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  • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
  • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
• • G—PHYSICS
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  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • A—HUMAN NECESSITIES
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• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70532—B7 molecules, e.g. CD80, CD86
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/26—Infectious diseases, e.g. generalised sepsis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present disclosure provides novel macrocyclic peptides which inhibit the PD- 1/PD-Ll and CD80/PD-L1 protein/protein interaction, and are thus useful for the amelioration of various diseases, including cancer and infectious diseases.
• PD-L1 B7-H1
• PD-L2 b7- DC
• the activation of T cells expressing PD-1 has been shown to be downregulated upon interaction with cells expressing PD-L1 or PD-L2 (Freeman et al, J. Exp. Med., 192: 1027-1034 (2000); Latchman et al., Nat. Immunol, 2:261-268 (2001); Carter et al, Eur. J. Immunol, 32:634-643 (2002)).
• Both PD-L1 and PD-L2 are B7 protein family members that bind to PD-1, but do not bind to other CD28 family members.
• T cells When PD-1 expressing T cells contact cells expressing its ligands, functional activities in response to antigenic stimuli, including proliferation, cytokine secretion, and cytotoxicity, are reduced.
• PD-1/PD-L1 or PD-L2 interactions down regulate immune responses during resolution of an infection or tumor, or during the development of self tolerance (Keir, M.E. et al, Annu. Rev. Immunol, 26:Epub (2008)).
• Chronic antigen stimulation such as that which occurs during tumor disease or chronic infections, results in T cells that express elevated levels of PD-1 and are dysfunctional with respect to activity towards the chronic antigen (reviewed in Kim et al., Curr. Opin. Imm. (2010)). This is termed "T cell exhaustion”.
• B cells also display PD-l/PD-ligand suppression and "exhaustion”.
• A is selected from a bond
• R d is hydrogen or methyl, or, R d and R 4 , together with the atoms to which they are attached, can form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, hydroxy, and phenyl;
• R k is methyl or, R k and R 11 , together with the atoms to which they are attached, form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one or two groups independently selected from amino, cyano, methyl, halo, and hydroxy.
• R d is selected from hydrogen and methyl, or, R d and R 4 , together with the atoms to which they are attached, form a ring selected from azetidine, pyrollidine, morpholine, piperidine, piperazine, and tetrahydrothiazole; wherein each ring is optionally substituted with one to four groups independently selected from amino, cyano, methyl, halo, halomethyl, and hydroxy;
• the present disclosure provides a method blocking the interaction of PD-L1 with PD-1 and/or CD80 in a subject, said method comprising administering to the subject a therapeutically effective amount of at least one macrocyclic peptide described herein.
• R 4 is not part of a ring
• preferred R 4 side chains are: valine, alanine, isoleucine, and glycine.
• R 10 side chains are: tryptophan,
• R 11 side chains are tyrosine
• X aa i 5 is a naturally or nonnaturally occurring amino acid or a spacer followed by a tag or a spacer followed by a solubilizing or a PK-enhancing element.
• the subject matter described herein is directed to a polypeptide comprising a sequence of Formula 1(b):
• A is an electrophilic moiety such as a Michael acceptor or a chloro- or bromoacetyl group which is capable of reacting with a sulfhydryl group present on residue X aa i 4 to form a covalent thioether bond, thereby yielding a macrocyclic peptide; wherein such thioether bond may or may not be oxidized to the corresponding diastereomeric sulfoxides;
• X aa 2 is a naturally or nonnaturally occurring amino acid selected from the group consisting of L-Ala, L- m Ala, m Gly, L- m Val;
• X aa 4 is a naturally or nonnaturally occurring amino acid comprising L-Pro, L-Ala,
• the subject matter described herein is directed to a polypeptide comprising a sequence of Formula 1(c):
• X aa i2 is a naturally or nonnaturally occurring amino acid comprising L- m Nle,
• X aa 8 is a naturally or nonnaturally occurring aromatic or heteroaromatic or arylalkyl or heteroarylalkyl amino acid or an alkyl amino acid;
• X aa i2 is a naturally or nonnaturally occurring aromatic or heteroaromatic or arylalkyl or heteroarylalkyl or alkyl or heteroalkyl amino acid;
• A is an electrophilic moiety such as a Michael acceptor or a chloro- or bromoacetyl group which is capable of reacting with a sulfhydryl group present on residue X aa i3 to form a covalent thioether bond, thereby yielding a macrocyclic peptide; wherein such thioether bond may or may not be oxidized to the corresponding diastereomeric sulfoxides;
• A can be a Gly or other spacer with a free amino terminus, which can be used to cyclize the peptide via amide bond formation with the C-terminal a-carboxyl group of X aa i 4 , thereby providing a head-to-tail cyclic peptide; and wherein, if A is not present, the N-terminal amino group of the X aa i amino acid can be used to cyclize the peptide via amide bond formation with the C-terminal a-carboxyl group of X aa i4, thereby providing a head-to-tail cyclic peptide;
• X aa 8 is a naturally or nonnaturally occurring positively charged amino acid
• Xaai i is a naturally or nonnaturally occurring aromatic or heteroaromatic or arylalkyl or heteroarylalkyl or heteroalkyl or a positively charged amino acid;
• Xaal is selected from Phe and D-Phe;
• Ri and R2 is an alkyl or arylalkyl group
• X aa 2 is selected from the group consisting of L-Ala, L- m Ala, m Gly;
• X aa 9 is selected from the group consisting of L-Ala, L-Arg and L-Ser;
• Xaai i is selected from the group consisting of L-Nle, L- m Nle, L- m Ala, L-Phe, L- m Phe and L- m Leu and L- m Ser;
• X aa i4 is selected from the group consisting of L-Cys and D-Cys;
• A is a chloroacetyl group attached to the a-amine of the N-terminal Xaai residue which is capable of reacting with a sulfhydryl group present on the Cys 13 residue to form a covalent thioether bond, thereby yielding a macrocyclic peptide; wherein such thioether bond may or may not be oxidized to the corresponding diastereomeric sulfoxides;
• X aa 8 is selected from the group consisting of L-Ala and L-Tyr;
• X aa 9 is selected from L-Leu, L-Phe, L-Tyr and L-Val;
• the present disclosure is also directed to macrocyclic peptides comprising a sequence provided in Formula 1(d).
• the present disclosure is also directed to macrocyclic peptides comprising a sequence provided in Formula IV.
• the present disclosure is also directed to a method of modulating an immune response in a subject comprising administering to the subject one or more macrocyclic peptides comprising the sequence selected from the peptides described herein.
• Figures lOA-C - Anti-PD-Ll antibody and peptides both promote IFNy secretion by CMV-specific T cells in a dose-dependent manner.
• the most robust response was generated by the antibody directed to PD-Ll (MDX-1 105) (Anti-PD-1 Ab#l, EC 50 0.6nM; Figure 10A) followed by Compound No. 71 (EC 50 300nM; Figure 10B),
• Ci-C 3 alkoxy refers to aCi-C 3 alkyl group attached to the parent molecular moiety through an oxygen atom.
• Ci-C 3 alkoxyCi-C 3 alkyl refers to a Ci-C 3 alkoxy group attached to the parent molecular moiety through a Ci-C 3 alkyl group.
• Ci-C 6 alkoxycarbonylCi-C 3 alkyl refers to a
• Ci-C 4 alkyl refers to a group derived from a straight or branched chain saturated hydrocarbon containing from one to four carbon atoms.
• Ci-C 3 alkylsulfonyl refers to a Ci-C 3 alkyl group attached to the parent molecular moiety through a sulfonyl group.
• Ci-C 3 alkylsulfonylamino refers to a
• aminoCi-Csalkyl refers to an amino group attached to the parent molecular moiety through a Ci-C 3 alkyl group.
• the term as used herein refers to a furanyl group attached to the parent molecular moiety through a Ci-C 3 alkyl group.
• the furanyl group can be attached to the alkyl moiety through any substitutable atom in the group.
• halomethyl refers to a methyl group substituted with one, two, or three halogen atoms.
• hydroxy refers to -OH.
• NR c R d carbonylCi-C 3 alkyl refers to an NR c R d carbonyl group attached to the parent molecular moiety through a Ci-C 3 alkyl group.
• arylalkyl refers, without limitation, to alkyl groups as defined above having an aryl substituent, such as benzyl, phenethyl or naphthylpropyl, wherein said aryl and/or alkyl groups may optionally be substituted as defined above.
• heterocyclic groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl, piperidinyl, piperazinyl, oxopyrrolidinyl, oxopiperazinyl, oxopiperidinyl and oxadiazolyl.
• a heterocyclo group may be substituted with one or more functional groups, such as those described for "alkyl” or "aryl".
• heteroarylalkyl refers, without limitation, to alkyl groups as defined above having a heteroaryl substituent, wherein said heteroaryl and/or alkyl groups may optionally be substituted as defined above.
• the "inhibitory concentration" of PD- 1/PD-L1 inhibitor is intended to mean the concentration at which a compound screened in an assay of the disclosure inhibits a measurable percentage of the interaction of PD- 1 with PD-Ll .
• Examples of “inhibitory concentration” values range from IC50 to IC90, and are preferably, IC50, ICeo, IC70, ICso, or IC 90 , which represent 50%, 60%, 70%, 80% or 90% reduction in PD- 1/PD-L1 binding activity, respectively. More preferably, the "inhibitory concentration" is measured as the IC5 0 value. It is understood that another designation for IC5 0 is the half-maximal inhibitory concentration.
• Binding of the macrocyclic peptides to PD-Ll can be measured, for example, by methods such as homogeneous time-resolved fluorescence (HTRF), Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC), nuclear magnetic resonance spectroscopy (NMR), and the like. Further, binding of the macrocyclic peptides to PD-Ll expressed on the surface of cells can be measured as described herein in cellular binding assays.
• HTRF homogeneous time-resolved fluorescence
• SPR Surface Plasmon Resonance
• ITC isothermal titration calorimetry
• NMR nuclear magnetic resonance spectroscopy
• Administration of a therapeutic agent described herein includes, without limitation, administration of a therapeutically effective amount of therapeutic agent.
• therapeutically effective amount refers, without limitation, to an amount of a therapeutic agent to treat or prevent a condition treatable by administration of a composition of the PD- 1/PD-L1 binding inhibitors described herein. That amount is the amount sufficient to exhibit a detectable therapeutic or preventative or ameliorative effect. The effect may include, for example and without limitation, treatment or prevention of the conditions listed herein.
• the precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance.
• cytotoxic T lymphocyte-associated antigen-4 "CTLA-4", “CTLA4", “CTLA-4 antigen” and "CD 152"
• CTLA-4 cytotoxic T lymphocyte-associated antigen-4
• CTLA-4 CTL-4
• CTLA4 CTL-4 antigen
• CD 152 CD 152
• CTLA-4 nucleic acid sequence can be found under GENBANK® Accession No. LI 5006.
• immune response refers to the action of, for example, lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including macrocyclic peptides, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
• treatment refers to administering an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a condition (e.g., a disease), the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a disease, or otherwise arrest or inhibit further development of the disease, condition, or disorder in a statistically significant manner.
• a condition e.g., a disease
• An “adverse event” (AE) as used herein is any unfavorable and generally unintended, even undesirable, sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medical treatment.
• hyperproliferative disease refers to conditions wherein cell growth is increased over normal levels.
• hyperproliferative diseases or disorders include malignant diseases (e.g., esophageal cancer, colon cancer, biliary cancer) and non-malignant diseases (e.g., atherosclerosis, benign hyperplasia, and benign prostatic hypertrophy).
• the present disclosure is also directed to macrocyclic peptides that are capable of competing with the binding of a reference anti-PD-Ll antibody (MDX-1105) by 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%, and at least about 100%.
• Such macrocyclic peptides may share structural homology with one or more macrocyclic peptides disclosed herein, including mutant, conservative substitution, functional substitution, and deletion forms, provided they specific bind to PD-Ll .
• a macrocyclic peptide binds substantially to the same region of PD-Ll as a reference anti- PD-Ll antibody
• the macrocyclic peptide should bind to an epitope of PD-Ll that at least overlaps with the PD-Ll epitope that the anti-PD-Ll monoclonal antibody binds to.
• the overlapping region can range from one amino acid residue to several hundred amino acid residues.
• the macrocyclic peptide should then compete with and/or block the binding of the anti-PD-Ll monoclonal antibody to PD-Ll and thereby decrease the binding of the anti-PD-Ll monoclonal antibody to PD-Ll, preferably by at least about 50% in a competition assay. (See Figure 9).
• Anti-PD-Ll antibodies that may be used as reference antibodies for competition assay purposes are known in the art.
• the following representative anti-PD- Ll antibodies may be used: MDX-1 105 (BMS); L01X-C (Serono), L1X3 (Serono), MSB- 0010718C (Serono), and PD-Ll Probody (CytomX), and the PD-Ll antibodies disclosed in co-owned WO 2007/005874.
• a macrocyclic peptide of the disclosure comprises amino acid sequences that are homologous to the amino acid sequences of the macrocyclic peptides described herein, and wherein the macrocyclic peptides retain the desired functional and/or biological properties of the macrocyclic peptide of the disclosure.
• the disclosure provides a macrocyclic peptide, or antigen-binding portion thereof, comprising: an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the compounds described herein; and the macrocyclic peptide exhibits one or more of the following properties:
• the macrocyclic peptide does not substantially bind to human CD28, CTLA-4 or ICOS;
• Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
• nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
• nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
• hyperproliferative disease such as cancer as described herein
• anti-viral disease such as cancer as described herein
• Blockade of PD- 1 by macrocyclic peptides can enhance the immune response to cancerous cells in the patient.
• the ligand for PD-1, PD-L1 is not expressed in normal human cells, but is abundant in a variety of human cancers (Dong et al, Nat. Med., 8:787- 789 (2002)).
• the interaction between PD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and immune evasion by the cancerous cells (Dong et al, J. Mol. Med., 81 :281-287 (2003); Blank et al, Cancer Immunol.
• the disclosure provides a method of inhibiting growth of tumor cells in a subject, comprising administering to the subject a
• immunogenic agent such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune stimulating cytokines (He et al, J. Immunol, 173 :4919-
• PD-L1 blockade may also be combined with standard cancer treatments. PD-L1 blockade may be effectively combined with chemotherapeutic regimes. In these instances, it may be possible to reduce the dose of chemotherapeutic reagent administered (Mokyr, M. et al, Cancer Res., 58:5301-5304 (1998)). An example of such a
• PD-L1 blocking macrocyclic peptides can also be used in combination with bispecific macrocyclic peptides that target Fc alpha or Fc gamma receptor-expressing effectors cells to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243).
• Bispecific macrocyclic peptides can be used to target two separate antigens.
• anti-Fc receptor/anti tumor antigen e.g., Her-2/neu
• bispecific macrocyclic peptides have been used to target macrophages to sites of tumor. This targeting may more effectively activate tumor specific responses.
• the T cell arm of these responses would be augmented by the use of PD-Ll blockade.
• antigen may be delivered directly to DCs by the use of bispecific macrocyclic peptides which bind to tumor antigen and a dendritic cell specific cell surface marker.
• Tumors evade host immune surveillance by a large variety of mechanisms. Many of these mechanisms may be overcome by the inactivation of proteins which are expressed by the tumors and which are immunosuppressive. These include among others TGF-beta (Kehrl, J. et al, J. Exp. Med., 163: 1037-1050 (1986)), IL-10 (Howard, M. et al, Immunology Today, 13: 198-200 (1992)), and Fas ligand (Hahne, M. et al, Science, 274: 1363-1365 (1996)). Macrocyclic peptides to each of these entities may be used in combination with anti-PD-Ll to counteract the effects of the immunosuppressive agent and favor tumor immune responses by the host.
• macrocyclic peptides which may be used to activate host immune responsiveness can be used in combination with anti-PD-Ll. These include molecules on the surface of dendritic cells which activate DC function and antigen presentation. Anti- CD40 macrocyclic peptides are able to substitute effectively for T cell helper activity (Ridge, J. et al, Nature, 393:474-478 (1998)) and can be used in conjunction with PD-1 antibodies (Ito, N. et al, Immunobiology , 201(5):527-540 (2000)). Activating macrocyclic peptides to T cell costimulatory molecules such as CTLA-4 (e.g., U.S. Patent No. 5,811,097), OX-40 (Weinberg, A.
• CTLA-4 e.g., U.S. Patent No. 5,811,097
• OX-40 Weinberg, A.
• Bone marrow transplantation is currently being used to treat a variety of tumors of hematopoietic origin. While graft versus host disease is a consequence of this treatment, therapeutic benefit may be obtained from graft vs. tumor responses.
• PD-Ll blockade can be used to increase the effectiveness of the donor engrafted tumor specific T cells.
• another aspect of the disclosure provides a method of treating an infectious disease in a subject comprising administering to the subject a macrocyclic peptide of the present disclosure, or antigen-binding portion thereof, such that the subject is treated for the infectious disease.
• the antibody is a human anti-human PD-L1 macrocyclic peptide (such as any of the macrocyclic peptides described herein). Additionally or alternatively, the antibody can be a chimeric or humanized antibody.
• antibody mediated PD-L1 blockade can be used alone, or as an adjuvant, in combination with vaccines, to stimulate the immune response to pathogens, toxins, and self-antigens.
• pathogens for which this therapeutic approach may be particularly useful include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to HIV, Hepatitis (A, B, and C), Influenza, Herpes, Giardia, Malaria (Butler, N.S. et al, Nature Immunology, 13: 188-195 (2012); Hafalla, J.C.R., et al, P LOS Pathogens (February 2, 2012)),
• PD-L1 blockade is particularly useful against established infections by agents such as HIV that present altered antigens over the course of the infections. These novel epitopes are recognized as foreign at the time of anti-human PD-L1 administration, thus provoking a strong T cell response that is not dampened by negative signals through PD-L1.
• pathogenic bacteria causing infections treatable by methods of the disclosure include chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria.
• pathogenic fungi causing infections treatable by methods of the disclosure include Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.
• pathogenic parasites causing infections treatable by methods of the disclosure include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,
• the macrocyclic peptides may provoke and amplify autoimmune responses.
• Trp-2 vaccinated mice (Overwijk, W. et al, Proc. Natl. Acad. Sci. USA, 96:2982-2987 (1999)); autoimmune prostatitis evoked by TRAMP tumor cell vaccines (Hurwitz, A., supra (2000)), melanoma peptide antigen vaccination and vitiligo observed in human clinical trials (Rosenberg, S.A. et al, J. Immunother. Emphasis Tumor
• Alzheimer's disease involves inappropriate accumulation of A.beta. peptide in amyloid deposits in the brain; antibody responses against amyloid are able to clear these amyloid deposits (Schenk et al, Nature, 400: 173-177 (1999)).
• Neutralizing antibody responses to reproductive hormones may be used for contraception.
• Neutralizing antibody response to hormones and other soluble factors that are required for the growth of particular tumors may also be considered as possible vaccination targets.
• cytokines such as TNF. alpha.
• compositions e.g., macrocyclic peptides, multispecific and bispecific molecules and immunoconjugates
• the compositions can be administered by injection (e.g., intravenous or subcutaneous).
• Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the composition.
• Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, decarbazine and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient.
• anti-neoplastic agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, decarbazine and cyclophosphamide hydroxyurea which, by themselves, are only effective at levels which are toxic or subtoxic to a patient.
• Cisplatin is intravenously administered as a 100 mg/dose once every four weeks and adriamycin is intravenously administered as a 60-75 mg/ml dose once every 21 days.
• Co-administration of the macrocyclic peptides, or antigen binding fragments thereof, of the present disclosure with chemotherapeutic agents provides two anti-cancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the peptides.
• kits can further contain at least one additional reagent, or one or more additional macrocyclic peptides of the disclosure (e.g., a human antibody having a complementary activity which binds to an epitope in PD-L1 antigen distinct from the macrocycle).
• Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
• a vaccine is prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccination (Dranoff et al, Proc. Natl. Acad. Sci. USA, 90:3539-3543 (1993)).
• tumor specific antigens are differentiation antigens expressed in the tumors and in the cell from which the tumor arose, for example melanocyte antigens gplOO, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be the targets of tumor specific T cells found in the host.
• Another example is a combination of a macrocyclic peptide and anti-CTLA-4 agent further in combination with interleukin-2 (IL-2) for the treatment of melanoma.
• IL-2 interleukin-2
• the scientific rationale behind the combined use of PD-L1 macrocyclic peptide and CTLA-4 blockade with chemotherapy is that cell death, which is a consequence of the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway.
• Other combination therapies that may result in synergy with a combined anti-PD-Ll macrocyclic peptide and CTLA-4 blockade through cell death include radiation, surgery, or hormone deprivation. Each of these protocols creates a source of tumor antigen in the host.
• Angiogenesis inhibitors may also be combined with a combined PD-L1 and CTLA- 4 blockade. Inhibition of angiogenesis leads to tumor cell death, which may also be a source of tumor antigen to be fed into host anti
• antigen may be delivered directly to DCs by the use of bispecific macrocyclic peptides which bind to tumor antigen and a dendritic cell specific cell surface marker.
• Tumors evade host immune surveillance by a large variety of mechanisms. Many of these mechanisms may be overcome by the inactivation of proteins, which are expressed by the tumors and which are immunosuppressive. These include, among others, TGF-.beta. (Kehrl, J. et al, J. Exp. Med., 163: 1037-1050 (1986)), IL-10 (Howard, M. et al, Immunology Today, 13 : 198-200 (1992)), and Fas ligand (Hahne, M. et al,
• agents that may be used to activate host immune responsiveness can be further used in combination with a macrocyclic peptide of the present disclosure.
• macrocyclic peptide of the present disclosure include molecules on the surface of dendritic cells that activate DC function and antigen presentation.
• Anti-CD40 macrocyclic peptides are able to substitute effectively for T cell helper activity (Ridge, J. et al, Nature, 393 :474-478 (1998)) and can be used in conjunction with the macrocyclic peptides of the present disclosure, either alone or in combination with an anti-CTLA-4 combination (Ito, N. et al, Immunobiology ,
• T cell costimulatory molecules such as OX-40 (Weinberg, A. et al, Immunol, 164:2160-2169 (2000)), 4- IBB (Melero, I. et al, Nat. Med., 3:682-685 (1997), and ICOS (Hutloff, A. et al, Nature, 397:262-266 (1999)) may also provide for increased levels of T cell activation.
• steroids have been administered to treat inflammatory bowel disease (IBD) and prevent exacerbations of IBD, they have not been used to prevent (decrease the incidence of) IBD in patients who have not been diagnosed with IBD.
• IBD inflammatory bowel disease
• a macrocyclic peptide of the present disclosure can be further combined with the use of any non-absorbable steroid.
• a "non-absorbable steroid" is a non-absorbable steroid
• the daily oral dosage of the active ingredient when used for the indicated effects, will range between about 0.001 to 1000 mg/kg of body weight, preferably between about 0.01 to 100 mg/kg of body weight per day, and most preferably between about 0.6 to 20 mg/kg/day.
• the daily dosage of the active ingredient when used for the indicated effects will range between O.OOlng to 100.0 ng per min/per Kg of body weight during a constant rate infusion.
• Such constant intravenous infusion can be preferably administered at a rate of 0.01 ng to 50 ng per min per Kg body weight and most preferably at 0.01 ng to 10.0 mg per min per Kg body weight.
• compositions are typically administered in a mixture with suitable pharmaceutical diluents, excipients, or carriers (collectively referred to herein as pharmaceutical carriers) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, aerosol sprays generated with or without propellant and syrups, and consistent with conventional pharmaceutical practices.
• suitable pharmaceutical diluents, excipients, or carriers suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, aerosol sprays generated with or without propellant and syrups, and consistent with conventional pharmaceutical practices.
• the compounds described herein may be delivered in prodrug form.
• the subject matter described herein is intended to cover prodrugs of the presently claimed compounds, methods of delivering the same, and compositions containing the same.
• a mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil may be prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient.
• the capsules should be washed and dried.
• Tablets may be prepared by conventional procedures so that the dosage unit, for example is 100 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 1 1 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.
• Ph phenyl
• Prelude Method A All manipulations were performed under automation on a Prelude peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a 10 or 45 mL polypropylene tube fitted with a bottom frit. The tube connects to the Prelude peptide synthesizer through both the bottom and the top of the tube. DMF and DCM can be added through the top of the tube, which washes down the sides of the tube equally. The remaining reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube.
• Period agitation describes a brief pulse of 2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 30 seconds. Amino acid solutions were generally not used beyond three weeks from preparation.
• DMF dimethylformamide
• DIC ⁇ , ⁇ '-diisopropylcarbodiimide
• HOAt 1 -hydroxy 7-azabenzotriazole
• Sieber Fmoc-amino-xanthen-3-yloxy, where "3-yloxy” describes the position and type of connectivity to the polystyrene resin.
• Coupling of Fmoc-N-methyl amino acids and coupling to a secondary amine N-terminus used the "Secondary amine-coupling procedure" described below. Coupling of chloroacetyl group to the N-terminus of the peptide is described by the "Chloroacetyl chloride coupling procedure” or “Chloroacetic acid coupling procedure” detailed below.
• Chloroacetic acid coupling procedure B To the reaction vessel containing the resin from the previous step was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 5.0 mL). The mixture was periodically agitated for 3 minutes and then the solution was drained through the frit. The resin was washed successively five times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• DMF 4.0 mL
• the mixture was shaken at room temperature for 12 to 18 hours, then the solution was drained through the frit.
• the resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was agitated for 90 seconds before the solution was drained through the frit.
• the resin was washed successively four times as follows: for each wash, CH2CI2 (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.
• the mixture was periodically agitated for 15 minutes, then the solution was drained through the frit.
• the resin was twice washed as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• DMF 4.0 mL
• DMF acetic anhydride
• DMF 10: 1 :89 v/v/v, 5.0 mL
• the mixture was periodically agitated for 15 minutes, then the solution was drained through the frit.
• the resin was washed successively two times as follows: for each wash, DMF (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.
• the resin was washed successively four times as follows: for each wash, DCM (4.0 mL) was added through the top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.
• the resulting resin was used directly in the next step.
• the mixture was shaken at room temperature for 12 to 18 hours, then the solution was drained through the frit.
• the resin was washed successively three times as follows: for each wash, DMF (4.0 mL) was added to top of the vessel and the resulting mixture was agitated for 90 seconds before the solution was drained through the frit.
• the resin was washed successively four times as follows: for each wash, CH2CI2 (4.0 mL) was added to top of the vessel and the resulting mixture was periodically agitated for 90 seconds before the solution was drained through the frit.
• Resin-swelling procedure To 50 niL polypropylene conical tube was added Merrifield:Sieber resin (140 mg, 0.100 mmol). Then DMF (7 niL) was added to the tube followed by DCM (7 niL). The resin was then transferred to the reaction vessel from top of the vessel. The procedure is repeated additionally two times. DMF (7 mL) was added followed by DCM (7 mL). The resin was allowed to swell with 2 bubbling from the bottom of the reaction vessel for 15 minutes before the solvent was drained through the frit.
• the mixture was mixed by N2 bubbling for 5 minutes at 75 °C for all amino acids (50 °C for Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH), followed by 6 hrs with no heating. After draining, the resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. To the reaction vessel was added a solution of acetic anhydride :DIEA: DMF (10: 1 :89 v/v/v, 5.0 mL). The mixture was periodically bubbled for 2 minutes at 65 °C, then the solution was drained. The resin was washed successively three times as follows: DMF (7 mL) wash from top, followed by DMF (7 mL) wash from bottom and finally with DMF (7 mL) wash from top. The resulting resin was used directly in the next step.
• Symphony Method A All manipulations were performed under automation on a Symphony peptide synthesizer (Protein Technologies). All procedures unless noted were performed in a Symphony polypropylene tube fitted with a bottom frit. The tube connects to a the Symphony peptide synthesizer through both the bottom and the top of the tube. All Solvents, DMF, DCM, amino acids and reagents are added through the bottom of the tube and pass up through the frit to contact the resin. All solutions are removed through the bottom of the tube. "Periodic agitation" describes a brief pulse of 2 gas through the bottom frit; the pulse lasts approximately 5 seconds and occurs every 15 seconds. Amino acid solutions were generally not used beyond three weeks from preparation. HATU solution were used within 5 days of preparation.
• DMF dimethylformamide
• HCTU 2- (6-Chloro-l-H-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium
• HATU 1- [Bis(dimethylamino)methylene]- 1H- 1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid
• the resin was washed three times as follows: to the reaction vessel was added
• the resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with 2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit.
• DMF dimethyl methacrylate
• To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit.
• the resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• the resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with 2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit.
• DMF dimethyl methacrylate
• To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit.
• the resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• the synthesis was paused by the Symphony software to add to the reaction vessel manually the custom amino acid (0.2M in DMF, 1.25 mL, 5 eq), then restart automation: to add HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq).
• the mixture was periodically agitated for 300 minutes, then the reaction solution was drained through the frit.
• the resin was washed six times as follows with DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• DMF dimethylformamide
• HCTU 2- (6-Chloro-l-H-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium
• HATU 1- [Bis(dimethylamino)methylene]- 1H- 1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid
• Sieber Fmoc-amino-xanthen-3-yloxy, where "3-yloxy” describes the position and type of connectivity to the polystyrene resin.
• the resin used is Merrifield polymer (polystyrene) with a Sieber linker (Fmoc -protected at nitrogen); 100- 200 mesh, 1% DVB, 0.71 mmol/g loading.
• Other common Acid sensitive resins can also be used in the synthesis such as Rink or functionalized Chloro trityl Resin. Common amino acids used are listed below with side-chain protecting groups indicated inside parenthesis.
• the resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with 2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit.
• DMF dimethyl methacrylate
• To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit.
• the resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• the resin was washed three times as follows: to the reaction vessel was added
• the resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with N2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit. To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit. The resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• the System was paused by the system for the manual addition of the custom amino acid to the reaction vessel (0.2M in DMF, 1.25 mL, 5 eq), then the automation was restarted to add to the reaction vesicle HATU (0.2M in DMF, 1.25 mL, 5 eq), and finally NMM (0.8M in DMF, 1.25 mL, 10 eq).
• the mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit.
• the resin was washed 6 times as follows: DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• the resin was washed three times as follows: to the reaction vessel was added DMF (2.5 mL) upon which the mixture was periodically agitated with 2 bubbling from the bottom of the reaction vessel for 30 seconds before the solvent was drained through the frit.
• DMF dimethyl methacrylate
• To the reaction vessel was added piperidine:DMF (20:80 v/v, 2.5 mL). The mixture was periodically agitated for 2.5 minutes and then the solution was drained through the frit.
• the resin was washed 6 times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• the mixture was periodically agitated for 15 minutes, then the reaction solution was drained through the frit.
• the resin was washed 6 times as follows: DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• AC2O/DIPEA/DMF v/v/v 1 : 1 :3 2.5 mL
• the mixture was periodically agitated for 10 minutes, then the reaction solution was drained through the frit.
• the resin was washed successively six times as follows: for each wash, DMF (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• the resin was washed successively four times as follows: for each wash, DCM (2.5 mL) was added through the bottom of the vessel and the resulting mixture was periodically agitated for 30 seconds before the solution was drained through the frit.
• the resulting resin was then dried with a stream of Nitrogen for 10 mins.

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