of human and primate constant regions.

The protein scaffold may be an Ig scaffold, for example an IgG, or IgA scaffold. The IgG scaffold may comprise some or all the domains of an antibody (/.e. CHI, CH2, CH3, VH, VL). The antigen binding protein may comprise an IgG scaffold selected from IgGl, IgG2, IgG3, IgG4 or IgG4PE. For example, the scaffold may be IgGl. The scaffold may consist of, or comprise, the Fc region of an antibody, or is a part thereof.

The subclass of an antibody in part determines secondary effector functions, such as complement activation or Fc receptor (FcR) binding and antibody dependent cell cytotoxicity (ADCC) (Huber et al. Nature 229(5284): 419-20 (1971); Brunhouse et al. Mol Immunol 16(11): 907-17 (1979)). In identifying the optimal type of antibody for a particular application, the effector functions of the antibodies can be taken into account. For example, hlgGl antibodies have a relatively long half life, are very effective at fixing complement, and they bind to both FcyRI and FcyRII. In contrast, human IgG4 antibodies have a shorter half life, do not fix complement and have a lower affinity for the FcRs. Replacement of serine 228 with a proline (S228P) in the Fc region of IgG4 reduces heterogeneity observed with hIgG4 and extends the serum half life (Kabat et al. "Sequences of proteins of immunological interest" 5.sup.th Edition (1991); Angal et al. Mol Immunol 30(1): 105-8 (1993)). A second mutation that replaces leucine 235 with a glutamic acid (L235E) eliminates the residual FcR binding and complement binding activities (Alegre et al. J Immunol 148(11): 3461-8 (1992)). The numbering of the hIgG4 amino acids was derived from EU numbering reference: Edelman et al. Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969.

The term "donor antibody" refers to an antibody that contributes the amino acid sequences of its variable regions, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner. The donor, therefore, provides the altered immunoglobulin coding region and resulting expressed altered antibody with the antigenic specificity and neutralising activity characteristic of the donor antibody.

The term "acceptor antibody" refers to an antibody that is heterologous to the donor antibody, which contributes all (or any portion) of the amino acid sequences encoding its heavy and/or light chain framework regions and/or its heavy and/or light chain constant regions to the first immunoglobulin partner. A human antibody may be the acceptor antibody.

Affinity, also referred to as "binding affinity", is the strength of binding at a single interaction site, /.e. of one molecule, e.g. an antigen binding protein of the invention, to another molecule, e.g. its target antigen, at a single binding site. The binding affinity of an antigen binding protein to its target may be determined by equilibrium methods (e.g. enzyme-linked immunoabsorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics (e.g. BIACORE analysis). Avidity, also referred to as functional affinity, is the cumulative strength of binding at multiple interaction sites, e.g. the sum total of the strength of binding of two molecules (or more, e.g. in the case of a bispecific or multispecific molecule) to one another at multiple sites, e.g. taking into account the valency of the interaction.

As used herein an "immuno-modulator" or "immuno-modulatory agent" refers to any substance including monoclonal antibodies that affects the immune system. In some embodiments, the immuno-modulator or immuno-modulatory agent upregulates an aspect of the immune system. Immuno-modulators can be used as anti-neoplastic agents for the treatment of cancer. For example, immuno-modulators include, but are not limited to, anti-ICOS antibodies.

As used herein the term "agonist" refers to an antigen binding protein including, but not limited to, an antibody, that upon contact with a co-signalling 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 and/or (3) 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 signalling, 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. Cytokine analysis may be performed according to the methods known in the art and, for example, as disclosed in US20160215059, which is incorporated by reference herein. In one embodiment, the ICOS binding protein is an agonist ICOS binding protein.

As used herein the term "antagonist" refers to an antigen binding protein including, but not limited to, an antibody, that upon contact with a co-signalling receptor causes one or more of the following (1) attenuates, blocks or inactivates the receptor and/or blocks activation of a receptor by its natural ligand, (2) reduces, decreases or shortens the activity, function or presence of the receptor and/or (3) reduces, descrease, abrogates the expression of the receptor. Antagonist activity can be measured in vitro by various assays know in the art such as, but not limited to, measurement of an increase or decrease in cell signalling, cell proliferation, immune cell activation markers, cytokine production. Antagonist 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.

By "isolated" it is intended that the molecule, such as an antigen binding protein or nucleic acid, is removed from the environment in which it may be found in nature. For example, the molecule may be purified away from substances with which it would normally exist in nature. For example, the mass of the molecule in a sample may be 95% of the total mass.

As used herein, the term "responder" refers someone who is identified (using a particular test or method) to be more likely to derive benefit in response to a particular treatment (e.g. treatment with an ICOS binding protein of the invention). It is understood that not all people who have been identified as a responder will necessarily derive benefit, but as a patient class, they are more likely to do so. For example, it may be that out of the total untested diseased population, approximately 80% of that population derive benefit from a drug, but out of the group of "Responders" (i.e. those individuals who have been tested, and identified as a Responder according to the set criteria) approximately 99% will derive benefit.

As used herein, the term "biomarker" refers to a characteristic, or combination of characteristics (sometimes referred to as a "signature"), for use as a tool for the diagnosis of a particular disease, or for predicting disease outcome/progression, or for predicting the response to, or monitoring treatment subsequent to treatment with, for example, a particular drug (e.g. treatment with an ICOS binding protein of the invention). An example of a biomarker includes, but is not limited to, soluble ICOS.

As used herein, the term "characteristic" is used to refer to all types of indicia, including nucleic acids, proteins, polypeptides and chemicals (e.g. degradation products, hormones, modified biological compounds), and further may include measurements on the body such as blood pressure, blood oxygen levels, MRI scans etc. The characteristic may be the presence or absence of one or more markers, the absolute amount or level of one or more markers, or may be comparative, for example from placebo-treated patients as compared to drug-treated patients, or by comparison between samples from healthy people as compared to drug-treated patients.

In one aspect of the invention, there is provided a method of treating cancer in a human in need thereof, the method comprising determining the level of soluble ICOS (sICOS) in a sample from the human and administering to the human an effective amount of an agent directed to human ICOS if the level of the sICOS is increased relative to a reference level, thereby treating the cancer in the human. In one embodiment, the agent directed to human ICOS is an ICOS binding protein or antigen binding portion thereof. In one embodiment, the agent directed to human ICOS is an anti-ICOS antibody or antigen binding portion thereof. In one embodiment, the anti-ICOS antibody is an ICOS agonist. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7; and a VL domain comprising an amino acid sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:8. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL domain comprising the amino acid sequence as set forth in SEQ ID NO:8. In one embodiment, the anti-ICOS antibody comprises one or more of: CDRH1 as set forth in SEQ ID NO: l; CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set forth in SEQ ID NO:5 and/or CDRL3 as set forth in SEQ ID NO: 6 or a direct equivalent of each CDR wherein a direct equivalent has no more than two amino acid substitutions in said CDR.

In one embodiment of any one of the aspects described herein, the ICOS binding proteins or antigen binding portions comprise one or more of: CDRH1 as set forth in SEQ ID NO: l; CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set forth in SEQ ID NO: 5 and/or CDRL3 as set forth in SEQ ID NO:6 or a direct equivalent of each CDR wherein a direct equivalent has no more than two amino acid substitutions in said CDR.

In one embodiment of any one of the aspects described herein, the ICOS binding proteins or antigen binding portions thereof specifically bind to human ICOS wherein the ICOS binding protein comprises a VH domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7 and/or a VL domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:8.

In one embodiment, the ICOS binding proteins or antigen binding portions thereof are humanized monoclonal antibodies or antigen binding portions that comprise heavy chain CDRs having the amino acid sequences set forth in SEQ ID NO: l; SEQ ID NO:2; and SEQ ID NO:3 and light chain CDRs having the amino acid sequences set forth in SEQ ID NO:4; SEQ ID NO:5; and SEQ ID NO:6. In one embodiment, the humanized monoclonal antibodies comprise a hIgG4PE scaffold; a VH domain comprising an amino acid sequence set forth in SEQ ID NO:7; and a VL domain comprising an amino acid sequence set forth in SEQ ID NO:8. The antibodies of the present invention may stimulate cytokine production when contacted with a T cell.

In one embodiment of any one of the aspects described herein, the ICOS binding proteins compete for binding to human ICOS with any one of the ICOS binding proteins or antigen binding portions thereof of the invention.

In one embodiment of any one of the aspects described herein, the cancer is colorectal cancer (CRC), gastric, esophageal, cervical, bladder, urothelial, breast, head and neck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell, non-small cell lung carcinoma, head and neck squamous cell carcinoma, mesothelioma, pancreatic, or prostate cancer. In one aspect, the sample comprises serum. In one aspect, the level of sICOS is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4- fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold relative to the reference level.

In another aspect of the invention there is provided a method of inhibiting proliferation of a cancer cell in a human in need thereof, the method comprising administering to the human an effective amount of an agent directed to human ICOS, thereby inhibiting proliferation of the cancer cell in the human, wherein a sample from the human has an increased level of sICOS relative to a reference level. In one embodiment, the agent directed to human ICOS is an ICOS binding protein or antigen binding portion thereof. In one embodiment, the agent directed to human ICOS is an anti-ICOS antibody or antigen binding portion thereof. In one embodiment, the anti-ICOS antibody is an ICOS agonist. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7; and a VL domain comprising an amino acid sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:8. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL domain comprising the amino acid sequence as set forth in SEQ ID NO: 8. In one embodiment, the anti-ICOS antibody comprises one or more of: CDRH1 as set forth in SEQ ID NO: l; CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set forth in SEQ ID NO:5 and/or CDRL3 as set forth in SEQ ID NO: 6 or a direct equivalent of each CDR wherein a direct equivalent has no more than two amino acid substitutions in said CDR. In one embodiment of any one of the aspects described herein, the cancer is colorectal cancer (CRC), gastric, esophageal, cervical, bladder, urothelial, breast, head and neck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell, non-small cell lung carcinoma, head and neck squamous cell carcinoma, mesothelioma, pancreatic, or prostate cancer. In one embodiment, the sample comprises serum. In one embodiment, the level of sICOS is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold relative to the reference level.

In yet another aspect of the invention, there is provided a method of predicting whether a human having cancer will be sensitive to treatment with an agent directed to human ICOS, the methods comprising determining the level of soluble ICOS (sICOS) in a sample from the human, wherein an increased level of sICOS relative to a reference level indicates the human will be sensitive to treatment with an agent directed to human ICOS. In one embodiment, the agent directed to human ICOS is an ICOS binding protein or antigen binding portion thereof. In one embodiment, the agent directed to human ICOS is an anti-ICOS antibody or antigen binding portion thereof. In one embodiment, the anti-ICOS antibody is an ICOS agonist. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7; and a VL domain comprising an amino acid sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:8. In one embodiment, the anti- ICOS antibody comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 7 and a VL domain comprising the amino acid sequence as set forth in SEQ ID NO:8. In one aspect, the anti-ICOS antibody comprises one or more of: CDRH1 as set forth in SEQ ID NO: l; CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set forth in SEQ ID NO: 5 and/or CDRL3 as set forth in SEQ ID NO:6 or a direct equivalent of each CDR wherein a direct equivalent has no more than two amino acid substitutions in said CDR. In one embodiment of any one of the aspects described herein, the cancer is colorectal cancer (CRC), gastric, esophageal, cervical, bladder, urothelial, breast, head and neck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell, non-small cell lung carcinoma, head and neck squamous cell carcinoma, mesothelioma, pancreatic, or prostate cancer. In one embodiment, the sample comprises serum. In one embodiment, the level of sICOS is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100- fold, at least about 500-fold, or at least about 1000-fold relative to the reference level.

In another aspect of the invention, there is provided an agent directed to human ICOS for use in the treatment of cancer in a human classified as a responder, wherein a responder is characterized by the presence of an increased level of soluble ICOS (sICOS) in a sample from the human relative to a reference level. In one embodiment, the agent directed to human ICOS is an ICOS binding protein or antigen binding portion thereof. In one embodiment, the agent directed to human ICOS is an anti- ICOS antibody or antigen binding portion thereof. In one embodiment, the anti-ICOS antibody is an ICOS agonist. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7; and a VL domain comprising an amino acid sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:8. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL domain comprising the amino acid sequence as set forth in SEQ ID NO: 8. In one embodiment, the anti-ICOS antibody comprises one or more of: CDRH1 as set forth in SEQ ID NO: l; CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set forth in SEQ ID NO:5 and/or CDRL3 as set forth in SEQ ID NO: 6 or a direct equivalent of each CDR wherein a direct equivalent has no more than two amino acid substitutions in said CDR. In one embodiment of any one of the aspects described herein, the cancer is colorectal cancer (CRC), gastric, esophageal, cervical, bladder, urothelial, breast, head and neck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell, non-small cell lung carcinoma, head and neck squamous cell carcinoma, mesothelioma, pancreatic, or prostate cancer. In one embodiment, the sample comprises serum. In one embodiment, the level of sICOS is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold relative to the reference level.

In another aspect, the present invention provides an increased level of soluble ICOS (sICOS) in a sample from a human relative to a reference level for use as a biomarker in the treatment/diagnosis of a cancer responsive to an agent directed to human ICOS. In one embodiment, the agent directed to human ICOS is an ICOS binding protein or antigen binding portion thereof. In one embodiment, the agent directed to human ICOS is an anti-ICOS antibody or antigen binding portion thereof. In one embodiment, the anti-ICOS antibody is an ICOS agonist. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7; and a VL domain comprising an amino acid sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO:8. In one embodiment, the anti-ICOS antibody comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL domain comprising the amino acid sequence as set forth in SEQ ID NO:8. In another embodiment, the anti-ICOS antibody comprises one or more of: CDRH1 as set forth in SEQ ID NO: l; CDRH2 as set forth in SEQ ID NO:2; CDRH3 as set forth in SEQ ID NO:3; CDRL1 as set forth in SEQ ID NO:4; CDRL2 as set forth in SEQ ID NO:5 and/or CDRL3 as set forth in SEQ ID NO:6 or a direct equivalent of each CDR wherein a direct equivalent has no more than two amino acid substitutions in said CDR. In one embodiment of any one of the aspects described herein, the cancer is colorectal cancer (CRC), gastric, esophageal, cervical, bladder, urothelial, breast, head and neck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell, non-small cell lung carcinoma, head and neck squamous cell carcinoma, mesothelioma, pancreatic, or prostate cancer. In one embodiment, the sample comprises serum. In one embodiment, the level of sICOS is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2- fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold relative to the reference level.

In another aspect, the present invention provides increased level of soluble ICOS (sICOS) relative to a reference level for use in a diagnostic method. In another aspect, the present invention provides an increased level of soluble ICOS (sICOS) relative to a reference level for use in therapy. In one embodiment of any one of the aspects described herein, the cancer is colorectal cancer (CRC), gastric, esophageal, cervical, bladder, urothelial, breast, head and neck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell, non-small cell lung carcinoma, head and neck squamous cell carcinoma, mesothelioma, pancreatic, or prostate cancer. In one aspect, the level of sICOS is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, or at least about 1000-fold relative to the reference level.

In one aspect of the invention, a kit for the treatment of cancer is provided, comprising a kit for determining the level of soluble ICOS (sICOS) in a sample from a human, and a means for determining the level of sICOS in a sample from a human. In one embodiment, the means is a capture antibody.

In one aspect, the present invention provides a pharmaceutical composition comprising an agent directed to human ICOS, for use in treating cancer in a human wherein at least a first sample from the human is determined to have an increased level of soluble ICOS (sICOS) relative to a reference level.

In one aspect of the invention, there is provided use of an agent directed to human ICOS in the manufacture of a medicament for the treatment of cancer in a human, wherein one or more samples from the human is determined to have an increased level of soluble ICOS (sICOS) relative to a reference level.

Samples, e.g. biological samples, for testing or determining of levels of soluble ICOS may be any bodily fluid or tissue, including, but not limited to, serum, blood, blood components, urine, and saliva. Testing for soluble ICOS levels may be conducted by several techniques known in the art and/or described herein. In some embodiments, the sample is serum. A control or reference level can be any one of skill in the art would choose, such as the level of soluble ICOS of a sample from a healthy human or a particular level of soluble ICOS that correlates with responsiveness to ICOS activation, as described for example, in Example 1. In one embodiment, the reference level is the level of soluble ICOS of a sample from a healthy human. A healthy human is a human that does not have cancer.

Conventional molecular biology, microbiology, and recombinant DNA techniques including sequencing techniques are well known among those skilled in the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein "Sambrook, et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames & S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

In some embodiments, the ICOS binding protein is administered at a dose of about 0.08 - 800 mg (e.g. a dose about 0.08 mg; a dose about 0.24 mg; a dose about 0.8 mg; a dose about 2.4 mg; a dose about 8 mg; a dose about 16 mg; a dose about 24 mg; a dose about 32 mg; a dose about 40 mg; a dose about 48 mg; a dose about 56 mg; a dose about 64 mg; a dose about 72 mg; a dose about 80 mg; a dose about 88 mg; a dose about 96 mg; a dose about 100 mg; a dose about 160 mg; a dose about 200 mg; a dose about 240 mg; a dose about 300 mg; a dose about 400 mg; a dose about 500 mg; a dose about 600 mg; a dose about 700 mg or a dose about 800 mg). In some embodiments, the ICOS binding protein is administered at a dose of about 0.08 - 240 mg. In further embodiments, the ICOS binding protein is administered at a dose of about 0.001 - 10 mg/kg (e.g. a dose about 0.001 mg/kg, a dose about 0.003 mg/kg, a dose about 0.01 mg/kg, a dose about 0.03 mg/kg, a dose about 0.1 mg/kg, a dose about 0.3 mg/kg, a dose about 0.6 mg/kg, a dose about 1.0 mg/kg, a dose about 2.0 mg/kg, a dose about 3.0 mg/kg, a dose about 6 mg/kg or a dose about 10 mg/kg). In some embodiments, the ICOS binding protein is administered at a dose of about 0.001 - 3 mg/kg. In some embodiments, the ICOS binding protein is administered at a dose of about 0.3 mg/kg. In some embodiments, the ICOS binding protein is administered at a dose of about 1 mg/kg. In some embodiments, the ICOS binding protein is administered at a dose of about 3 mg/kg. In some embodiments, the ICOS binding protein is administered at a dose of about 24 mg. In some embodiments, the ICOS binding protein is administered at a dose of about 48 mg. In some embodiments, the ICOS binding protein is administered at a dose of about 72 mg. In some embodiments, the ICOS binding protein is administered at a dose of about 80 mg. In some embodiments, the ICOS protein is administered at a dose of about 96 mg. In some embodiments, the ICOS protein is administered at a dose of about 120 mg. In some embodiments, the ICOS protein is administered at a dose of about 148 mg. In some embodiments, the ICOS binding protein is administered at a dose of about 160 mg. In some embodiments, the ICOS binding protein is administered at a dose of about 240 mg. In some embodiments, the ICOS protein is administered at a dose of about 320 mg. In some embodiments, the ICOS protein is administered at a dose of about 480 mg.

In one embodiment, the dose of the ICOS binding protein is in the range of about 0.08 mg to about 800 mg. In another embodiment, the dose of the ICOS binding protein is in the range of about 0.8 mg to about 240 mg.

In another embodiment, the dose of the ICOS binding protein is in the range of about 8 mg to about 80 mg. In another embodiment, the dose of the ICOS binding protein is about 0.08 mg, about 0.24 mg, about 0.48 mg, about 0.8 mg, about 1.6 mg, about 2.4 mg, about 8 mg, about 24 mg, about 48 mg, about 80 mg, about 160 mg or about 240 mg. In one embodiment, the dose of ICOS binding protein is about 24 mg, about 48 mg, about 80 mg or about 160 mg. In one embodiment, the dose of the ICOS binding protein is at least about 24 mg. In one embodiment, the dose of the ICOS binding protein is at least about 48 mg.

In one embodiment, the ICOS binding protein is administered once every 2-6 weeks ( e.g . 2, 3 or 4 weeks, in particular 3 weeks). In one embodiment the ICOS binding protein is administered once every 3 weeks. In one embodiment the ICOS binding protein is administered once every 3 weeks for 2-6 dosing cycles (e.g. the first 3, 4, or 5 dosing cycles, in particular, the first 4 dosing cycles). In one mebodiment, the ICOS binding protein is administered once every 6 weeks.

In some embodiments, the ICOS binding protein is administered at an administration interval (or treatment cycle) of once a week (Q1W), once every 2 weeks (Q2W), once every 3 weeks (Q3W), once every 4 weeks (Q4W), once every 5 weeks (Q5W), or once every 6 weeks (Q6W). In some embodiments, the ICOS binding protein is administered at an administration interval (or treatment cycle) of once a week (Q1W). In some embodiments, the ICOS binding protein is administered at an administration interval (or treatment cycle) of once every 2 weeks (Q2W). In some embodiments, the ICOS binding protein is administered at an administration interval (or treatment cycle) of once every three weeks (Q3W). In some embodiments, the ICOS binding protein is administered at an administration interval (or treatment cycle) of once every 4 weeks (Q4W). In some embodiments, the ICOS binding protein is administered at an administration interval (or treatment cycle) of once every 5 weeks (Q5W). In some embodiments, the ICOS binding protein is administered at an administration interval (or treatment cycle) of once every 6 weeks (Q6W). In some embodiments, the ICOS binding protein is administered for a period of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks, or more. In some embodiments, the ICOS binding protein is administered on the first day of a treatment cycle or within 1, 2, or 3 days of the first day of a treatment cycle.

In one embodiment, the ICOS binding protein is vopratelimab. In one embodiment, vopratelimab is administered at 0.03 mg/kg, 0.3 mg/kg or 0.1 mg/kg. In one embodiment, vopratelimab is administered every 3 weeks. In another embodiment, the dosing amount and interval between doses of vopratelimab is pulsatile.

In some embodiments, the combination described herein is administered according to dosing regimens demonstrated to achieve a clinical benefit for the patient. In some embodiments, a clinical benefit is stable disease ("SD"), a partial response ("PR") and/or a complete response ("CR"). In some embodiments, a clinical benefit is stable disease ("SD"). In some embodiments, a clinical benefit is a partial response ("PR"). In some embodiments, a clinical benefit is a complete response ("CR"). In some embodiments, PR or CR is determined in accordance with Response Evaluation Criteria in Solid Tumors (RECIST). In some embodiments, the combination is administered for a longer period to maintain clinical benefit.

In one embodiment, the cancer is selected from: brain cancer, glioblastomas, glioma (such as diffuse intrinsic pontine glioma), Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast cancer ( e.g . inflammatory breast cancer), Wilm's tumor, ependymoma, medulloblastoma, cardiac tumors, colon cancer, colorectal cancer, head and neck cancer (e.g. squamous cell carcinoma of the head and neck, cancer of the mouth (/.e. oral cancer), salivary gland cancer, buccal cancer, pharyngeal cancer, oropharyngeal cancer, nasopharangeal cancer, hypopharyngeal cancer, laryngeal cancer), eye cancer (e.g. retinoblastoma), lung cancer (e.g. nonsmall cell lung cancer, small cell cancer), liver cancer (/.e. hepatocellular cancer), skin cancer (e.g. basal cell carcinoma, merkel cell carcinoma, squamous cell carcinoma), melanoma, ovarian cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, prostate cancer, sarcoma (e.g. soft tissue sarcoma, Ewing's sarcoma, Kaposi sarcoma, rhabdomyosarcoma), bone cancer, osteosarcoma, giant cell tumor of bone, thyroid cancer, parathyroid cancer, thymoma, blood cancer (which may be broadly categorised as leukemias, lymphomas or myelomas, and include examples such as lymphoblastic T- cell leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, 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, erythroleukemia, malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic T cell lymphoma, Burkitt's lymphoma, and follicular lymphoma), neuroblastoma, pituitary tumor, adrenocortical cancer, anal cancer (/.e. rectal cancer), bladder cancer, urothelial cancer, urethral cancer, vaginal cancer, vulvar cancer, cervical cancer, endometrial cancer, uterine cancer, fallopian tube cancer, renal cancer (/.e. kidney cancer, e.g. renal cell carcinoma), mesothelioma (e.g. malignant pleural mesothelioma), esophageal cancer (e.g. esophageal squamous cell carcinoma), gastric cancer (/.e. stomach cancer), gastroinstestinal carcinoid tumor, GIST (gastrointestinal stromal tumor), appendicial cancer, penile cancer, testicular cancer, germ cell tumors.

In one embodiment, the cancer is a solid tumor. In one embodiment, the tumor is selected from head and neck cancer, gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, non-small cell lung carcinoma, prostate cancer, colorectal cancer, ovarian cancer and pancreatic cancer. In another embodiment the human has a liquid tumor such as diffuse large B cell lymphoma (DLBCL), multiple myeloma, chronic lyphomblastic leukemia (CLL), follicular lymphoma, acute myeloid leukemia and chronic myelogenous leukemia.

In one embodiment, the cancer exhibits microsatellite instability (MSI). Microsatellite instability ("MSI") is or comprises a change that in the DNA of certain cells (such as tumor cells) in which the number of repeats of microsatellites (short, repeated sequences of DNA) is different than the number of repeats that was contained in the DNA from which it was inherited. Microsatellite instability arises from a failure to repair replication-associated errors due to a defective DNA mismatch repair (MMR) system. This failure allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, leading to increased mutational load. It has been demonstrated that at least some tumors characterized by MSI-H have improved responses to certain anti-PD-1 agents (Le etal. (2015) N. Engl. J. Med. 372(26):2509-2520; Westdorp etal. (2016) Cancer Immunol. Immunother. 65(10): 1249-1259).

In some embodiments, a cancer has a microsatellite instability status of high microsatellite instability (e.g. MSI-H status). In some embodiments, a cancer has a microsatellite instability status of low microsatellite instability (e.g. MSI-L status). In some embodiments, a cancer has a microsatellite instability status of microsatellite stable (e.g. MSS status). In some embodiments microsatellite instability status is assessed by a next generation sequencing (NGS)-based assay, an immunohistochemistry (IHC)-based assay, and/or a PCR-based assay. In some embodiments, microsatellite instability is detected by NGS. In some embodiments, microsatellite instability is detected by IHC. In some embodiments, microsatellite instability is detected by PCR. In some embodiments, the cancer is associated with a high tumor mutation burden (TMB). In some embodiments, the cancer is associated with high TMB and MSI-H. In some embodiments, the cancer is associated with high TMB and MSI-L or MSS. In some embodiments, the cancer is endometrial cancer associated with high TMB. In some related embodiments, the endometrial cancer is associated with high TMB and MSI-H. In some related embodiments, the endometrial cancer is associated with high TMB and MSI-L or MSS.

In some embodiments, a cancer is a mismatch repair deficient (dMMR) cancer. Microsatellite instability may arise from a failure to repair replication-associated errors due to a defective DNA mismatch repair (MMR) system. This failure allows persistence of mismatch mutations all over the genome, but especially in regions of repetitive DNA known as microsatellites, leading to increased mutational load that may improve responses to certain therapeutic agents.

In some embodiments, a cancer is a hypermutated cancer. In some embodiments, a cancer harbors a mutation in polymerase epsilon (POLE). In some embodiments, a cancer harbors a mutation in polymerase delta (POLD).

In some embodiments, a cancer is endometrial cancer (e.g. MSI-H or MSS/MSI-L endometrial cancer). In some embodiments, a cancer is a MSI-H cancer comprising a mutation in POLE or POLD (e.g. a MSI-H non-endometrial cancer comprising a mutation in POLE or POLD).

In some embodiments, the cancer is an advanced cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a recurrent cancer (e.g. a recurrent gynecological cancer such as recurrent epithelial ovarian cancer, recurrent fallopian tube cancer, recurrent primary peritoneal cancer, or recurrent endometrial cancer). In one embodiment, the cancer is recurrent or advanced.

In one embodiment, the cancer is selected from: appendiceal cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer (in particular esophageal squamous cell carcinoma), fallopian tube cancer, gastric cancer, glioma (such as diffuse intrinsic pontine glioma), head and neck cancer (in particular head and neck squamous cell carcinoma and oropharyngeal cancer), leukemia (in particular acute lymphoblastic leukemia, acute myeloid leukemia) lung cancer (in particular non small cell lung cancer), lymphoma (in particular Hodgkin's lymphoma, non-Hodgkin's lymphoma), melanoma, mesothelioma (in particular malignant pleural mesothelioma), Merkel cell carcinoma, neuroblastoma, oral cancer, osteosarcoma, ovarian cancer, prostate cancer, renal cancer, salivary gland tumor, sarcoma (in particular Ewing's sarcoma or rhabdomyosarcoma) squamous cell carcinoma, soft tissue sarcoma, thymoma, thyroid cancer, urothelial cancer, uterine cancer, vaginal cancer, vulvar cancer or Wilms tumor. In a further embodiment, the cancer is selected from: appendiceal cancer, bladder cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, melanoma, mesothelioma, non-small- cell lung cancer, prostate cancer and urothelial cancer. In a further embodiment, the cancer is selected from cervical cancer, endometrial cancer, head and neck cancer (in particular head and neck squamous cell carcinoma and oropharyngeal cancer), lung cancer (in particular non small cell lung cancer), lymphoma (in particular non-Hodgkin's lymphoma), melanoma, oral cancer, thyroid cancer, urothelial cancer or uterine cancer. In another embodiment, the cancer is selected from head and neck cancer (in particular head and neck squamous cell carcinoma and oropharyngeal cancer), lung cancer (in particular non small cell lung cancer), urothelial cancer, melanoma or cervical cancer.

In one embodiment, the cancer is a solid tumor. In one embodiment, the solid tumor is advanced solid tumor. In one embodiment, the cancer is selected from head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN or HNSCC), gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, non-small cell lung carcinoma, prostate cancer, colorectal cancer, ovarian cancer and pancreatic cancer. In one embodiment, the cancer is selected from the group consisting of: colorectal cancer, cervical cancer, bladder cancer, urothelial cancer, head and neck cancer, melanoma, mesothelioma, non-small cell lung carcinoma, prostate cancer, esophageal cancer, and esophageal squamous cell carcinoma. In one aspect the human has one or more of the following: SCCHN, colorectal cancer, esophageal cancer, cervical cancer, bladder cancer, breast cancer, head and neck cancer, ovarian cancer, melanoma, renal cell carcinoma (RCC), esophageal squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma ( e.g . pleural malignant mesothelioma), and prostate cancer. In one embodiment, the cancer is selected from breast cancer, head and neck cancer, kidney cancer, lung cancer and melanoma.

In another embodiment the human has a liquid tumor such as diffuse large B cell lymphoma (DLBCL), multiple myeloma, chronic lymphoblastic leukemia, follicular lymphoma, acute myeloid leukemia and chronic myelogenous leukemia.

In one embodiment, the cancer is head and neck cancer. In one embodiment, the cancer is HNSCC. Squamous cell carcinoma is a cancer that arises from particular cells called squamous cells. Squamous cells are found in the outer layer of skin and in the mucous membranes, which are the moist tissues that line body cavities such as the airways and intestines. Head and neck squamous cell carcinoma (HNSCC) develops in the mucous membranes of the mouth, nose, and throat. HNSCC is also known as SCCHN and squamous cell carcinoma of the head and neck.

HNSCC can occur in the mouth (oral cavity), the middle part of the throat near the mouth (oropharynx), the space behind the nose (nasal cavity and paranasal sinuses), the upper part of the throat near the nasal cavity (nasopharynx), the voicebox (larynx), or the lower part of the throat near the larynx (hypopharynx). Depending on the location, the cancer can cause abnormal patches or open sores (ulcers) in the mouth and throat, unusual bleeding or pain in the mouth, sinus congestion that does not clear, sore throat, earache, pain when swallowing or difficulty swallowing, a hoarse voice, difficulty breathing, or enlarged lymph nodes.

HNSCC can metastasize to other parts of the body, such as the lymph nodes, lungs or liver.

Tobacco use and alcohol consumption are the two most important risk factors for the development of HNSCC, and their contributions to risk are synergistic. In addition, the human papillomavirus (HPV), especially HPV-16, is now a well-established independent risk factor. Patients with HNSCC have a relatively poor prognosis. Recurrent/metastatic (R/M) HNSCC is especially challenging, regardless of human papillomavirus (HPV) status, and currently, few effective treatment options are available in the art. HPV-negative HNSCC is associated with a locoregional relapse rate of 19-35% and a distant metastatic rate of 14-22% following standard of care, compared with rates of

9-18% and 5-12%, respectively, for HPV-positive HNSCC. The median overall survival for patients with R/M disease is 10-13 months in the setting of first-line chemotherapy and 6 months in the second-line setting. The current standard of care is platinum-based doublet chemotherapy with or without cetuximab. Second-line standard of care options include cetuximab, methotrexate, and taxanes. All of these chemotherapeutic agents are associated with significant side effects, and only

10-13% of patients respond to treatment. HNSCC regressions from existing systemic therapies are transient and do not add significantly increased longevity, and virtually all patients succumb to their malignancy.

In one embodiment, the cancer is head and neck cancer. In one embodiment the cancer is head and neck squamous cell carcinoma (HNSCC). In one embodiment, the cancer is recurrent/metastatic (R/M) HNSCC. In one embodiment, the cancer is recurring/refractory (R/R) HNSCC. In one embodiment, the cancer is HPV-negative or HPV-positive HNSCC. In one embodiment, the cancer is a locally advanced HNSCC. In one embodiment, the cancer is (R/M) HNSCC in PD-L1 CPS (Combined Positive Score) positive (CPS >1) patients. The combined positive score is as determined by an FDA-approved test. PD-L1 CPS is the number of PD-L1 staining cells (tumor cells, lymphocytes, macrophages) divided by the total number of viable tumor cells, multiplied by 100. In one embodiment, PD-L1 CPS is determined using PharmDx 22C3 In one embodiment, the cancer is HNSCC in PD-1 binding protein/PD-Ll binding protein experienced or PD-1 binding protein/PD-Ll binding protein na^'ive patients. In one embodiment, the cancer is HNSCC in PD-1 binding protein/PD- Ll binding protein experienced or PD-1 binding protein/PD-Ll binding protein na^'ive patients.

In one embodiment, the head and neck cancer is oropharyngeal cancer. In one embodiment, the head and neck cancer is an oral cancer (/^'.e. a mouth cancer).

In one embodiment, the cancer is lung cancer. In some embodiments, the lung cancer is a squamous cell carcinoma of the lung. In some embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as squamous NSCLC. In some embodiments, the lung cancer is an ALK-translocated lung cancer ( e.g . ALK-translocated NSCLC). In some embodiments, the cancer is NSCLC with an identified ALK translocation. In some embodiments, the lung cancer is an EGFR-mutant lung cancer (e.g. EGFR- mutant NSCLC). In some embodiments, the cancer is NSCLC with an identified EGFR mutation.

In one embodiment, the cancer is melanoma. In some embodiments, the melanoma is an advanced melanoma. In some embodiments, the melanoma is a metastatic melanoma. In some embodiments, the melanoma is a MSI-H melanoma. In some embodiments, the melanoma is a MSS melanoma. In some embodiments, the melanoma is a POLE-mutant melanoma. In some embodiments, the melanoma is a POLD-mutant melanoma. In some embodiments, the melanoma is a high TMB melanoma.

In one embodiment, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is an advanced colorectal cancer. In some embodiments, the colorectal cancer is a metastatic colorectal cancer. In some embodiments, the colorectal cancer is a MSI-H colorectal cancer. In some embodiments, the colorectal cancer is a MSS colorectal cancer. In some embodiments, the colorectal cancer is a POLE-mutant colorectal cancer. In some embodiments, the colorectal cancer is a POLD- mutant colorectal cancer. In some embodiments, the colorectal cancer is a high TMB colorectal cancer.

In some embodiments, the cancer is a gynecologic cancer (/.e. a cancer of the female reproductive system such as ovarian cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar cancer, uterine cancer, or primary peritoneal cancer, or breast cancer). In some embodiments, cancers of the female reproductive system include, but are not limited to, ovarian cancer, cancer of the fallopian tube(s), peritoneal cancer, and breast cancer.

In some embodiments, the cancer is ovarian cancer ( e.g . serous or clear cell ovarian cancer). In some embodiments, the cancer is fallopian tube cancer (e.g. serous or clear cell fallopian tube cancer). In some embodiments, the cancer is primary peritoneal cancer (e.g. serous or clear cell primary peritoneal cancer).

In some embodiments, the ovarian cancer is an epithelial carcinoma. Epithelial carcinomas make up 85% to 90% of ovarian cancers. While historically considered to start on the surface of the ovary, new evidence suggests at least some ovarian cancer begins in special cells in a part of the fallopian tube. The fallopian tubes are small ducts that link a woman's ovaries to her uterus that are a part of a woman's reproductive system. In a normal female reproductive system, there are two fallopian tubes, one located on each side of the uterus. Cancer cells that begin in the fallopian tube may go to the surface of the ovary early on. The term "ovarian cancer" is often used to describe epithelial cancers that begin in the ovary, in the fallopian tube, and from the lining of the abdominal cavity, call the peritoneum. In some embodiments, the cancer is or comprises a germ cell tumor. Germ cell tumors are a type of ovarian cancer develops in the egg- producing cells of the ovaries. In some embodiments, a cancer is or comprises a stromal tumor. Stromal tumors develop in the connective tissue cells that hold the ovaries together, which sometimes is the tissue that makes female hormones called estrogen. In some embodiments, the cancer is or comprises a granulosa cell tumor. Granulosa cell tumors may secrete estrogen resulting in unusual vaginal bleeding at the time of diagnosis. In some embodiments, a gynecologic cancer is associated with homologous recombination repair deficiency/homologous repair deficiency (HRD) and/or BRCAl/2 mutation(s). In some embodiments, a gynecologic cancer is platinum-sensitive. In some embodiments, a gynecologic cancer has responded to a platinum-based therapy. In some embodiments, a gynecologic cancer has developed resistance to a platinum-based therapy. In some embodiments, a gynecologic cancer has at one time shown a partial or complete response to platinum-based therapy ( e.g . a partial or complete response to the last platinum-based therapy or to the penultimate platinum-based therapy). In some embodiments, a gynecologic cancer is now resistant to platinum-based therapy.

In some embodiments, the cancer is breast cancer. Usually breast cancer either begins in the cells of the milk producing glands, known as the lobules, or in the ducts. Less commonly breast cancer can begin in the stromal tissues. These include the fatty and fibrous connective tissues of the breast. Over time the breast cancer cells can invade nearby tissues such the underarm lymph nodes or the lungs in a process known as metastasis. The stage of a breast cancer, the size of the tumor and its rate of growth are all factors which determine the type of treatment that is offered. Treatment options include surgery to remove the tumor, drug treatment which includes chemotherapy and hormonal therapy, radiation therapy and immunotherapy. The prognosis and survival rate varies widely; the five year relative survival rates vary from 98% to 23% depending on the type of breast cancer that occurs. Breast cancer is the second most common cancer in the world with approximately 1.7 million new cases in 2012 and the fifth most common cause of death from cancer, with approximately 521,000 deaths. Of these cases, approximately 15% are triple-negative, which do not express the estrogen receptor, progesterone receptor (PR) or HER2. In some embodiments, triple negative breast cancer (TNBC) is characterized as breast cancer cells that are estrogen receptor expression negative (<1% of cells), progesterone receptor expression negative (< 1% of cells), and HER2-negative.

In some embodiments, the cancer is estrogen receptor(ER)-positive breast cancer, ER- negative breast cancer, PR-positive breast cancer, PR-negative breast cancer, HER2-positive breast cancer, HER2-negative breast cancer, BRCAl/2-positive breast cancer, BRCAl/2-negative cancer, or TNBC. In some embodiments, the breast cancer is a metastatic breast cancer. In some embodiments, the breast cancer is an advanced breast cancer. In some embodiments, the cancer is a stage II, stage III or stage IV breast cancer. In some embodiments, the cancer is a stage IV breast cancer. In some embodiments, the breast cancer is a triple negative breast cancer.

In one embodiment, the cancer is endometrial cancer. Endometrial carcinoma is the most common cancer of the female genital, tract accounting for 10-20 per 100,000 person-years. The annual number of new cases of endometrial cancer (EC) is estimated at about 325 thousand worldwide. Further, EC is the most commonly occurring cancer in post-menopausal women. About 53% of endometrial cancer cases occur in developed countries. In 2015, approximately 55,000 cases of EC were diagnosed in the U.S. and no targeted therapies are currently approved for use in EC. There is a need for agents and regimens that improve survival for advanced and recurrent EC in 1L and 2L settings. Approximately 10,170 people are predicted to die from EC in the U.S. in 2016. The most common histologic form is endometrioid adenocarcinoma, representing about 75-80% of diagnosed cases. Other histologic forms include uterine papillary serous (less than 10%), clear cell 4%, mucinous 1%, squamous less than 1% and mixed about 10%. From the pathogenetic point of view, EC falls into two different types, so-called types I and II. Type I tumors are low-grade and estrogen-related endometrioid carcinomas (EEC) while type II are non-endometrioid (NEEC) (mainly serous and clear cell) carcinomas. The World Health Organization has updated the pathologic classification of EC, recognizing nine different subtypes of EC, but EEC and serous carcinoma (SC) account for the vast majority of cases. EECs are estrogen- related carcinomas, which occur in perimenopausal patients, and are preceded by precursor lesions (endometrial hyperplasia/endometrioid intraepithelial neoplasia). Microscopically, lowgrade EEC (EEC 1-2) contains tubular glands, somewhat resembling the proliferative endometrium, with architectural complexity with fusion of the glands and cribriform pattern. High-grade EEC shows solid pattern of growth. In contrast, SC occurs in postmenopausal patients in absence of hyperestrogen ism. At the microscope, SC shows thick, fibrotic or edematous papillae with prominent stratification of tumor cells, cellular budding, and anaplastic cells with large, eosinophilic cytoplasms. The vast majority of EEC are low grade tumors (grades 1 and 2), and are associated with good prognosis when they are restricted to the uterus. Grade 3 EEC (EEC3) is an aggressive tumor, with increased frequency of lymph node metastasis. SCs are very aggressive, unrelated to estrogen stimulation, mainly occurring in older women. EEC 3 and SC are considered high-grade tumors. SC and EEC3 have been compared using the surveillance, epidemiology and End Results (SEER) program data from 1988 to 2001. They represented 10% and 15% of EC respectively, but accounted for 39% and 27% of cancer death respectively. Endometrial cancers can also be classified into four molecular subgroups: (1) ultramutated/POLE-mutant; (2) hypermutated MSI+ (e.g., MSI-H or MSI-L); (3) copy number low/micro satellite stable (MSS); and (4) copy number high/serous -like. Approximately 28% of cases are MSI-high. (Murali, Lancet Oncol. (2014). In some embodiments, the patient has a mismatch repair deficient subset of 2L endometrial cancer. In some embodiments, the endometrial cancer is metastatic endometrial cancer. In some embodiments, the patient has a MSS endometrial cancer. In some embodiments, the patient has a MSI-H endometrial cancer.

In one embodiment, the cancer is cervical cancer. In some embodiments, the cervical cancer is an advanced cervical cancer. In some embodiments, the cervical cancer is a metastatic cervical cancer. In some embodiments, the cervical cancer is a MSI-H cervical cancer. In some embodiments, the cervical cancer is a MSS cervical cancer. In some embodiments, the cervical cancer is a POLE- mutant cervical cancer. In some embodiments, the cervical cancer is a POLD-mutant cervical cancer. In some embodiments, the cervical cancer is a high TMB cervical cancer.

In one embodiment, the cancer is uterine cancer. In some embodiments, the uterine cancer is an advanced uterine cancer. In some embodiments, the uterine cancer is a metastatic uterine cancer. In some embodiments, the uterine cancer is a MSI-H uterine cancer. In some embodiments, the uterine cancer is a MSS uterine cancer. In some embodiments, the uterine cancer is a POLE- mutant uterine cancer. In some embodiments, the uterine cancer is a POLD-mutant uterine cancer. In some embodiments, the uterine cancer is a high TMB uterine cancer. In one embodiment, the cancer is urothelial cancer. In some embodiments, the urothelial cancer is an advanced urothelial cancer. In some embodiments, the urothelial cancer is a metastatic urothelial cancer. In some embodiments, the urothelial cancer is a MSI-H urothelial cancer. In some embodiments, the urothelial cancer is a MSS urothelial cancer. In some embodiments, the urothelial cancer is a POLE-mutant urothelial cancer. In some embodiments, the urothelial cancer is a POLD- mutant urothelial cancer. In some embodiments, the urothelial cancer is a high TMB urothelial cancer.

In one embodiment, the cancer is thyroid cancer. In some embodiments, the thyroid cancer is an advanced thyroid cancer. In some embodiments, the thyroid cancer is a metastatic thyroid cancer. In some embodiments, the thyroid cancer is a MSI-H thyroid cancer. In some embodiments, the thyroid cancer is a MSS thyroid cancer. In some embodiments, the thyroid cancer is a POLE- mutant thyroid cancer. In some embodiments, the thyroid cancer is a POLD-mutant thyroid cancer. In some embodiments, the thyroid cancer is a high TMB thyroid cancer.

Tumors may be a hematopoietic (or hematologic or hematological or blood-related) cancer, for example, cancers derived from blood cells or immune cells, which may be referred to as "liquid tumors". Specific examples of clinical conditions based on hematologic tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS) and Waldenstrom's macroglobulinemia; lymphomas such as non-Hodgkin's lymphoma, Hodgkin's lymphoma, and the like.

The cancer may be any cancer in which an abnormal number of blast cells or unwanted cell proliferation is present or that is diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia. These leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia. Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid or myelocytic) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polcythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.

In one embodiment, the cancer is non-Hodgkin's lymphoma. Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes, spleens, bone marrow, peripheral blood, and/or extranodal sites. Lymphoid cancers include B-cell malignancies, which include, but are not limited to, B-cell non-Hodgkin's lymphomas (B-NHLs). B-NHLs may be indolent (or low-grade), intermediate-grade (or aggressive) or high-grade (very aggressive). Indolent B cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large B cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS related) lymphomas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castleman's disease. NHL may also include T-cell non-Hodgkin's lymphomas (T-NHLs), which include, but are not limited to T-cell non-Hodgkin's lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma/delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides, and Sezary syndrome.

Hematopoietic cancers also include Hodgkin's lymphoma (or disease) including classical Hodgkin's lymphoma, nodular sclerosing Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma, lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocyte depleted Hodgkin's lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM) including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's Macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. Tissues which include hematopoietic cells referred herein to as "hematopoietic cell tissues" include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, lymphoid tissues associated with mucosa (such as the gut-associated lymphoid tissues), tonsils, Peyer's patches and appendix, and lymphoid tissues associated with other mucosa, for example, the bronchial linings.

In one embodiment, the treatment is first-line or second line treatment of HNSCC. In one embodiment, the treatment is first-line or second line treatment of recurrent/metastatic HNSCC. In one embodiment the treatment is first line treatment of recurrent/metastatic (1L R/M) HNSCC. In one embodiment, the treatment is first line treatment of 1L R/M HNSCC in a PD-L1 CPS (combined positive score) positive (CPS >1) patients. In one embodiment the treatment is second line treatment of recurrent/metastatic (2L R/M) HNSCC.

In one embodiment, the treatment is first-line, second-line, third-line, fourth-line or fifth-line treatment of PD-l/PD-Ll-naive HNSCC. In one embodiment, the treatment first-line, second-line, third-line, fourth-line or fifth-line treatment of PD-1/PD-L1 experienced HNSCC.

In some embodiments, the treatment of cancer is first-line treatment of cancer. In one embodiment, the treatment of cancer is second-line treatment of cancer. In some embodiments, the treatment is third-line treatment of cancer. In some embodiments, the treatment is fourth-line treatment of cancer. In some embodiments, the treatment is fifth-line treatment of cancer. In some embodiments, prior treatment to said second-line, third-line, fourth-line or fifth-line treatment of cancer comprises one or more of radiotherapy, chemotherapy, surgery or radiochemotherapy.

In one embodiment, the prior treatment comprises treatment with diterpenoids, such as paclitaxel, nab-paclitaxel or docetaxel; vinca alkaloids, such as vinblastine, vincristine, or vinorelbine; platinum coordination complexes, such as cisplatin or carboplatin; nitrogen mustards such as cyclophosphamide, melphalan, or chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; triazenes such as dacarbazine; actinomycins such as dactinomycin; anthrocyclins such as daunorubicin or doxorubicin; bleomycins; epipodophyllotoxins such as etoposide or teniposide; anti metabolite anti-neoplastic agents such as fluorouracil, methotrexate, cytarabine, mecaptopurine, thioguanine, or gemcitabine; methotrexate; camptothecins such as irinotecan or topotecan; rituximab; ofatumumab; trastuzumab; cetuximab; bexarotene; sorafenib; erbB inhibitors such as lapatinib, erlotinib or gefitinib; pertuzumab; ipilimumab; nivolumab; FOLFOX; capecitabine; FOLFIRI; bevacizumab; atezolizumab; selicrelumab; obinotuzumab or any combinations thereof. In one embodiment, prior treatment to said second line treatment, third-line, fourth-line or fifth-line treatment of cancer comprises ipilimumab and nivolumab. In one embodiment, prior treatment to said second line treatment, third-line, fourth-line or fifth-line treatment of cancer comprises FOLFOX, capecitabine, FOLFIRI/bevacizumab and atezolizumab/selicrelumab. In one embodiment, prior treatment to said second line treatment, third-line, fourth-line or fifth-line treatment of cancer comprises carboplatin/Nab-paclitaxel. In one embodiment, prior treatment to said second line treatment, third-line, fourth-line or fifth-line treatment of cancer comprises nivolumab and electrochemotherapy. In one embodiment, prior treatment to said second line treatment, third-line, fourth-line or fifth-line treatment of cancer comprises radiotherapy, cisplatin and carboplatin/paclitaxel.

In one embodiment, the treatment is first-line or second line treatment of head and neck cancer (in particular head and neck squamous cell carcinoma and oropharyngeal cancer). In one embodiment, the treatment is first-line or second line treatment of recurrent/metastatic HNSCC. In one embodiment the treatment is first line treatment of recurrent/metastatic (1L R/M) HNSCC. In one embodiment, the treatment is first line treatment of 1L R/M HNSCC in a PD-L1 CPS (combined positive score) positive (CPS >1) patients. In one embodiment the treatment is second line treatment of recurrent/metastatic (2L R/M) HNSCC.

In some embodiments, the treatment results in one or more of increased tumor infiltrating lymphocytes including cytotoxic T cells, helper T cell and NK cells, increased T cells, increased granzyme B+ cells, reduced proliferating tumor cells and increased activated T cells as compared to levels prior to treatment ( e.g . baseline level). Activated T cells may be observed by greater 0X40 and human leukocyte antigen DR expression. In some embodiments, treatment results in upregulation of PD-1 and/or PD-L1 as compared to levels prior to treatment (e.g. baseline level).

The present disclosure also relates to a method for treating or lessening the severity of a cancer selected from: brain (gliomas), glioblastomas, 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, 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, erythroleukemia, 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.

By the term "treating" and grammatical variations thereof as used herein, is meant therapeutic therapy. In reference to a particular condition, treating means: (1) to ameliorate the condition of 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 treatment thereof, or (4) to slow the progression of the condition or one or more of the biological manifestations of the condition. Prophylactic therapy is also contemplated herein. The skilled artisan will appreciate that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug 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. 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.

An "effective amount" means that amount of a drug or pharmaceutical 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 which, 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.

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 that makes them pathological to the host organism. Primary cancer cells can be readily distinguished from non-cancerous cells by well-established techniques, particularly 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 computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation on physical examination, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. Tumors may be a hematopoietic (or hematologic or hematological or blood-related) cancer, for example, cancers derived from blood cells or immune cells, which may be referred to as "liquid tumors." Specific examples of clinical conditions based on hematologic tumors include leukemias such as chronic myelocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia; plasma cell malignancies such as multiple myeloma, MGUS and Waldenstrom's macroglobulinemia; lymphomas such as non-Hodgkin's lymphoma, Hodgkin's lymphoma; and the like.

The cancer may be any cancer in which an abnormal number of blast cells or unwanted cell proliferation is present or that is diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia (undifferentiated or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia. These leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polcythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.

In one embodiment, the methods of the present invention further comprise administering at least one neo-plastic agent or cancer adjuvant to said human. The methods of the present invention may also be employed with other therapeutic methods of cancer treatment.

Typically, any anti-neoplastic agent or cancer adjuvant that has activity versus a tumor, such as a susceptible tumor being treated may be co-administered in the treatment of cancer in the present invention. Examples of such agents can be found in Cancer Principles and Practice of Oncology by V.T. Devita, T.S. Lawrence, and S.A. Rosenberg (editors), 10^th edition (December 5, 2014), Lippincott Williams & Wilkins Publishers.

A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved. Typical anti-neoplastic agents useful in the present invention include, but are not limited to, anti-microtubule or anti-mitotic 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 actinomycins, anthracyclins, and bleomycins; topoisomerase I inhibitors such as camptothecins; topoisomerase II inhibitors such as epipodophyllotoxins; anti metabolites such as purine and pyrimidine analogues and anti-folate compounds; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; cell cycle signalling inhibitors; proteasome inhibitors; heat shock protein inhibitors; inhibitors of cancer metabolism; and cancer gene therapy agents such as genetically modified T cells.

Examples of a further active ingredient or ingredients for use in combination or co administered with the present methods or combinations are anti-neoplastic agents. Examples of anti neoplastic agents include, but are not limited to, chemotherapeutic agents; immuno-modulatory agents; immuno-modulators; and immunostimulatory adjuvants.

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, 5 ,20-epoxy-l,2a,4,7 ,10 ,13a-hexa-hydroxytax-ll-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. It was first isolated in 1971 by Wani M.C., et al., J. Am. Chem. Soc., 93(9): 2325-2327 (1971), who characterized its structure by chemical and X-ray crystallographic methods. One mechanism for its activity relates to paclitaxel's capacity to bind tubulin, thereby inhibiting cancer cell growth (Schiff P.B. and Horwitz S.B., Proc. Natl. Acad. Sci. USA, 77: 1561-1565 (1980); Schiff P.B., et al., Nature, 277: 665-667 (1979); Kumar N., J. Biol. Chem., 256: 10435-10441 (1981)). For a review of synthesis and anticancer activity of some paclitaxel derivatives see: D. G. I. Kingston et a/., Studies in Organic Chemistry vol. 26, entitled "New Trends in Natural Products Chemistry 1986", Atta-ur-Rahman, P.W. Le Quesne, Eds. (Elsevier, Amsterdam, 1986) pp 219-235.

Paclitaxel has been approved for clinical use for the treatment of refractory ovarian cancer in the United States (Markman M., Yale J. Biol. Med., 64(6): 583-590 (1991); McGuire W.P., et al., Ann. Intern. Med., 111(4): 273-279 (1989)) and for the treatment of breast cancer (Holmes F.A., et al., J. Natl. Cancer Inst., 83(24): 1797-1805 (1991)). It is a potential candidate for treatment of neoplasms in the skin (Einzig A. I., et. al., Cancer Treat. Res., 58: 89-100 (1991)) and head and neck carcinomas (Forastiere A.A., Semin. Oncol., 20(4 Suppl. 3): 56-60 (1993). The compound also shows potential for the treatment of polycystic kidney disease (Woo D.D., et. al., Nature, 368(6473): 750-753 (1994)), lung cancer and malaria. Treatment of patients with paclitaxel results in bone marrow suppression (Ignoffo R.J. et. al, Cancer Chemotherapy Pocket Guide, (1998)) related to the duration of dosing above a threshold concentration (50nM) (Kearns, C.M., et. al., Semin. Oncol., 22(3 Suppl. 6): 16-23 (1995)).

Docetaxel, (2R,3S)- N-carboxy-3-phenylisoserine,N-te/t-butyl ester, 13-ester with 5b-20- epoxy-l,2a,4,7 ,10 ,13oc-hexahydroxytax-ll-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, prepared using a natural precursor, 10-deacetyl-baccatin III, extracted from the needle of the European Yew tree. The main dose limiting toxicity of docetaxel treatment is neutropenia.

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 indications as a second line therapy of various solid tumors, it is primarily indicated for 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 effects of vincristine and to a lesser extent myelosupression and gastrointestinal mucositis effects occur.

Vinorelbine, 3',4'-didehydro -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 semisynthetic vinca alkaloid. Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, for the treatment of various solid tumors, particularly 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 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 crosslinks with DNA causing adverse biological effects to the tumor. Examples of platinum coordination complexes include, but are not limited to, cisplatin and carboplatin.

Cisplatin, cis-diamminedichloroplatinum, is commercially available as PLATINOL® as an injectable solution. Cisplatin is primarily indicated for the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer. The primary dose limiting side effects of cisplatin are nephrotoxicity, which may be controlled by hydration and diuresis, and ototoxicity.

Carboplatin, platinum, diammine [l,l-cyclobutane-dicarboxylate(2-)-0,0'], is commercially available as PARAPLATIN® as an injectable solution. Carboplatin is primarily indicated in the first and second line treatment of advanced ovarian carcinoma. Bone marrow suppression is the dose limiting toxicity of carboplatin.

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, for the treatment of malignant lymphomas, multiple myeloma, and leukemias. Alopecia, nausea, vomiting and leukopenia are the most common dose limiting side effects of cyclophosphamide.

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

Busulfan, 1,4-butanediol dimethanesulfonate, is commercially available as MYLERAN® TABLETS. Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia. Bone marrow suppression is the most common dose limiting side effects of busulfan.

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. Delayed myelosuppression is the most common dose limiting side effects of carmustine.

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. Nausea, vomiting, and anorexia are the most common dose limiting side effects of dacarbazine.

Antibiotic anti-neoplastics are non-phase specific agents, which bind or intercalate with DNA. This action disrupts the 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. Nausea, vomiting, and anorexia are the most common dose limiting side effects of dactinomycin.

Daunorubicin, (8S-cis-)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]- 7,8,9,10-tetrahydro-6,8,ll-trihydroxy-l-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 for the treatment of acute nonlymphocytic leukemia and advanced HIV associated Kaposi's sarcoma. Myelosuppression is the most common dose limiting side effect of daunorubicin.

Doxorubicin, (8S, 10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl, 7,8,9,10-tetrahydro-6,8,ll-trihydroxy-l-methoxy-5,12 naphthacenedione hydrochloride, is commercially available as an injectable form as RUBEX® or ADRIAMYCIN RDF®. Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblastic leukemia, but is also a useful component for the treatment of some solid tumors and lymphomas. Myelosuppression is the most common dose limiting side effect of doxorubicin.

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. Pulmonary and cutaneous toxicities are the most common dose limiting side effects of bleomycin.

Topoisomerase I inhibitors include, but are not limited to, camptothecins. The cytotoxic activity of camptothecins 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)-10,ll-ethylenedioxy-20-camptothecin.

Irinotecan, (4S)-4,ll-diethyl-4-hydroxy-9-[(4-piperidinopiperidino) carbonyloxy]-lH- pyrano[3',4',6,7]indolizino[l,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 : irinotecan or SN-38 ternary complex with replication enzymes. Irinotecan is indicated for treatment of metastatic cancer of the colon or rectum. The dose limiting side effects of irinotecan are myelosuppression, including neutropenia, and GI effects, including diarrhea.

T opoteca n, (S)-10-[(di methyla mi nojmethyl] -4-ethyl-4,9-d i hyd roxy- 1 H- pyrano[3',4',6,7]indolizino[l,2-b]quinoline-3,14-(4H,12H)-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 religation 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. The dose limiting side effect of topotecan is myelosuppression, primarily neutropenia.

Also of interest, is the camptothecin derivative of formula A' following, currently under development, including the racemic mixture (R,S) form as well as the R and S enantiomers:

known by the chemical name "7-(4-methylpiperazino-methylene)-10,ll-ethylenedioxy- 20(R,S)-camptothecin (racemic mixture) or "7-(4-methylpiperazino-methylene)-10,ll-ethylenedioxy- 20(R)-camptothecin (R enantiomer) or "7-(4-methylpiperazino-methylene)-10,ll-ethylenedioxy- 20(S)-camptothecin (S enantiomer). Such compound, as well as related compounds, is described, including methods of making, in U.S. Patent Nos. 6,100,273, 6,063,923; 5,342,947; 5,559,235; and 5,491,237.

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- -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 for the treatment of testicular and non-small cell lung cancers. Myelosuppression is the most common side effect of etoposide. The incidence of leucopenia tends to be more severe than thrombocytopenia.

Teniposide, 4'-demethyl-epipodophyllotoxin 9[4,6-0-(R )-thenylidene- -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 for the treatment of acute leukemia in children. Myelosuppression is the most common dose limiting side effect of teniposide. Teniposide can induce both leucopenia and thrombocytopenia.

Anti metabolite 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, mercaptopurine, thioguanine, and gemcitabine.

5-fluorouracil, 5-fluoro-2,4- (1H,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 for the treatment of carcinomas of the breast, colon, rectum, stomach and pancreas. Myelosuppression and mucositis are dose limiting side effects of 5-fluorouracil. Other fluoropyrimidine analogs include 5-fluoro deoxyuridine (floxuridine) and 5- fluorodeoxyuridine monophosphate.

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 dihydrofolic 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 for the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphoma, and carcinomas of the breast, head, neck, ovary and bladder. Myelosuppression (leucopenia, thrombocytopenia, and anemia) and mucositis are expected side effects of methotrexate administration.

Cytarabine, 4-amino-l- -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 for the treatment of acute leukemia. Other cytidine analogs include 5- azacytidine and 2',2'-difluorodeoxycytidine (gemcitabine). Cytarabine induces leucopenia, thrombocytopenia, and mucositis.

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 for the treatment of acute leukemia. Myelosuppression and gastrointestinal mucositis are expected side effects of mercaptopurine at high doses. 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 for the treatment of acute leukemia. Myelosuppression, including leucopenia, thrombocytopenia, and anemia, is the most common dose limiting side effect of thioguanine administration. However, gastrointestinal side effects occur and can be dose limiting. 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 for the treatment of locally advanced non-small cell lung cancer and alone for the treatment of locally advanced pancreatic cancer. Myelosuppression, including leucopenia, thrombocytopenia, and anemia, is the most common dose limiting side effect of gemcitabine administration.

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 for the treatment of malignant lymphoma and acute leukemia in children; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrazole, vorazole, and exemestane, which are useful for the treatment of adrenocortical carcinoma and hormone dependent breast carcinoma containing estrogen receptors; progestrins such as megestrol acetate, which are useful for 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, which are useful for 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 U.S. Patent Nos. 5,681,835, 5,877,219, and 6,207,716, which are useful for 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 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/SH3domain blockers, serine/threonine kinases, phosphatidyl inositol-3 kinases, myo-inositol signalling, and Ras oncogenes.

Several protein tyrosine kinases catalyze 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, vascular endothelial growth factor receptor (VEGFR), tyrosine kinase with immunoglobulin-like and epidermal growth factor homology 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 J.C., Exp. Opin. Ther. Patents, 10(6):803-818 (2000); Shawver L.K., et al., Drug Discov. Today, 2(2): 50-63 (1997); and Lofts, F. J. and Gullick W.J., "Growth factor receptors as targets." in New Molecular Targets for Cancer Chemotherapy, Kerr D.J. and Workman P. (editors), (June 27, 1994), CRC Press. Non-limiting examples of growth factor receptor inhibitors include pazopanib and sorafenib.

Pazopanib, 5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2- methylbenzenesulfonamide, is a VEGFR inhibitor and is commercially available as VOTRIENT® tablets. Pazopanib was disclosed and claimed in International Application No. PCT/US01/49367, having an International filing date of December 19, 2001, International Publication Number W002/059110 and an International Publication date of August 1, 2002, the entire disclosure of which is hereby incorporated by reference. Pazopanib is indicated for the treatment of advanced renal cell carcinoma and advanced soft tissue sarcoma. Grade 3 fatigue and hypertension are the most common dose limiting side effects of pazopanib.

Sorafenib, 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino] phenoxy]-N-methyl- pyridine-2-carboxamide, is a multikinase inhibitor, and is commercially available as NEXAVAR® tablets. Sorafenib is indicated for the treatment of renal cell carcinoma, hepatocellular carcinoma, and certain differentiated thyroid carcinomas.

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 Sinha S. and Corey S.J., J. Hematother. Stem Cell Res., 8(5): 465-480 (2004) and Bolen, J.B., Brugge, J.S., Annu. Rev. Immunol., 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., J. Pharmacol. Toxicol. Methods, 34(3): 125-32 (1995).

Inhibitors of serine/threonine kinases include, but are not limited to, MAP kinase cascade blockers which include blockers of Raf kinases (rafk), Mitogen or Extracellular Regulated Kinase (MEKs), and Extracellular Regulated Kinases (ERKs); Protein kinase C family member blockers including blockers of PKCs (alpha, beta, gamma, epsilon, mu, lambda, iota, zeta); IkB kinases (IKKa, IKKb); PKB family kinases; AKT kinase family members; TGF beta receptor kinases;and mammaliam target of rapamycin (mTOR) inhibitors, including, but not limited to rapamycin (FK506) and rapalogs, RAD001 or everolimus (Afinitor), CCI-779 or temsirolimus, AP23573, AZD8055, WYE-354, WYE-600, WYE-687 and Ppl21. Examples of inhibitors of serine/threonine kinases include, but are not limited to, trametinib, dabrafenib, and Akt inhibitors afuresertib and N-{(lS)-2-amino-l-[(3,4- difluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-l-methyl-lH-pyrazol-5-yl)-2-furancarboxamide.

Trametinib, N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodo-phenylamino)-6,8-dimethyl-2,4,7-trioxo- 3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-l-yl]phenyl}acetamide, is a MEK inhibitor and is commercially available as MEKINIST® tablets. Trametinib was disclosed and claimed in International Application No. PCT/JP2005/011082, having an International filing date of June 10, 2005; International Publication Number WO 2005/121142 and an International Publication date of December 22, 2005, the entire disclosure of which is hereby incorporated by reference. Trametinib is indicated for the treatment of some unresectable or metastatic melanomas.

Dabrafenib, N-{3-[5-(2-Amino-4-pyrimidinyl)-2-(l,l-dimethylethyl)-l,3-thiazol-4-yl]-2- fluorophenyl}-2,6-difluorobenzenesulfonamide, is a B-Raf inhibitor and is commercially available as TAFINLAR® capsules. Dabrafenib was disclosed and claimed, in International Application No. PCT/US2009/042682, having an International filing date of May 4, 2009, the entire disclosure of which is hereby incorporated by reference. Dabrafenib is indicated for the treatment of some unresectable or metastatic melanomas.

Afuresertib, N-{(lS)-2-amino-l-[(3-fluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-l- methyl-lH-pyrazol-5-yl)-2-thiophenecarboxamide or a pharmaceutically acceptable salt thereof, is an Akt inhibitor, and was disclosed and claimed in International Application No. PCT/US2008/053269, having an International filing date of February 7, 2008; International Publication Number WO 2008/098104 and an International Publication date of August 14, 2008, the entire disclosure of which is hereby incorporated by reference. Afuresertib can be prepared as described in International Application No. PCT/US2008/053269.

N-{(lS)-2-amino-l-[(3,4-difluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-l-methyl-lH- pyrazol-5-yl)-2-furancarboxamide or a pharmaceutically acceptable salt thereof, is an Akt inhibitor, and was disclosed and claimed in International Application No. PCT/US2008/053269, having an International filing date of February 7, 2008; International Publication Number WO 2008/098104 and an International Publication date of August 14, 2008, the entire disclosure of which is hereby incorporated by reference. N-{(lS)-2-amino-l-[(3,4-difluorophenyl)methyl]ethyl}-5-chloro-4-(4- chloro-l-methyl-lH-pyrazol-5-yl)-2-furancarboxamide can be prepared as described in International Application No. PCT/US2008/053269.

Inhibitors of phosphatidyl 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., Curr. Opin. Immunol., 8(3): 412-418 (1996); Canman C.E., and Lim D.S., Oncogene, 17(25): 3301-3308 (1998); Jackson S.P., Int. J. Biochem. Cell Biol., 29(7): 935-938 (1997); and Zhong H., et al., Cancer Res., 60(6): 1541-1545 (2000).

Also useful in the present invention are myo-inositol signalling inhibitors such as phospholipase C blockers and myo-inositol analogs. Such signal inhibitors are described in Powis G., and Kozikowski A., "Inhibitors of Myo-inositol Signaling." in New Molecular Targets for Cancer Chemotherapy, Kerr D.J. and Workman P. (editors), (June 27, 1994), CRC Press.

Another group of signal transduction pathway inhibitors 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 other immunotherapies. 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 O.G., et al., J. Biomed. Sci., 7(4): 292-298 (2000); Ashby M.N., Curr. Opin. Lipidol., 9(2): 99-102 (1998); and Bennett C.F. and Cowsert L.M., Biochim. Biophys. Acta., 1489(1): 19-30 (1999).

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 or other antagonists to the extracellular ligand binding domain of receptor tyrosine kinases. Examples of antibody or other antagonists to receptor kinase ligand binding include, but are not limited to, cetuximab (ERBITUX®); trastuzumab (HERCEPTIN®); trastuzumab emtansine (KADCYLA®); pertuzumab (PERJETA®); ErbB inhibitors including lapatinib, erlotinib, and gefitinib; and 2C3 VEGFR2 specific antibody (see Brekken R.A., et al., Cancer Res., 60(18): 5117-5124 (2000)).

Cetuximab is a chimeric mouse human antibody which is commercially available as ERBITUX®. Cetuximab inhibits epidermal growth factor receptor (EGFR). Ceteximab in combination with radiation therapy is indicated for the treatment of squamous cell carcinoma of the head and neck, and is also indicated for the treatment of some colorectal cancers.

Trastuzumab is a humanized monoclonal antibody which is commercially available as HERCEPTIN®. Trastuzumab binds to the HER2 (also known as ErbB2) receptor. The original indication for trastuzumab is HER2 positive breast cancer.

Trastuzumab emtansine is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab (Herceptin®) linked to the cytotoxic agent emtansine (DM1), and is commercially available as an injectable solution KADCYLA®. Trastuzumab emtansine is indicated for the treatment of some HER2-positive metastatic brease breast cancers.

Pertuzumab is a monoclonal antibody which is commercially available as PERJETA®. Pertuzumab is a HER dimerization inhibitor, binding to HER2 to inhibit it from dimerizing with other HER receptors, which is hypothesized to result in slowed tumor growth. Pertuzumab is indicated in combination with trastuzumab (Herceptin®) and docetaxel (TAXOTERE®) for the treatment of some HER2-positive metastatic breat cancers.

Lapatinib, /V-(3-chloro-4-{[(3-fluorophenyl)methyl]oxy}phenyl)-6-[5-({[2-

(methylsulfonyl)ethyl]amino}methyl)-2-furanyl]-4-quinazolinamine is a dual inhibitor of ErbB-1 and ErbB-2 (EGFR and HER2) tyrosine kinases, and is commercially available as TYKERB® tablets. Lapatinib is indicated in combination with capecitabine (XELODA®) for the treatment of HER2-positive metastatic breast cancer.

Erlotinib, /V-(3-ethynylphenyl)-6,7-bis{[2-(methyloxy)ethyl]oxy}-4-quinazolinamine, is an ErbB inhibitor, and is commercially available as TARCEVA® tablets. Erlotinib is indicated for the treatment of some locally advanced or metastatic non-small cell lung cancers, and for the treatment of some locally advanced, unresectable or metastatic pancreatic cancers, in combination with gemcitabine.

Gefitinib, N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4- amine, is an ErbB-1 inhibitor, and is commercially available as IRESSA® tablets. Gefitinib is indicated as monotherapy for the treatment of patients with locally advanced or metastatic non-small-cell lung cancer after failure of both platinum-based and docetaxel chemotherapies.

Non-receptor kinase angiogenesis inhibitors may also find use in the present invention. Inhibitors of angiogenesis related VEGFR and TIE2 are discussed above in regard to signal transduction inhibitors (both receptors are receptor tyrosine kinases). Angiogenesis in general is linked to erbB2/EGFR signaling since inhibitors of erbB2 and EGFR have been shown to inhibit angiogenesis, primarily VEGF expression. Accordingly, non-receptor tyrosine kinase inhibitors may be used in combination with the EGFR/erbB2 inhibitors of the present invention. For example, anti-VEGF antibodies, which do not recognize VEGFR (the receptor tyrosine kinase), but bind to the ligand; small molecule inhibitors of integrin (alphav betas) that will inhibit angiogenesis; endostatin and angiostatin (non-RTK) may also prove useful in combination with the disclosed compounds. (See Bruns C.J., et al., Cancer Res., 60(11): 2926-2935 (2000); Schreiber A.B., et al., Science, 232(4755): 1250-1253 (1986); Yen L., et al., Oncogene, 19(31): 3460-3469 (2000)).

Agents used in immunotherapeutic regimens may also be useful in combination with the compounds of formula (I). There are a number of immunologic strategies to generate an immune response against erbB2 or EGFR. These strategies are generally in the realm of tumor vaccinations. The efficacy of immunologic approaches may be greatly enhanced through combined inhibition of erbB2/EGFR signaling pathways using a small molecule inhibitor. Discussion of the immunologic/tumor vaccine approach against erbB2/EGFR are found in Reilly R.T., et al., Cancer Res., 60(13): 3569-3576 (2000); and Chen Y., et al., Cancer Res., 58(9): 1965-1971 (1998).

Agents used in proapoptotic regimens (e.g., Bcl-2 antisense oligonucleotides) may also be used in the combination of the present invention. Members of the Bcl-2 family of proteins block apoptosis. Upregulation of Bcl-2 has therefore been linked to chemoresistance. Studies have shown that the epidermal growth factor (EGF) stimulates anti-apoptotic members of the Bcl-2 family (i.e., Mcl-1). Therefore, strategies designed to downregulate the expression of Bcl-2 in tumors have demonstrated clinical benefit. Such proapoptotic strategies using the antisense oligonucleotide strategy for Bcl-2 are discussed in Waters J.S., et al., J. Clin. Oncol., 18(9): 1812-1823 (2000); and Kitada S., et al., Antisense Res. Dev., 4(2): 71-79 (1994).

Cell cycle signalling 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 signalling 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 G.R., and Chang Y.T., Exp. Opin. Ther. Patents, 10(2): 215- 230 (2000). Further, p21WAFl/CIPl has been described as a potent and universal inhibitor of cyclin- dependent kinases (Cdks) (Ball K.L., Prog. Cell Cycle Res., 3: 125-134 (1997)). Compounds that are known to induce expression of p21WAFl/CIPl have been implicated in the suppression of cell proliferation and as having tumor suppressing activity (Richon V.M., et al., Proc. Natl. Acad. Sci. USA, 97(18): 10014-10019 (2000)), and are included as cell cycle signaling inhibitors. Histone deacetylase (HDAC) inhibitors are implicated in the transcriptional activation of p21WAFl/CIPl (Vigushin D.M., and Coombes R.C., Anticancer Drugs, 13(1): 1-13 (2002)), and are suitable cell cycle signaling inhibitors for use in combination herein. Examples of such HDAC inhibitors include, but are not limited to vorinostat, romidepsin, panobinostat, valproic acid, and mocetinostat.

Vorinostat, N-hydroxy-N'-phenyl-octanediamide, is a HDAC inhibitor, and is commercially available as ZOLINZA® capsules. Vorinostat is indicated for the treatment of cutaneous T-cell lymphoma (CTCL).

Romidepsin, (lS,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-di(propan-2-yl)-2-oxa-12,13-dithia- 5,8,20,23-tetrazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone, is a HDAC inhibitor, and is commercially available as an injectable solution as ISTODAX®. Romidepsin is indicated for the treatment of CTCL.

Panobinostat, (2E)-N-hydroxy-3-[4-({[2-(2-methyl-lH-indol-3- yl)ethyl]amino}methyl)phenyl]acrylamide, is a non-selective HDAC inhibitor, and is commercially available as FARYDAK® capsules. Panobinostat, in combination with bortezomib and dexamethasone, is indicated for the treatment of multiple myeloma. Valproic acid, 2-propylpentanoic acid, is a HDAC inhibitor, and is commercially available as DEPAKENE® capsules, among others. Valproic acid is indicated as monotherapy and adjunctive therapy for the treatment of some seizures and has been explored for the treatment of various cancers.

Mocetinostat, N-(2-Aminophenyl)-4-[[(4-pyridin-3-ylpyrimidin-2-yl)amino]methyl] benzamide, is a benzamide HDAC inhibitor. Mecetinostat is currently undergoing clinical trials for the treatment of various cancers.

Proteasome inhibitors are drugs that block the action of proteasomes, cellular complexes that break down proteins, like the p53 protein. Several proteasome inhibitors are marketed or are being studied for the treatment of cancer. Suitable proteasome inhibitors for use in combination herein include, but are not limited to bortezomib, disulfiram, epigallocatechin gallate, salinosporamide A, and carfilzomib.

Bortezomib, [(lR)-3-methyl-l-({(2S)-3-phenyl-2-[(pyrazin-2- ylcarbonyl)amino]propanoyl}amino)butyl]boronic acid, is a proteasome inhibitor, and is commercially available as an injectable solution as VELCADE® . Bortezomib is indicated for the treatment of multiple myeloma and mantle cell lymphoma.

Disulfiram, l,l^,,l",l'"-[disulfanediylbis(carbonothioylnitrilo)]tetraethane, is commercially available as ANTABUSE® tablets. Disulfiram is indicated as an aid in the management of sobriety in selected chronic alcohol patients. When disulfiram is complexed with metals to form dithiocarbamate complexes, it is a proteasome inhibitor, and such dithiocarbamate complexes have been explored for the treatment of various cancers (Cheriyan V.T., et al., PLoS One, 9(4): e93711 (2014)).

Epigallocatechin gallate (EGCG), [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman- 3-yl]3,4,5-trihydroxybenzoate, is the most abundant catechin in tea, and is a proteasome inhibitor. EGCG has been explored for the treatment of various cancers (Yang H., et al., Curr. Cancer Drug Targets, 11(3): 296-306 (2011)).

Salinosporamide A, (4R,5S)-4-(2-chloroethyl)-l-((lS)-cyclohex-2-enyl(hydroxy)methyl)-5- methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione, also known as marizomib, is a proteasome inhibitor. Salinosporamide A has been explored for the treatment of various cancers.

Carfilzomib, (2S)-4-Methyl-N-[(2S)-l-[[(2S)-4-methyl-l-[(2R)-2-methyloxiran-2-yl]-l- oxopentan-2-yl]amino]-l-oxo-3-phenylpropan-2-yl]-2-[[(2S)-2-[(2-morpholin-4-ylacetyl)amino]-4- phenylbutanoyljaminojpentanamide, is a selective proteasome inhibitor, and is commercially available as an injectable solution as KYPROLIS®. Carfilzomib is indicated for the treatment of certain multiple myelomas.

The 70 kilodalton heat shock proteins (Hsp70s) and 90 kilodalton heat shock proteins (Hsp90s) are a family of ubiquitously expressed heat shock proteins. Hsp70s and Hsp90s are over expressed certain cancer types. Several Hsp70 and Hsp90 inhibitors are being studied in the treatment of cancer. Examples of Hsp70 and Hsp90 inhibitors for use in combination herein include, but are not limited to tanespimycin and radicicol.

Tanespimycin, 17-N-allylamino-17-demethoxygeldanamycin, is a derivative of the antibiotic geldanamycin, and is a Hsp90 inhibitor. Tanespimyicn has been explored for the treatment of various cancers.

Radicicol, [laS-(laR*,2Z,4E, 14*, 15aR*)]-8-Chloro-la, 14,15, 15a-tetrahydro-9,ll-dihydroxy- 14-methyl-6H-oxireno[e][2]benzoxacyclotetradecin-6,12(7H)-dione, also known as monorden, is a Hsp90 inhibitor . Radicicol has been explored for the treatment of various cancers.

Many tumor cells show a markedly different metabolism from that of normal tissues. For example, the rate of glycolysis, the metabolic process that converts glucose to pyruvate, is increased, and the pyruvate generated is reduced to lactate, rather than being further oxidized in the mitochondria via the tricarboxylic acid (TCA) cycle. This effect is often seen even under aerobic conditions and is known as the Warburg Effect.

Lactate dehydrogenase A (LDH-A), an isoform of lactate dehydrogenase expressed in muscle cells, plays a pivotal role in tumor cell metabolism by performing the reduction of pyruvate to lactate, which can then be exported out of the cell. The enzyme has been shown to be upregulated in many tumor types. The alteration of glucose metabolism described in the Warburg effect is critical for growth and proliferation of cancer cells and knocking down LDH-A using RNA-i has been shown to lead to a reduction in cell proliferation and tumor growth in xenograft models (Tennant D.A., et al., Nat. Rev. Cancer, 10(4): 267-277 (2010); Fantin V.R., et al., Cancer Cell, 9(6): 425-434 (2006)).

High levels of fatty acid synthase (FAS) have been found in cancer precursor lesions. Pharmacological inhibition of FAS affects the expression of key oncogenes involved in both cancer development and maintenance. Alii P.M., et al., Oncogene, 24(1): 39-46 (2005).

Inhibitors of cancer metabolism, including inhibitors of LDH-A and inhibitors of fatty acid biosynthesis (or FAS inhibitors), are suitable for use in combination herein.

Cancer gene therapy involves the selective transfer of recombinant DNA/RNA using viral or nonviral gene delivery vectors to modify cancer calls for therapeutic purposes. Examples of cancer gene therapy include, but are not limited to suicide and oncolytic gene therapies, as well as adoptive T-cell therapies.

Additional examples of a further active ingredient or ingredients (anti-neoplastic agent) for use in combination or co-administered with the present methods or combinations are antibodies or other antagonists to CD20, retinoids, or other kinase inhibitors. Examples of such antibodies or antagonists include, but are not limited to rituximab (RITUXAN® and MABTHERA®), ofatumumab (ARZERRA®), and bexarotene (TARGRETIN®).

Rituximab is a chimeric monoclonal antibody which is commercially available as RITUXAN® and MABTHERA®. Rituximab binds to CD20 on B cells and causes cell apoptosis. Rituximab is administered intravenously and is approved for treatment of rheumatoid arthritis and B-cell non- Hodgkin's lymphoma.

Ofatumumab is a fully human monoclonal antibody which is commercially available as ARZERRA®. Ofatumumab binds to CD20 on B cells and is used to treat chronic lymphocytic leukemia CLL; a type of cancer of the white blood cells) in adults who are refractory to treatment with fludarabine (FLUDARA®) and alemtuzumab (CAMPATH®).

Bexarotene, 4-[l-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)ethenyl]benzoic acid, is commercially available as TARGRETIN® capsules. Bexarotene is a member of a subclass of retinoids that selectively activate retinoid X receptors (RXRs). These retinoid receptors have biologic activity distinct from that of retinoic acid receptors (RARs). Bexarotene is indicated for the treatment of certain CTCLs.

Additional examples of a further active ingredient or ingredients (anti-neoplastic agent) for use in combination or co-administered with the present methods or combinations are Toll-like Receptor 4 (TLR4) antagonists.

Aminoalkyl glucosaminide phosphates (AGPs) are known to be useful as vaccine adjuvants and immunostimulatory agents for stimulating cytokine production, activating macrophages, promoting innate immune response, and augmenting antibody production in immunized animals. Aminoalkyl glucosaminide phosphates (AGPs) are synthetic ligands of the Toll-like Receptor 4 (TLR4). AGPs and their immunomodulating effects via TLR4 are disclosed in patent publications such as WO 2006016997, WO 2001090129, and/or US Patent No. 6,113,918 and have been reported in the literature. Additional AGP derivatives are disclosed in US Patent No. 7,129,219, US Patent No. 6,911,434, and US Patent No. 6,525,028. Certain AGPs act as agonists of TLR4, while others are recognized as TLR4 antagonists.

Select anti-neoplastic agents that may be used in combination with the present methods or combinations, include but are not limited to: abarelix, abemaciclib, abiraterone, afatinib, aflibercept, aldoxorubicin, alectinib, alemtuzumab, arsenic trioxide, asparaginase, axitinib, AZD-9291, belinostat, bendamustine, bevacizumab, blinatumomab, bosutinib, brentuximab vedotin, cabazitaxel, cabozantinib, capecitabine, ceritinib, clofarabine, cobimetinib, crizotinib, daratumumab, dasatinib, degarelix, denosumab, dinutuximab, docetaxel, elotuzumab, entinostat, enzalutamide, epirubicin, eribulin, filgrastim, flumatinib, fulvestrant, fruquintinib, gemtuzumab ozogamicin, ibritumomab, ibrutinib, idelalisib, imatinib, irinotecan, ixabepilone, ixazomib, lenalidomide, lenvatinib, leucovorin, mechlorethamine, necitumumab, nelarabine, netupitant, nilotinib, obinutuzumab, olaparib, omacetaxine, osimertinib, oxaliplatin, paclitaxel, palbociclib, palonosetron, panitumumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, plerixafor, pomalidomide, ponatinib, pralatrexate, quizartinib, radium-223, ramucirumab, regorafenib, rolapitant, rucaparib, sipuleucel-T, sonidegib, sunitinib, talimogene laherparepvec, tipiracil, topotecan, trabectedin, trifluridine, triptorelin, uridine, vandetanib, velaparib, vemurafenib, venetoclax, vincristine, vismodegib, and zoledronic acid. EXAMPLES

The following examples illustrate various non-limiting aspects of this invention.

Example 1

The mechanism and function of soluble ICOS (sICOS) is not currently known. At least two isoforms of ICOS have been identified, as shown in FIG. 1. Previously, sICOS has been detected in serum of patients with autoimmune diseases such as diffused cutaneous systemic sclerosis (SS) (Hasegawa et al., Rheumatology 2013;52:242-251 and Yanaba et al., Arch Dermatol Res 2013; 305: 17-23).

The study described herein sought to determine whether sICOS level in serum correlates with parameters such as cancer type, treatment status, and response to anti-ICOS antibody treatment. Response to anti-ICOS antibody treatment was measured by measuring T cell activation during anti- ICOS antibody treatment, with IFN-g (y) levels used as a read-out of T cell activation. Measurement of IFN-y levels as a read-out of T cell activation are further described in US20160215059, which is incorporated by reference herein.

The relative levels of soluble ICOS expression in different cancer types compared to healthy cohorts

Detectable sICOS levels has previously been shown in patients from systemic sclerosis (SS). It was first investigated whether sICOS expression could be detected in patients of different cancer types, and whether there was an increase of sICOS concentration in serum from various cancer types/stage of progression compared to healthy individuals.

A total of 223 healthy and cancer donors were evaluated for sICOS level detection. Donors included healthy donors, donors with a disease (e.g., cancer, autoimmune disease), and donors untreated or treated with a cancer therapy (e.g., chemotherapy, radiation, signal transduction inhibitors). Heterogeneous expression of total sICOS expression were detected in serum from all cancer types/autoimmune diseases (FIG. 3). The following seven cancer types were included in the study: breast cancer (N=34); head and neck (H/N) (N= 18); kidney (N=21); lung (N=27); melanoma (N=29); chronic lymphocyte leukemia (CLL) (N=26); diffuse large B-cell lymphoma (DLBCL) (N=20). Systemic sclerosis (N=7) was included as positive control group. A different type of autoimmune diease, systemic lupus erythematosus (SLE) (N= 16), was included for comparsion with SS. A total of 25 healthy donors were screened for comparsion of "normal" range of sICOS expression.

Overall, heterogeneous expression of total sICOS expression were detected in serum from all cancer types/autoimmune diseases (FIG 3). Comparing to healthy cohorts (Median sICOS concentration =2.49 pg/mL), all cancer types except DLBCL and CLL expressed relatively higher sICOS (FIG.3 and Table 1): Breast (Median sICOS=1889.4pg/mL); H/N (Median sICOS= 1128.2 pg/mL); Kidney (Median sICOS=548.193 pg/mL), Melanoma (Median sICOS=339.313 pg/mL); Lung (Median sICOS= 119.948 pg/mL). On the other hand, DLBCL (Median sICOS=14 pg/mL) and CLL (Median sICOS=0 pg/mL) had little or no sICOS detection. 2 autoimmune diseases, Systemic Sclerosis (Median sICOS=2608.56 pg/mL) and SLE Median sICOS= 1788.81 pg/mL) appeared to express relatively high levels of sICOS.

Table 1

Competiton assay for evaluation of the potential interference of H2L5 IQG4PE binding to surface ICOS antigen in the presence of serum matrix containing sICOS.

One hypothesis for the potential role of sICOS is that it could act as a sink to sequester H2L5 IgG4PE to prevent it from binding to surface ICOS antigen on T cells, and subsequently affect its efficacy in T cell activation. To evaluate whether the presence of sICOS will potentially sequester anti- ICOS antibody from binding to ICOS surface antigen on T cells, Baf/3hICOS cell line, which was created by Ba/F3 cell line transfected with human ICOS in bDNA4 vector, was used in a competition assay to test whether binding of H2L5 IgG4PE-PE (H2L5 IgG4PE conjugated with PE) would be affected in the presence of sICOS (using either serum from donors tested with high, medium and low range of sICOS, or recombinant ICOS-Fc protein).

The competition assay was set up with either 2-Step incubation or 1-Step incubation (FIGS 4 and 5). 1 Step incuation - H2L5 IgG4PE was incubated with sICOS, from either source, and Baf/3hICOS cells for 2 hours. Incubation was performed on ice in the dark. 2 Step incubation - H2L5 IgG4PE was incubated with sICOS from either source for 1 hr before adding Baf/3hICOS cells and incubating for a further 1 hr. Incubations were performed on ice in the dark.

Overall, a dose-dependent competition of recombinant ICOS-Fc protein and H2L5 IgG4PE was observed ranging from the higher-end concentration of 350 ng/mL to 8750 ng/mL, but not with ICOS- Fc protein concentration below 350 ng/mL. The serum from donors being tested fell into a range of sICOS concentration between 0 to 230 ng/mL and showed undetectable competition with H2L5 IgG4PE. These results suggested a much higher range of sICOS concentrations than that currently detected in the serum/plasma samples from the 223 cancer donors tested would be required to exhibit potential interference of H2L5 IgG4PE binding to surface ICOS antigen.

The correlation of sICOS level and IFN-g level induced through activation of T cells

The mechanism of soluble ICOS production is currently unknown. It was investigated whether the sICOS level correlated with T cell activation using IFN-y production as the readout. The samples were taken from donors having head and neck cancer or lung cancer.

T cell activation assay was carried out using isolated PBMC that were "overnight-rested", which were activated using the following conditions: 0.6pg/ml_ of plate-bound anti-CD3 mAb (or isotype control antibody) and 10 pg/mL of plate-bound H2L5 IgG4PE (or isotype control antibody). Other control conditions such as the use of anti-CD3 mAb alone ("CD3") or No treatment (Baseline) was included for comparsion. The response was calculated based on the change (increase) of IFN-y production of "CD3+bICOS" activation from "CD3" or fronY'Baseline" control conditions.

Serum isolated from the respective PBMC samples were evaluated for sICOS level detection as described above.

Matching sets of sICOS and IFN-g data from 5 donors were evaluated. As shown in Figures 6 and 7, a positive correlation was observed in the level of sICOS in serum vs. response to ICOS treatment (IFN-y). Without intending to be bound by theory, the positive correlation observed indicates that patients having higher levels of sICOS in serum are more likely to be responsive to treatment by an agonist anti-ICOS antibody such as H2L5 IgG4PE than patients having lower levels of sICOS in serum.

The results described in Example 1 were obtained using the following materials and methods.

Human serum/plasma collection

The collection population for early stage comparison of soluble ICOS levels relative to healthy cohorts included total of 223 human serum or plasma samples. 211 plasma or serum samples were purchased from either Conversant Bio, Avaden BioSciences, BioreclamationIVT, Asterand or processed from blood ordered from Blood Donation Unit (BDU) within GSK facility at Upper Providence. Overall, the following indicate the number of samples (211) from each cancer type: Healthy (25) Breast (34), Head and Neck, H/N (18), Kidney (21), Lung (21), Melanoma (23), Chronic Lymphocytic Leukemia (CLL) (26), Diffused large B cell lymphoma, DLBCL (20), Systemic Lupus Erythematosus, SLE (16), Systemic Sclerosis, SS (7). The inclusion criteria for the patients included patients diagnosed with cancer catergories of interest at any stage of treatment or progression. A further set of samples from prospective collections that included 6 patient donors with lung cancer or melanoma from pre-therapy (visit 1) and 90 days post-anti-PD-1 mAb treatment (Visit 2) were also included in the analysis.

Total sICOS detection by MSD

MSD standard bind 96-well assay plate (Meso Scale Discovery Cat#L15XA) was coated with 40pl per well of 2pg/ml_ of an anti-ICOS Capturing Ab developed in-house in PBS overnight at 4°C. Plates were washed 3 times with 150pL/well of MSD Wash Buffer (PBS plus 0.1% Tween 20) and blocked with 150pL/well of Assay Diluent/Blocking Casein Buffer in PBS (Thermo Scientific Cat#37582) at room temperature for lhr 30 min. This was followed by 2hr incubation with 25pL/well of serum or plasma (in triplicates) or 25pl per well (in duplicates) of 2-fold serial titrated human recombinant ICOS- Fc protein standards that were further 2 fold diluted in assay diluent, on a plate shaker at room temperature. The plates were then washed three times with 150pL of MSD Wash Buffer, followed by lhr 30min incubation with 25pl/well of 2pg/ml_ of Sulfo-Tag Labeled Anti-Human ICOS Detection Ab (generated by GSK Immunogenicity and Clinical Immunology group (ICI)) that did not compete with the capture antibody for binding to the soluble ICOS protein. The plates were then washed three times with 150pL of MSD Wash Buffer before 150pl of 2X Read buffer (Meso Scale Discovery Cat #R92TC-3) was added to each well for detection of signals in MSD Plate Reader (Sector 600).

The raw ECL signals and calculated concentration (in pg/mL) were generated and exported using MSD Discovery Workbench Version 4.0 software. The detection range of soluble ICOS concentration was generated automatically by the software based on the signals detected from serialtitrated recombinant ICOS-Fc protein standards. The minimum linear dectection range of soluble ICOS concentration based on recombinant ICOS-Fc standard curve was approximately 200 pg/mL. Concentration below 200 pg/mL were labeled as " Below Detection Range"; Concentration above the detection range were labeled as "Above Dectection Range" in raw data. TIBCO Spotfire was used for MSD data analysis and generation of graphs.

Statistic analysis was performed using 1-way Anova.

Correlation of soluble ICOS expression to functional responses

Correlation evaluation of the level of soluble ICOS to several parameters such as cancer types, treatment status, and the functional response to agonist anti-ICOS mAb H2L5 IgG4PE treatment was performed using TIBCO Spotfire software.

Functional response to agonist anti-ICOS mAb H2L5 IgG4PE treatment was measured by measuring IFN-y secretion.

Pre-T reatment Rest For overnight rest of cells, PBMCs were cultures in complete media in a non-tissue culture treated 96- well plate, cells were rested at 100,000 cells per well in 100 pL.

Plate coating for cell activation

The day prior to activation, 96-well plates were coated with 0.6 pg/mL anti-CD3 (eBioscience) or its isotype control antibody overnight at 4°C in 100 pL/well lx ELISA coating buffer (Biolegend) as per the manufacturer's protocol. On the day of treatment, the plates were washed three times with 150 pL lx PBS, carefully to prevent wells from drying. Selected wells were coated with 10 pg/mL H2L5 IgG4PE or IgG4 istoype antibody in 100 pL/well lx coating buffer. The plates were incubated at room temperature for 4 hours. Wells which were not being coated were filled with lx PBS until use. Following the incubation, the wells were washed and prepared for cells and treatments.

Activation and Treatment

Cells were collected and counted using CELLOMETER (Nexcelom Bioscience). The cells were plated in the appropriate wells for antibody treatment. Cells were plated at a density of 100,000 cells/well in 300 pL complete media. Complete media is made in RPMI 1640 media with autologous plasma and growth supplements (RPMI 1640 media (Gibco), 10% Autologus plasma, 1% Glutamax (Gibco), 1% NEAA (non-essential amino acids) (Gibco), 1% Sodium Pyruvate (Gibco), 25 mM Hepes (Gibco), 50 pM Beta-mercaptoethanol (Sigma), 1: 1000 M-CSF (macrophage colony stimulating factor) (Pepro-Tech) - Beta-mercaptoethanol is made fresh to a workable concentration in RPMI 1640 media. M-CSF is resuspended in distilled water as lOOx concentration to use as 1: 1000).

0.6pg/mL of plate-bound anti-CD3 mAb (or isotype control antibody) and 10 pg/mL of plate-bound H2L5 IgG4PE (or isotype control antibody) were prepared. After plating with treatment, cells were incubated at 37°C/5% CO2 for 72-96 hours. Approximately 100 pL of the cell supernatants were collected after 24 hours or 48 hours, 72 hours or 96 hours for cytokine analysis. If supernatents were collected twice before the final collection, additional media was replaced after the second collection (200 pL).

Human IFNY Cytokine Assay

Samples and calibrators were diluted in Diluent 2 (Meso Scale Discovery, Cat No. R51BB-3). One millileter of the diluent was added to the calibrator. After vortexing, the calibrator was incubated on ice for a minimum of 5 minutes and labelled as Calibrator 1. A 1:4 serial diluation was used to prepare th 6 additional calibrator dilutions. Diluent 2 was used as the plate background. Fifty microliters of prepared samples (in triplicate) and calibrators (in duplicate) were added to the MSD plate. Plates were sealed and incubated at room temperature with shaking for 2 hours. Plates were washed 3 times. Detection antibody solution was prepared in Diluent 3 (Meso Scale Discovery, Cat No. R51BA- 5). For each plate, 60 pL of each of the detection antibody was added to the diluent for a total of 3 mL of detection reagent. Following the addition of 25 pL of detection antibodyes, the plates were sealed and incubated at room temperature, in the dark, with shaking for 2 hours. Please were washed 3 times. Read Buffer (Meso Scale Discovery Cat No. R92TC-3) and they were read on the QUICKPLEX MSD plate reader.

Competition Assay to evaluate whether the presence of sICOS will potentially sequester anti-ICOS antibody from binding to ICOS surface antigen

Baf/3hICOS cell line, which is a mouse pro-B cell line expressing recombinant Human ICOS, was used in a competition assay to test whether binding of H2L5 IgG4PE-PE (H2L5 IgG4PE conjugated with phycoerythrin (PE) would be affected in the presence of sICOS (using serum from selected donors tested with high, medium and low range of sICOS). Serial titrations of recombinant ICOS-Fc chimera protein (R&D Systems, Cat#169-CS-050)) was used at doses ranging from 35 pg/mL to 0 pg/mL (35 pg/mL 5 fold diluted down 6 times to 7 pg/mL, 1.4 pg/mL, 0.28 pg/mL, 0.056 pg/mL, 0.0112 pg/mL and 0.00224 pg/mL, and 0 pg/mL) to create a standard for comparison .

The incubation was performed using either 1-Step or 2-Step incubations. For 2-Step incubation - 50 pL of H2L5 IgG4PE-PE (4 pg/mL) or isotype-PE (4 pg/mL) was incubated with 50 pL of recombinant ICOS-Fc protein (serial titrations identified above) or 50 pL human serum, as a source of soluble ICOS, for lhr on ice in the dark. After lhr, 100 pL of Baf/3hICOS cells (at 1.3c10^L6 cells/mL) were added, mixed well and incubated for an additional lhr. For 1-Step incubation - 50 pL of H2L5 IgG4PE-PE (4 pg/mL) or isotype-PE (4 ug/mL) was incubated with 50 pL of recombinant ICOS-Fc protein (serial titrations outlined above) or 50 pL human serum, as a source of soluble ICOS, in the presence of 100 pL Baf/3hICOS cells (1.3c10^L6 cells/mL) for 2 hrs on ice in the dark.

Following the incubations, the cells were spun down, washed twice with FACS buffer (200 pL for each wash). Finally, 200 pL of FACS buffer was added prior to cell acquision by flow cytometry (BD Canto II flow cytometer). The quantification of binding of PE-conjugated H2L5 IgG4PE anti-ICOS mAb or IgG4 isotype antibody was calculated using QuantiBRITE Beads (BD Bioscience, Cat#340495) to calibrate. The Geometric Mean and calculated PE/Molecules per cell were graphed using GraphPad Prism Version 7.

Claims

1. A method of treating cancer in a human in need thereof, the method comprising determining the level of soluble ICOS (sICOS) in a sample from the human and administering to the human an effective amount of an agent directed to human ICOS if the level of the sICOS is increased relative to a reference level, thereby treating the cancer in the human.

2. An agent directed to human ICOS for use in the treatment of cancer in a human classified as a responder, wherein the responder is characterized by the presence of an increased level of soluble ICOS (sICOS) in a sample from the human relative to a reference level.

3. The method of claim 1 or the agent of claim 2, wherein the agent directed to human ICOS is an anti-ICOS antibody or antigen binding portion thereof.

4. The method or agent of claim 3, wherein the anti-ICOS antibody or antigen binding portion thereof is an ICOS agonist.

5. The method or the agent of claim 3 or 4, wherein the anti-ICOS antibody comprises a VH domain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7; and a VL domain comprising an amino acid sequence at least 90% identical to the amino acid sequence as set forth in SEQ ID NO: 8.

6. The method or the agent of claim 4, wherein the anti-ICOS antibody comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 7 and a VL domain comprising the amino acid sequence as set forth in SEQ ID NO:8.

7. The method or the agent of claim claim 4, wherein the anti-ICOS antibody comprises a heavy chain comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9; and a light chain comprising an amino acid sequence at least 90% identical to the amino acid seqsuence as set forth in SEQ ID NO: 10.

8. The method or the agent of claim 4, wherein the anti-ICOS antibody comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:9; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 10.

9. The method or the agent of any one of claims 1 to 8, wherein the cancer is selected from the group consisting of colorectal cancer (CRC), gastric, esophageal, cervical, bladder, breast, head and neck, ovarian, melanoma, renal cell carcinoma (RCC), EC squamous cell, non-small cell lung carcinoma, mesothelioma, pancreatic, and prostate cancer.

10. The method or the agent of any one of claims 1 to 9, wherein the sample comprises serum.

11. The method or the agent of any one of claims 1 to 10, further comprising administering one or more additional anti-neoplastic agents.

12. A kit for the treatment of cancer comprising a means for determining the level of soluble ICOS (sICOS) in a sample from a human in any one of claims 1 to 11.

13. The kit of claim 12, wherein said means is a capture antibody.

14. A pharmaceutical composition comprising an agent directed to human ICOS, for use in treating cancer in a human wherein at least a first sample from the human is determined to have an increased level of soluble ICOS (sICOS) relative to a reference level.

15. Use of an agent directed to human ICOS in the manufacture of a medicament for the

treatment of cancer in a human wherein one or more samples from the human is determined to have an increased level of soluble ICOS (sICOS) relative to a reference level.