WO2016014799A1

WO2016014799A1 - Epidermal growth factor and blockade of immune checkpoints in cancer immunotherapy

Info

Publication number: WO2016014799A1
Application number: PCT/US2015/041738
Authority: WO
Inventors: George Weiner; Mohammed Milhem; Amani MAKKOUK; Zuhair K. BALLAS; Lyse NORIAN
Original assignee: University Of Iowa Research Foundation
Priority date: 2014-07-23
Filing date: 2015-07-23
Publication date: 2016-01-28
Also published as: WO2016014799A9

Abstract

Disclosed are methods, compositions, kits, and systems for diagnosing and/or treating a patient via immunotherapy. The diagnostic methods including detecting epidermal growth factor (EGF) and administering to the patient a therapeutic antibody that binds to a cell surface receptor of a regulatory T cell (T_reg) based on the detected EGF. The treatment methods include administering EGF or an agonist of the EGF receptor and administering the therapeutic antibody that binds to a cell surface receptor of a T_reg.

Description

EPIDERMAL GROWTH FACTOR AND BLOCKADE OF IMMUNE CHECKPOINTS

IN CANCER IMMUNOTHERAPY

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] The present application claims the benefit of priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Patent Application No. 62/028,109, filed on July 23, 2014, the content of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] The field of the invention relates to immunotherapy. In particular, the invention relates to the role of epidermal growth factor (EGF) in monoclonal antibody (mAB)-mediated immunotherapies for cancer related to blockade of immune checkpoints.

[0003] A promising new approach to cancer therapy is use of checkpoint blockade monoclonal antibodies that enhance the immune response to diseases such as cancer. However, these antibodies have significant toxicities and are very expensive, so predicting whether a patient is likely to respond to these antibodies is important prior to administering these antibodies to the patient. Furthermore, methods for enhancing a patient's sensitivity to these antibodies could enhance the efficacy of the antibodies.

SUMMARY

[0004] Disclosed are methods, compositions, kits, and systems for diagnosing and/or treating a patient via immunotherapy and blockade of immune checkpoints. The methods may include detecting epidermal growth factor (EGF) in a biological sample from the patient, and administering to the patient a therapeutic antibody that binds to a cell surface receptor of a regulatory T cell (Τ_Λ¾) or a therapeutic antibody that binds to a ligand for a cell surface receptor of a T_K£, based on the detected EGF in the biological sample. The treatment methods may include administering EGF or an agonist of the EGF receptor to a patient and administering atherapeutic antibody that binds to a cell surface receptor of a T_reg or a therapeutic antibody that binds to a ligand for a cell surface receptor of a T_reg to the patient.

[0005] In some embodiments, the disclosed diagnostic and/or treatment methods include: (a) detecting epidermal growth factor (EGF) in a biological sample from a patient; and based on the detected EGF (b) administering to the patient a therapeutic antibody that binds to a cell surface receptor of a T_reg or a therapeutic antibody that binds to a ligand for a cell surface receptor of a T_reg. In the methods, the biological sample may include blood or a fraction thereof (e.g., serum). The detected EGF may be detected at a concentration of greater than about 0.085 ng EGF /ml in the serum, prior to administering the therapeutic antibody.

[0006] Also disclosed are compositions, kits, and systems for performing these disclosed diagnostic and/or treatment methods. In some embodiments, the disclosed compositions, kits, and systems may include and/or utilize: (a) a diagnostic antibody for detecting EGF (e.g., a labeled anti-EGF antibody); and (b) a therapeutic antibody that binds to a cell surface receptor of a T_reg or a therapeutic antibody that binds to a ligand for a cell surface receptor of a T_reg.

[0007] In some embodiments, the disclosed treatment methods may include administering to a patient: (a) a pharmaceutical composition comprising EGF or an agonist for the EGF receptor; and (b) a pharmaceutical composition comprising a therapeutic antibody that binds to a cell surface receptor of a T_reg or at therapeutic antibody that binds to a ligand for a cell surface receptor of a T_reg. In the treatment methods, the pharmaceutical composition comprising EGF may be administered at a dose that achieves a concentration of greater than about 0.08S ng EGF/ml in the serum of the patient.

[0008] Also disclosed are compositions, kits, and systems for performing these disclosed treatment methods. In some embodiments, the compositions, kits, and systems may include and/or utilize: (a) a pharmaceutical agent comprising EGF or an agonist for the EGF receptor; and (b) a pharmaceutical agent comprising a therapeutic antibody that binds to a cell surface receptor of the T_reg or a therapeutic antibody that binds to a ligand for a cell surface receptor of a T_reg.

[0009] The disclosed methods, compositions, kits, and systems may utilize and/or include an antibody that binds to a cell surface receptor of a T_reg or an antibody that binds to a ligand for a cell surface receptor of a T_reg. In some embodiments, the cell surface receptor of the T_reg is an inhibitor receptor and the antibody is an antagonist for the cell surface receptor. For example, the therapeutic antibody may bind to cytotoxic T-lymphocyte antigen 4 (CTLA-4 or CD 152) or programmed cell death protein 1 (PD1 or CD279).

[0010] The disclosed methods, compositions, kits, and systems optionally may utilize and/or include an antigen, such as a tumor immunogen. For example, in the disclosed methods a tumor immunogen may be administered to a patient prior to, concurrently with, or after administering a therapeutic antibody and/or a pharmaceutical composition comprising EGF or an EGF agonist to the patient.

[0011] The disclosed methods, compositions, kits, and systems may be utilized to diagnose and/or treat a patient having cancer or a hyperproliferative disorder. In some embodiments, the patient has adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or teratocarcinoma, (including any of cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, prostate, skin, testis, thymus, and uterus).

BRIEF DESCRIPTION OF THE FIGURES

[0012] Figure I illustrates that addition of EGF results in increased expression of CTLA4 in T_reg cells having the phenotype CD3⁺CD4^÷CDl27^lowFoxp3⁺.

DETAILED DESCRIPTION

[0013] The disclosed subject matter further may be described utilizing terms as defined below. [0014] Unless otherwise specified or indicated by context, the terms "a", "an", and "the" mean "one or more." For example, "an antibody" and "agonist" should be interpreted to mean "one or antibodies" and "one or more agonists," respectively, unless otherwise specified or indicated by context

[0015] As used herein, "about", "approximately," "substantially," and "significantly" will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" and "approximately" will mean plus or minus <10% of the particular term and "substantially" and "significantly" will mean plus or minus > 10% of the particular term.

[0016] As used herein, the terms "include" and "including" have the same meaning as the terms "comprise" and "comprising." The terms "comprise" and "comprising" should be interpreted as being "open" transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms "consist" and "consisting of should be interpreted as being "closed" transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term "consisting essentially of should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0017] As used herein, the term "patient" may be used interchangeably with the term "subject" or "individual" and may include an "animal" and in particular a "mammal." Mammalian subjects may include humans and other primates, domestic animals, farm animals, and companion animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like.

[0018] As used herein, the term "biological sample" should be interpreted to include bodily fluids (e.g., blood, serum, plasma, saliva, urine samples) and body tissue samples. Suitable tissue samples may include tissue samples from cancerous tissues and tumors. [0019] The disclosed methods, compositions, and kits may be utilized to diagnose and/or treat a patient in need thereof. A "patient in need thereof is intended to include a patient having or at risk tor developing diseases and disorders such as cancer and hyperproliterative disorders. A patient in need thereof may include a patient having or at risk for developing any of adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, (including cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, prostate, skin, testis, thymus, and uterus).

[0020] In one aspect, the disclosed methods, compositions, kits, and systems are utilized for diagnosing and/or treating a patient via immunotherapy and blockade of immune checkpoints. The methods may include detecting epidermal growth factor (EGF) in a biological sample from the patient, and administering to the patient a therapeutic antibody that binds to a cell surface receptor of a regulatory T cell ( T_reg) or a therapeutic antibody that binds to a ligand for a cell surface receptor of a T_reg, based on the detected EGF in the biological sample (e.g., based on the detected concentration of EGF in the biological sample). EGF may be detected by any suitable method, including immunoassays. Preferably, EGF is detected by a method that determines the concentration of EGF in the biological sample.

[0021] In some embodiments, the disclosed diagnostic and/or treatment methods include: (a) detecting epidermal growth factor (EGF) in a biological sample from a patient (e.g., blood or a fraction thereof such as serum); and based on the detected EGF in the biological sample (e.g., based on the detected concentration of EGF in a serum sample from the patient) (b) administering to the patient a therapeutic antibody that binds to a cell surface receptor of a T_reg, or a therapeutic antibody that binds to a ligand for a cell surface receptor of a Τ^. The detected EGF may be detected at a concentration of greater than about 0.085 ng EGF/ml in the serum (preferably greater than about 0.100 ng EGF/ml, 0.200 ng EGF /ml, or 0.300 ng EGF /ml) by performing a diagnostic immunoassay for EGF (e.g., an enzyme linked immunoassay), prior to administering the therapeutic antibody. [0022] Also disclosed are compositions, kits, and systems for performing these disclosed diagnostic and/or treatment methods. In some embodiments, the disclosed compositions, kits, and systems may include and/or utilize: (a) a diagnostic antibody for detecting EGF (e.g., a labeled anti-EGF antibody); and (b) a therapeutic antibody that binds to a cell surface receptor of a T_reg or a therapeutic antibody that binds to a ligand for a cell surface receptor of a T_rCg. The diagnostic antibody utilized or present in the disclosed methods, compositions, kits, and systems may be labeled with a label suitable for performing a diagnostic immunoassay for determining the concentration of EGF in a biological sample. Suitable labels may include, but are not limited to, enzymes, chromophores, fluorophores, and radioisotopes.

[0023] The therapeutic antibody utilized or present in the disclosed methods, compositions, kits, and systems, may bind to a cell surface receptor of a Τ_Λ¾ or may bind to a ligand for a cell surface receptor of a T_Kg, where binding of the ligand to the cell surface receptor inhibits activity of the T_reg. As such, the therapeutic antibody may bind to the cell surface receptor and function as an antagonist for the cell surface receptor, or the therapeutic antibody may bind the ligand and prevent the ligand from binding to the ceil surface receptor as an agonist. The therapeutic antibody generally disrupts inhibition of the T_reg via activation of the inhibitory cell surface receptor.

[0024] In some aspects, the therapeutic antibody binds to a cell surface receptor of a T_reg where the receptor is an inhibitor receptor and the therapeutic antibody is an antagonist for the inhibitory cell surface receptor. For example, in some embodiments, the therapeutic antibody binds to cytotoxic T-lymphocyte antigen 4 (CTLA-4 or CD 152) (e.g., where the therapeutic antibody is ipilimumab (also known as MDX-010 and MDX-101 and marketed under the tradename Yervoy®) or tremelimumab). In other embodiments, the therapeutic antibody binds to programmed cell death protein 1 (PD1 or CD279) (e.g., where the therapeutic antibody is pembrolizumab (alternatively referred to as MK3475 and lambrolizumab and marketed under the tradename Ketruda®), nivolumab (alternatively referred to as MDX-1 106, BMS-936558, ONO- 4538 and marketed under the tradename Opdivo®), CT=01 1, or AMP-224). In other embodiments, the therapeutic antibody binds to lymphocyte activation gene 3 (LAG3 and also known as CD223) (e.g., where the therapeutic antibody is IMP321). In further embodiments, the therapeutic antibody binds to B and T lymphocyte attenuator (BTLA and also known as CD272). In even further embodiments, the therapeutic antibody binds to T cell membrane protein 3 (TIM3 and also known as HAVcr2). In even further embodiments, the therapeutic antibody binds to adenosine A2a receptor (A2aR) or any of the family of killer inhibitory receptors.

[0025] In other aspects, the therapeutic antibody binds to a ligand for a cell surface receptor of a T_reg, for example, where the receptor is an inhibitor receptor and the therapeutic antibody binds the ligand and prevents the ligand from binding to the receptor as an agonist. For example, in some embodiments, the therapeutic antibody binds to the ligand for PD1 (PDL1 or PDL2, e.g., where the therapeutic antibody is MDX-1 105). In other embodiments, the therapeutic antibody binds to the ligand B7-H3 (e.g., where the therapeutic antibody is MGA271 ), or the therapeutic antibody binds to B7-H4.

[0026] In some embodiments, the disclosed treatment methods may include administering to a patient: (a) a pharmaceutical composition comprising EGF or an agonist for the EGF receptor; and (b) a pharmaceutical composition comprising a therapeutic antibody that binds to a cell surface receptor of a T_rog or a therapeutic antibody that binds to a ligand for a cell surface receptor of a T_rtfg, as contemplated herein. Epidermal growth factor is the original member of the EGF-family of proteins which members have highly similar structural and functional characteristics. In addition to EGF, the EGF-family of proteins include heparin- binding EGF-like growth factor (HB-EGF), transforming growth factor-a (TGF-a), amphiregulin (AR), epiregulin (EPR), epigen, betacellulin (BTC), neuregulin-1 (NRG1), neuregulin-2 (NRG2), neuregulin-3 (NRG3), and neuregulin-4 (NRG4). EGF and other members of the EGF- family of proteins are ligands and agonists for the EGF receptor. The disclosed methods, compositions, kits, and systems may include or utilize EGF or an agonist for the EGF receptor. For example, suitable agonists may include EGF, other members of the EGF-family of proteins, or variants thereof, that bind to an EGF receptor (e.g., ErbB-1 ). [0027] The amino acid sequence of pro-EGF growth factor isoform 2 preproprotein (NCBI Reference Sequence: NP 001 171601 ) is provided as SEQ ID NO: l:

1141 phsllsanpl wqqraldpph qmeltq

[0028] The amino acid sequence of epidermal growth factor receptor (GenBank: CAA25240.1 ) is provided as SEQ ID NO:2:

1081 siddtflpvp eyinqsvpkr pagsvqnpvy hnqplnpaps rdphyqdphs tavgnpeyln 1141 tvqptcvnst fdspahwaqk gshqisldnp dyqqdffpke akpngifkgs taenaeylrv 1201 apqssefiga

[0029] Suitable EGF agonists may include polypeptides comprising the amino acid sequence of SEQ ID NO: 1 , the amino acid sequences of any of EGF, HB-EGF, TGF-a, AR, EPR, epigen, BTC, NRG 1, NRG2, NRG3, NRG4, or mutants or variants thereof. For example, EGF agonists may comprise the amino acid sequences of any of SEQ ID NO: l, EGF, HB-EGF, TGF-a, AR, EPR, epigen, BTC, NRG1, NRG2, NRG3, NRG4 or may comprise an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: l. Mutant polypeptides or variant polypeptides may include one or more amino acid substitutions, deletions, additions and/or amino acid insertions relative to the wild-type polypeptide, where preferably the mutant polypeptides or variant polypeptide exhibit the biological activity of the wild-type polypeptide (e.g., where the mutants or variants bind to an EGF receptor having the amino acid sequence of SEQ ID NO:2, or mutants or variants thereof, and function as agonists).

[0030] The terms "amino acid" and "amino acid sequence" refer to an oligopeptide, peptide, polypeptide, or protein sequence (which terms may be used interchangeably), or a fragment of any of these, and to naturally occurring or synthetic molecules. Where "amino acid sequence" is recited to refer to a sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.

[0031] The amino acid sequences contemplated herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, or derivative polypeptide may include conservative amino acid substitutions relative to a reference polypeptide. "Conservative amino acid substitutions" are those substitutions that are predicted to interfere least with the properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference protein. Table 1 provides a list of exemplary conservative amino acid substitutions.

[0032] Table 1

Original

Residue Conservative Substitution

Ala Gly. Scr

Arg His. Lys

Asn Asp. Gin. His

Asp Asn. Glu

eye Ala. Ser

Gin Asn. Glu, His

(ill) Asp. Gin. His

Gly Ala

His Asn. Arg, Gin. Glu

lie Leu. Val

Leu lie, Val

i,ys Arg. Gin, Glu

Met Leu. lie

Phe His. Met. Leu, Trp, Tyr

Ser C'ys. Thr

Thr Ser, Val

Trp Phe. Tyr

Tvr His, Phe. Trp

Val lie. Leu, Thr

[0033] Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.

[0034] A "deletion" refers to a change in the amino acid sequence that results in the absence of one or more amino acid residues. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acids residues. A deletion may include an internal deletion or a terminal deletion (e.g., an N-terminal truncation or a C -terminal truncation of a reference polypeptide).

[0035] The words "insertion" and "addition" refer to changes in an amino acid sequence resulting in the addition of one or more amino acid residues. An insertion or addition may refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues.

[0036] A "fragment" is a portion of an amino acid sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise up to the entire length of the reference sequence, minus at least one amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous amino acid residues of a reference polypeptide. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of a reference polypeptide. Fragments may be preferentially selected from certain regions of a molecule. The term "at least a fragment" encompasses the full length polypeptide.

[0037] "Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polypeptide sequences. Homology, sequence similarity, and percentage sequence identity may be determined using methods in the art and described herein.

[0038] The phrases "percent identity" and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well- known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide. Percent identity for amino acid sequences may be determined as understood in the art. (See, e.g., U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety). A suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources, including the NCBI, Bethesda, Md., at its website. The BLAST software suite includes various sequence analysis programs including "blastp," that is used to align a known amino acid sequence with other amino acids sequences from a variety of databases.

[0039] Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least IS, at least 20, at least 30, at least 40, at least SO, at least 70 or at least ISO contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0040] A "variant," "mutant," or "derivative" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250). Such a pair of polypeptides may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length of one of the polypeptides.

[0041] A "variant," "mutant" or a "derivative" may have substantially the same functional activity as a reference polypeptide. For example, a variant, mutant, or derivative of SEQ ID NO:l, EGF, HB-EGF, TGF-o, AR, EPR, epigen, BTC, NRG1, NRG2, NRG3, and NRG may bind to an EGF receptor, such as ErbB-1 (optionally comprising the amino acid sequence of SEQ ID NO:2 or mutants or variants thereof), and function as an agonist.

[0042] In the treatment methods, the pharmaceutical composition comprising EGF or an agonist for the EGF receptor may be administered at a dose that achieves a concentration of greater than about 0.085 ng EGF /ml in the serum of the patient (preferably greater than about 0.100 ng EGF /ml, 0.200 ng EGF /ml, or 0.300 ng EGF /ml). The pharmaceutical composition comprising EGF or the agonist for the EGF receptor may be administered prior to, concurrently with, or after administering the therapeutic antibody.

[0043] The disclosed methods, compositions, kits, and systems optionally may utilize and/or include an antigen, such as a tumor immunogen. For example, in the disclosed methods a tumor immunogen may be administered to a patient prior to, concurrently with, or after administering a therapeutic antibody and/or a pharmaceutical composition comprising EGF or an EGF agonist to the patient. Suitable tumor immunogens may include isolated antigens prepared from or a mixture of antigens present in a lysate prepared from a tumor isolated from a patient. Preferably, the tumor immunogen is tumor specific and is not expressed on non-cancer cells.

[0044] The compositions disclosed herein may include pharmaceutical compositions and/or vaccine composition formulated for administration to a subject in need thereof. Such compositions can be formulated and/or administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration.

[0045] The compositions may include pharmaceutical solutions comprising carriers, diluents, excipients, and surfactants, as known in the art. Further, the compositions may include preservatives {e.g., anti-microbial or anti-bacterial agents such as benzalkonium chloride). The compositions also may include buffering agents (e.g., in order to maintain the pH of the composition between 6.5 and 7.5). [0046] The pharmaceutical compositions may be administered prophylactically or therapeutically. In prophylactic administration, the vaccines may be administered in an amount sufficient to induce a cellular immune response tor protecting against disease or tor treating disease. In therapeutic applications, the compositions are administered to a patient in an amount sufficient to elicit a therapeutic effect (e.g., an immune response which cures or at least partially arrests or slows symptoms and/or complications of disease (i.e., a "therapeutically effective dose")).

[0047] The therapeutic compositions disclosed herein may be co-administered or sequentially administered to a patient in need thereof with other therapeutic compositions, which may include therapeutic compositions comprising chemotherapeutic agents. Suitable chemotherapeutic agents may include, but are not limited to Abiraterone Acetate, Abitrexate (Methotrexate), Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor (Everolimus), Aldesleukin, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Asparaginase Erwinia chrysanthemi, Axitinib, Azacitidine, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, Bexarotene, Bicalutamide, BiCNU (Carmustine), Bleomycin, Bortezomib, Bosulif (Bosutinib), Bosutinib, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabozantinib-S-Malate, Camptosar (Irinotecan Hydrochloride), Capecitabine, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Casodex (Bicalutamide), CeeNU (Lomustine), Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Chlorambucil, CHLORAMBUCIL-PREDNISONE, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), Cometriq (Cabozantinib-S-Malate), Cosmegen (Dactinomycin), Crizotinib, Cyclophosphamide, Cyfos (Ifosfamide), Cytarabine, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin Diftitox, Dexrazoxane Hydrochloride, Docetaxel, Doxorubicin Hydrochloride, DTIC-Dome (Dacarbazine), Efudex (Fluorouracil), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enzaiutamide, Epirubicin Hydrochloride, Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), Fulvestrant, Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemzar (Gemcitabine Hydrochloride), Giiotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hycamtin (Topotecan Hydrochloride), Ibrance (Palbociclib), Ibrutinib, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), Imatinib Mesylate, Imbruvica (Ibrutinib), Inlyta (Axitinib), Iressa (Gefitinib), Irinotecan Hydrochloride, Istodax (Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Keoxifene (Raloxifene Hydrochloride), Kyprolis (Carfilzoniib), Lanreotide Acetate, Lapatinib Ditosylate, Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), Lomustine, Lupron (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megace (Megestrol Acetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide), Nexavar (Sorafenib Tosylate), Nilotinib, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Olaparib, Omacetaxine Mepesuccinate, Ontak (Denileukin Diftitox), Oxaliplatin, Paclitaxel, Palbociclib, Palonosetron Hydrochloride, Pamidronate Disodium, Panobinostat, Parapiat (Carboplatin), Paraplatin (Carbopiatin), Pazopanib Hydrochloride, Pemetrexed Disodium, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone, Procarbazine Hydrochloride, Promacta (Eltrombopag Olamine), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Regorafenib, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Ruxolitinib Phosphate, Sorafenib Tosylate, Sprycel (Dasatinib), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Synovir (Thalidomide), Synribo (Omacetaxine Mepesuccinate), Tafinlar (Dabrafenib), Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thiotepa, Toposar (Etoposide), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Totect (Dexrazoxane Hydrochloride), Trametinib, Treanda (Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Vandetanib, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vinorelbine Tartrate, Vismodegib, Vorinostat, Votrient (Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Zaltrap (Ziv- Aflibercept), Zelboraf (Vemurafenib), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), Zytiga (Abiraterone Acetate), and combinations thereof. EXAMPLES

[0048] The following examples are illustrative and are not intended to limit the scope of the claimed subject matter.

[0049] Epidermal Growth Factor (EGF) and Blockade of Immune Checkpoints in Cancer Immunotherapy

[0050] Abstract

[0051] An exciting new approach to cancer therapy is the use of checkpoint blockade monoclonal antibodies that enhance the immune response to cancer, such as ipilimumab. (See Pardoll, Nature Reviews, Vol. 12, April 2012, pages 252-264, the content of which is incorporated herein by reference in its entirety). Using serum samples from melanoma patients, we found that elevated levels of the cytokine known as Epidermal Growth Factor (EGF) correlate with a clinical response to ipilimumab. In the laboratory, we found that the addition of EGF to lymphocytes results in increased expression by T regulatory cells Ow_gS) of the molecule known as cytotoxic T-lymphocyte antigen 4 (CTLA-4) which is the target for ipilimumab. Together, these findings suggest EGF increases expression of CTLA-4 by Tn_¾s. Thus, increased expression of CTLA-4 may cause ipilimumab to bind in greater amounts to the TngS, thereby enhancing the efficacy of checkpoint blockade therapy.

[0052] This discovery could have a significant impact on cancer immunotherapy in a number of ways. First, elevated levels of EGF could be used as a biomarker to indicate the likelihood that a given cancer patient will respond to ipilimumab or other checkpoint blockade antibodies. These antibodies have significant toxicities and are very expensive, so predicting whether or not a patient is likely to respond could be very valuable. Second, the ability to induce increased expression of CTLA-4 by T_regs could enhance sensitivity to ipilimumab. This could be done by signaling through the EGF receptor that is expressed by the T«gS. Thus, treatment with EGF or another agonist such as an agonistic anti-EGF receptor antibody could be used to improve response to checkpoint blockade therapy. [0053] Introduction

[0054] The immune system is under precise control which allows it to respond to infection while avoiding damage to normal tissues (autoimmunity). The process by which the immune system is suppressed has been defined in more detail in the past few years. T_regs play a major role in suppressing the immune response. The molecular pathways involved in suppressing the immune response are known broadly as immune checkpoints that signal T effector cells to turn off, thereby avoiding autoimmunity. In cancer, such checkpoints are often turned on by the cancer. This limits the ability of the immune system to respond to cancer. An exciting and promising new approach to cancer therapy is use of monoclonal antibodies that block these checkpoints and so enhance the immune response to cancer. Ipilimumab (anti- CTLA-4) is one such checkpoint blockade antibody that has been approved by the FDA for treatment of melanoma and is being tested in many different cancers.

[0055] CTLA-4 is required for T_reg function (Wing et al, Science 322( 10): 271. 2008, the content of which is incorporated herein by reference in its entirety) and ant i -CTLA-4 can eliminate intratumoral Τ_regS (Simpson et al, JEM 210(9): 1695. 2013, the content of which is incorporated herein by reference in its entirety). Thus, anti-CTLA-4 therapy may also work by inducing lysis of T_regs that express the CTLA-4 antigen. Antibodies that block other checkpoints including PD1 are showing considerable clinical promise as well. (See Pardoll, Nature Reviews, Vol. 12, April 2012, pages 252-264 the content of which is incorporated herein by reference in its entirety). These antibodies work in some patients, but not others.

[0056] Two important unmet areas of need related to checkpoint blockade therapy include the need to predict which patients will respond to checkpoint blockade, and the ability to enhance sensitivity to checkpoint blockade so more cancer patients respond to therapy. We have been working collaboratively to explore these unmet needs. [0057] Results and Discussion

[0058] Using serum samples obtained from melanoma patients before they received ipilimumab, we found that elevated levels of the cytokine known as Epidermal Growth Factor (EGF) correlate with a clinical response to ipilimumab. The results are presented in Table I .

[0059] Table 1

P-value of EGF levels in responders versus non-responders - 0.0042

[0060] Having identified EGF as a marker for responsiveness to ipilimumab therapy, we tested whether the addition of EGF to lymphocytes could increase expression by T_regS of CTLA- 4 which is the target for ipilimumab. The results are presented in Figure 1.

[0061] As shown in Figure 1, the addition of EGF increased expression of CTLA4 by cells having the T_reg phenotype (i.e., CD3'CD4'CD127^lowFoxp3' cells). We also observed that EGF had no detectable effect on CD4 cells that did not have the T_reg phenotype. Together, these findings suggest EGF can increase expression of CTLA4 by T_regs. This increased expression of CTLA4 could allow ipilimumab to bind in greater amounts to the T_reg , thereby enhancing the efficacy of ipilimumab-based checkpoint blockade therapy.

[0062] This discovery could have a significant impact on cancer immunotherapy in two ways. Elevated levels of EGF could be used as a biomarker to indicate the likelihood that a given cancer patient will respond to ipilimumab or other checkpoint blockade antibodies. These antibodies have significant toxicities and are very expensive, so predicting whether or not a patient is likely to respond could be very valuable. The ability to induce increased expression of CTLA4 by T^s could enhance sensitivity to ipilimumab. This could be done by signaling through the EGF receptor that is expressed by the T^s. Thus, treatment with EGF or another agonist such as an agonistic anti-EGF receptor antibody could be used to improve response to checkpoint blockade therapy.

[0063] In the foregoing description, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0064] Citations to a number of references are made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

Claims

CLAIMS We claim:

1. A method comprising:

(a) detecting epidermal growth factor (EGF) in a biological sample from a patient; and

(b) administering to the patient a therapeutic antibody that binds to a cell surface receptor of a regulatory T cell (Treg).

2. The method of claim 1 , wherein the biological sample is serum and the detected EGF is detected at a concentration of greater than about 0.085 ng EGF /ml in the serum by performing a diagnostic immunoassay for EGF.

3. The method of any of the foregoing claims, wherein the cell surface receptor is an inhibitor receptor and the therapeutic antibody is an antagonist for the cell surface receptor.

4. The method of any of the foregoing claims, wherein the therapeutic antibody binds to cytotoxic T-lymphocyte antigen 4 (CTLA-4).

5. The method of claim 4, wherein the therapeutic antibody is ipilimumab or tremelimumab.

6. The method of any of claims I -3, wherein the therapeutic antibody binds to programmed cell death protein 1 (PD1).

7. The method of claim 6, wherein the therapeutic antibody is pembrolizumab or nivolumab.

8. The method of any of the foregoing claims, wherein the patient has a cancer or hyperproliferative disorder selected from the group consisting of adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, including cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, prostate, skin, testis, thymus, and uterus.

9. A method comprising administering to a patient:

(a) a pharmaceutical composition comprising EGF or an agonist for the EGF receptor; and

(b) a pharmaceutical composition comprising a therapeutic antibody that binds to a cell surface receptor of a regulatory T cell (T_reg).

10. The method of claim 9, wherein the method comprises administering a pharmaceutical composition comprising EGF or an agonist for the EGF receptor at a dose that achieves a concentration of greater than about 0.08S ng EGF /ml in the serum of the patient.

1 1. The method of claim 9 or 10, wherein the cell surface receptor is an inhibitory receptor and the therapeutic antibody is an antagonist for the cell surface receptor.

12. The method of any of claims 9-1 1 , wherein the therapeutic antibody binds to cytotoxic T-lymphocyte antigen 4 (CTLA-4).

13. The method of claim 12, wherein the therapeutic antibody is ipilimumab or tremelimumab.

14. The method of any of claims 9-1 1 , wherein the therapeutic antibody binds to programmed cell death protein 1 (PD1).

15. The method of claim 14, wherein the therapeutic antibody is pembrolizumab or nivolumab.

16. The method of any of claims 9- IS, wherein the patient has a cancer or hyperproliferative disorder selected from the group consisting of adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, including cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, prostate, skin, testis, thymus, and uterus.

17. A kit or system comprising:

(a) a diagnostic antibody for detecting EGF; and

(b) a therapeutic antibody that binds to a cell surface receptor of a T_reg.

18. The kit or system of claim 17, wherein the cell surface receptor is an inhibitory receptor and the therapeutic antibody is an antagonist for the cell surface receptor.

19. The kit or system of claim 17 or 18, wherein the therapeutic antibody binds to cytotoxic T-lymphocyte antigen 4 (CTLA-4).

20. The kit or system of claim 19, wherein the therapeutic antibody is ipilimumab or tremelimumab.

21. The kit or system of claim 17 or 18, wherein the therapeutic antibody binds to programmed cell death protein 1 (PD1).

22. The kit or system of claim 21 , wherein the therapeutic antibody is pembrolizumab or nivolumab.

23. A kit or system comprising:

(a) a pharmaceutical agent comprising EGF or an agonist for the EGF receptor; and

(b) a pharmaceutical agent comprising a therapeutic antibody that binds to a cell surface receptor of a T_reg.

24. The kit or system of claim 23, wherein the cell surface receptor is an inhibitory receptor and the therapeutic antibody is an antagonist for the cell surface receptor.

25. The kit or system of claim 23 or 24, wherein the therapeutic antibody binds to cytotoxic T-lymphocyte antigen 4 (CTLA-4).

26. The kit or system of claim 25, wherein the therapeutic antibody is ipilimumab or tremelimumab.

27. The kit or system of claim 23 or 24, wherein the therapeutic antibody binds to programmed cell death protein 1 (PD1 ).

28. The kit or system of claim 27, wherein the therapeutic antibody is pembrolizumab or nivolumab.