Cancer immunotherapy

Abstract

To provide methods, compounds, compositions and kits for the treatment and / or prevention of cancer.SOLUTION: In particular, methods for the treatment of cancer are provided comprising the administration of a TLR2 agonist, such as a conjugate of dipalmitoyl-S-glyceryl-cysteine (Pam2Cys) and polyethylene glycol (PEG), more particularly a Pam2Cys-Ser-PEG compound, and an immunostimulant such as an anti-PD-1, anti-PDL1, anti-PL1, or anti-CTLA-4 immunotherapeutic.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to Australian Provisional Patent Application No. 2019903262, filed September 4, 2019, and Australian Provisional Patent Application No. 2019904864, filed December 20, 2019. The entire contents of each of AU2019903262 and AU2019904864 are incorporated herein by reference.

[0002] [Field of the Invention] The present invention relates to methods, compounds, compositions and kits for the treatment and / or prevention. In one aspect, the present invention relates to the use of immunotherapy for the treatment and / or prevention of cancer.

[0003] [Background of the invention] Immunotherapy has shown promise in the treatment of cancer due to its ability to slow the growth and metastasis of cancer cells and by helping the immune system destroy existing cancer cells. Immunotherapy can help the immune system by boosting or strengthening the immune system through stimulation of antigen-presenting cells, T cells, or innate immune cells, by reducing immunosuppression in the tumor environment by modulating inhibitory pathways, and / or by promoting adaptive or innate immune responses.

[0004] An example of immunotherapy is checkpoint inhibitors. Checkpoint inhibitors currently approved by the U.S. Food and Drug Administration (FDA) target cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death receptor 1 (PD-1), or programmed death ligand 1 (PD-L1). These checkpoint inhibitors work by preventing immune evasion from cancer cells. The first approved drug, ipilimumab, received FDA marketing approval in 2011 for metastatic melanoma. Since ipilimumab, five additional checkpoint inhibitors have been approved for a total of 14 different indications.

[0005] Between 2015 and 2017, the number of clinical trials using PD-1 and PD-L1 inhibitors increased by nearly 600%. Although the number of indications for which checkpoint inhibitors have been administered has increased significantly in recent years, the increase in benefit from these drugs in terms of the percentage of patients responding has slowed. Clinical responses to PD-1 immunotherapy have been positively correlated with tumor PD-L1 expression, along with other predictive biomarkers such as pre-existing CD8+ T cell infiltration and mutational / neoantigen burden, in other clinical trials. However, some patients with tumors that do not express PD-L1 have been demonstrated to respond to PD-1 pathway blockade. In fact, recent analyses suggest that checkpoint inhibitors may result in responses in fewer than 13% of patients with various types of cancer in the United States.

[0006] Thus, there remains a need for improved immunotherapies for the treatment of cancer.

[0007] The reference to any prior art herein is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art could reasonably be expected to be understood or regarded as relevant and / or in combination with other pieces of prior art by a person skilled in the art.

[0008] [Summary of the Invention] The present invention provides a method for treating, preventing, or minimizing the progression of cancer in a subject, comprising administering to the subject a therapeutically effective amount of a TLR2 agonist and an immunostimulant, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0009] In any aspect of the invention, the TLR2 agonist can be any one of those described herein. Preferably, the TLR2 agonist is a compound defined by any one of formulas (I), (IA1), (IA2), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), and (XIX) (collectively referred to herein as formulas (I)-(XIX)).

[0010] In any aspect of the invention, the TLR2 agonist may be a compound comprising a moiety A selected from A1' and A2 as defined herein and polyethylene glycol (PEG), wherein moiety A and PEG are linked by a glycine, serine, homoserine, threonine, phosphoserine, asparagine, or glutamine residue, or an ester of a glutamine residue.

[0011] In any embodiment of the invention, the compound has the moiety A1Y' or A2Y': [ka] wherein R and R are independently selected from the group consisting of H, -CHOH, -CHCHOH, -CH(CH)OH, -CHOPO(OH), -CHC(=O)NH, -CHCHC(=O)OH, and -CHCHC(=O)OR, wherein any one of the alkyl hydrogens may be replaced with a halogen; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; b and w are each independently an integer from 0 to 7, and v is an integer from 0 to 5, with the proviso that: the sum of b, v, and w is at least 3; The sum of b and w is 0 to 7; Z1 and Z2 are each independently selected from the group consisting of -O-, -NR-, -S-, -S(=O)-, -S(=O)2-, -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; R in each case of b, v, w, and z 11 , R 12 , R x , R y , R 14 , R 15 , R 16 , and R 17 are each independently H or a C1-C6 aliphatic; R, R 13 and R 18 are each independently H or a C1-C6 aliphatic; R 19 is H, a C1-C6 aliphatic group, an amino protecting group, L3-C(=O)-, or A2; L1 and L2 are each independently C5 to C 21 Aliphatic or C4~C 20 is heteroaliphatic; L3 is C1~C 21 Aliphatic or C2-C 20 is heteroaliphatic; A2 is an amino acid or a peptide; where R, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R x , R y Any aliphatic or heteroaliphatic group present in any of L1, L2, and L3 is optionally substituted; A1Y' or A2Y' is covalently linked to polyethylene glycol (PEG). may comprise or consist of or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0012] In one embodiment, the TLR2 agonist is a compound selected from any one of compounds 001 to 010, A101 to A114, and A201 to A232.

[0013] As used herein, reference to a "compound of the invention" may refer to any of the following: Compounds of formula (I) to (XIX); a compound selected from compounds 001 to 010, A101 to A114, and A201 to A232; a compound comprising a moiety A selected from A1′ and A2 as defined herein and polyethylene glycol (PEG), wherein the moiety A and the PEG are linked by a glycine, serine, homoserine, threonine, phosphoserine, asparagine or glutamine residue, or an ester of a glutamine residue; and / or A compound comprising a moiety of formula (A1Y') or (A2Y') covalently linked by PEG.

[0014] In any embodiment of the present invention, the immunostimulant may be, but is not limited to: - cellular immunotherapy (cytotoxic cellular immunotherapy or adoptive cellular immunotherapy); -Oncolytic viruses; -Cancer vaccines; -T cell engager; bispecific T cell engagers; and -Checkpoint inhibitors The hydroxybenzoates may be selected from any one of those described herein, including the group consisting of:

[0015] In a preferred embodiment, the immunostimulatory agent used in accordance with any of the methods described herein is a checkpoint inhibitor.

[0016] In another aspect, the invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and a checkpoint inhibitor, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0017] In another aspect, the invention provides a method for treating, preventing, or minimizing the progression of cancer in a subject that has been or is being administered an immunostimulant, comprising administering to the subject a therapeutically effective amount of a TLR2 agonist, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0018] In another aspect, the invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject who has been or is being administered a checkpoint inhibitor, comprising administering to the subject a therapeutically effective amount of a TLR2 agonist, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0019] In any aspect of the present invention, the TLR2 agonist and the immunostimulant may be administered simultaneously. Alternatively, they may be administered sequentially. For example, the immunostimulant may be administered before the TLR2 agonist, or the TLR2 agonist may be administered before the immunostimulant. Alternatively, treatment with the immunostimulant and / or the TLR2 agonist may be staggered. In a preferred embodiment, the immunostimulant is a checkpoint inhibitor. In this aspect of the present invention, the TLR2 agonist may be administered once or twice a week, and the checkpoint inhibitor may be administered once every three weeks.

[0020] In another aspect, the invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, comprising: - identifying a subject with cancer who has been or is being administered an immunostimulatory agent for the treatment of the cancer; - assessing whether the subject responds to an immunostimulant; - if the subject is unresponsive to the immunostimulant, administering to the subject a therapeutically effective amount of a TLR2 agonist; Thereby, methods are provided that include treating, preventing, or minimizing the progression of cancer in a subject.

[0021] In a preferred embodiment, the immunostimulatory agent is a checkpoint inhibitor, preferably a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. More preferably, the checkpoint inhibitor is a PD-1, PD-L1, or CTLA-4 antibody.

[0022] In another aspect, the invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, comprising: - identifying a subject who has cancer and is unresponsive to a treatment comprising an immunostimulant; administering to the subject a therapeutically effective amount of a compound that comprises, consists of, or consists essentially of a TLR2 agonist; Thereby, methods are provided that include treating, preventing, or minimizing the progression of cancer in a subject.

[0023] In another aspect, the invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, comprising: - identifying a subject with cancer; and administering to the subject a therapeutically effective amount of a TLR2 agonist and an immune stimulant; Thereby, methods are provided that include treating, preventing, or minimizing the progression of cancer in a subject.

[0024] In another aspect, the invention provides a method of treating, preventing, or minimizing the progression of cancer in a subject, comprising: - identifying a subject with cancer; and administering to the subject a therapeutically effective amount of a TLR2 agonist and a checkpoint inhibitor; Thereby, methods are provided that include treating, preventing, or minimizing the progression of cancer in a subject.

[0025] In any aspect of the present invention, the TLR2 agonist may be administered in a composition. Additionally, in any aspect of the present invention, the immunostimulant may be administered in a composition. In a preferred embodiment, the checkpoint inhibitor may be administered in a composition. Typically, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0026] In any embodiment of the present invention, the composition may be formulated for intravenous administration to a subject. In other words, the composition is suitable for intravenous administration. In another embodiment of the present invention, the composition is formulated for administration to the respiratory tract, preferably by inhalation or intranasally. In another preferred embodiment, the composition is formulated as a nasal spray or nasal drops. In another embodiment, the TLR2 agonist is formulated for administration to the respiratory tract, preferably by inhalation, and the immunostimulant, preferably any checkpoint inhibitor described herein, is formulated for intraperitoneal or intravenous administration. In this embodiment, the immunostimulant and the checkpoint inhibitor may be administered simultaneously or at different times.

[0027] In one embodiment, the TLR2 agonist is administered in the form of a TLR2 composition, which may not contain compounds that are agonists of other TLRs. Preferably, the only TLR agonist present in the TLR2 composition is an agonist of a TLR2 homodimer or heterodimer. Preferably, the composition contains only one TLR2 agonist.

[0028] In either embodiment, the composition comprises, consists essentially of, or consists of a TLR2 agonist and an immunostimulant, preferably a checkpoint inhibitor, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0029] In another aspect, the present invention further provides a composition comprising, consisting essentially of, or consisting of a TLR2 agonist and an immunostimulant, preferably a checkpoint inhibitor, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0030] In another aspect, the present invention further provides a method of increasing the survival rate of a subject having cancer, comprising administering to the subject a therapeutically effective amount of a TLR2 agonist and an immunostimulant, thereby increasing the survival rate of the subject having cancer.

[0031] In another aspect, the present invention further provides a method of increasing survival rate of a subject having cancer, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and a checkpoint inhibitor, thereby increasing survival rate of the subject having cancer.

[0032] In another aspect, the present invention further provides a method for minimizing, reducing, or preventing tumor growth in a subject with cancer, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and an immunostimulant, thereby minimizing, reducing, or preventing tumor growth in the subject with cancer.

[0033] In another aspect, the present invention further provides a method for minimizing, reducing, or preventing tumor growth in a subject with cancer, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and a checkpoint inhibitor, thereby minimizing, reducing, or preventing tumor growth in the subject with cancer.

[0034] In another aspect, the present invention further provides a method of minimizing, reducing, or preventing metastasis in a subject having cancer, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and an immunostimulant, thereby minimizing, reducing, or preventing metastasis in the subject having cancer. In a preferred embodiment, the method minimizes, reduces, or prevents metastasis to the lung.

[0035] In another aspect, the present invention further provides a method of minimizing, reducing, or preventing metastasis in a subject having cancer, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and a checkpoint inhibitor, thereby minimizing, reducing, or preventing metastasis in the subject having cancer. In a preferred embodiment, the method minimizes, reduces, or prevents metastasis to the lung.

[0036] In any embodiment, the present invention further provides a method of minimizing, reducing, or preventing cancer in a subject, comprising: - Identifying subjects with tumors capable of metastasizing; and administering to the subject a therapeutically effective amount of a TLR2 agonist and an immune stimulant; Thereby, methods are provided that include minimizing, reducing, or preventing cancer in a subject.

[0037] In any embodiment, the present invention further provides a method of minimizing, reducing, or preventing cancer in a subject, comprising: - Identifying subjects with tumors capable of metastasizing; and administering to the subject a therapeutically effective amount of a TLR2 agonist and a checkpoint inhibitor; Thereby, methods are provided that include minimizing, reducing, or preventing cancer in a subject.

[0038] In any embodiment, the present invention further provides a method of minimizing, reducing, or preventing metastasis in a subject having cancer, comprising: - To identify subjects with primary tumors capable of metastasizing; - Removal of the primary tumor from the subject; and administering to the subject a therapeutically effective amount of a TLR2 agonist and an immune stimulant; Thereby, methods are provided that include minimizing, reducing, or preventing metastasis in a subject with cancer.

[0039] In any embodiment, the present invention further provides a method of minimizing, reducing, or preventing metastasis in a subject having cancer, comprising: - To identify subjects with primary tumors capable of metastasizing; - Removal of the primary tumor from the subject; and administering to the subject a therapeutically effective amount of a TLR2 agonist and a checkpoint inhibitor; Thereby, methods are provided that include minimizing, reducing, or preventing metastasis in a subject with cancer.

[0040] In another aspect, the invention further provides a method for minimizing, reducing, or preventing the growth of a tumor at at least one site distant from the site of the primary tumor in a subject, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and an immunostimulatory agent, thereby minimizing, reducing, or preventing the growth of a tumor at at least one site distant from the site of the primary tumor in the subject.

[0041] In another aspect, the invention further provides a method for minimizing, reducing, or preventing the growth of a tumor at at least one site distant from the site of the primary tumor in a subject, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and a checkpoint inhibitor, thereby minimizing, reducing, or preventing the growth of a tumor at at least one site distant from the site of the primary tumor in the subject.

[0042] In any aspect of the invention, the methods described herein further comprise identifying a subject with cancer. In one embodiment, the cancer can be pre-malignant or non-metastatic. In another embodiment, the cancer can be malignant or metastatic.

[0043] In another aspect, the present invention further provides the use of a compound comprising, consisting of, or consisting essentially of a TLR2 agonist and an immunostimulant in the preparation of a medicament for treating, preventing, or minimizing the progression of cancer in a subject.

[0044] In another aspect, the present invention further provides the use of a compound comprising, consisting of, or consisting essentially of a TLR2 agonist in the manufacture of a first medicament, and an immune stimulant in the preparation of a second medicament, wherein the first and second medicaments are Treating, preventing, or minimizing the progression of cancer in a subject; minimize, reduce, or prevent the growth of tumors in a subject; minimize, reduce, or prevent metastasis in a subject; or Provides for use in increasing the survival rate of a subject.

[0045] Alternatively, the first and second agents are for any other method or use of the invention described herein.

[0046] In another aspect, the present invention further provides the use of a TLR2 agonist and an immunostimulant to treat, prevent, or prevent the progression of cancer in a subject.

[0047] In another aspect, the invention further provides a TLR2 agonist and an immune stimulant for use in treating, preventing, or preventing the progression of cancer in a subject, or the TLR2 agonist and checkpoint inhibitor for use in any other method or use of the invention described herein, including treating, preventing, or minimizing the progression of cancer in a subject, minimizing, reducing, or preventing tumor growth in a subject, minimizing, reducing, or preventing metastasis in a subject, or increasing the survival rate of a subject.

[0048] In another aspect, the present invention further provides a method for producing a pharmaceutical composition comprising: Treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving an immune stimulant; minimize, reduce, or prevent tumor growth in a subject who has received or is receiving an immune stimulant; minimize, reduce, or prevent metastasis in a subject who has received or is receiving an immunostimulant; or Increased survival rates in subjects receiving or currently receiving immune stimulants The present invention provides the use of a TLR2 agonist in the manufacture of a medicament for treating a patient suffering from atopic dermatitis.

[0049] In any aspect of the present invention, any of the agents described herein are suitable for administration intraperitoneally, intratumorally, topically, orally, intravenously, to the respiratory tract, preferably by inhalation, or intranasally, subcutaneously, or intramuscularly. Preferably, any of the agents described herein are suitable for administration intravenously or by inhalation. In this aspect, the agent may be formulated as a nasal spray or nasal drops.

[0050] In another aspect, the present invention further provides a TLR2 agonist for use in treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving an immunostimulant. Alternatively, the TLR2 agonist is for use in any other method or use of the present invention described herein. In one aspect of the present invention, the TLR2 agonist for use is suitable for intraperitoneal, intratumoral, topical, oral, or respiratory tract administration, preferably by inhalation or intranasal, intravenous, subcutaneous, or intramuscular administration. Preferably, the TLR2 agonist for use is suitable for intravenous administration or by inhalation administration. In another aspect, the TLR2 agonist for use can be formulated as a nasal spray or nasal drops for intranasal administration.

[0051] In another aspect, the present invention further provides the use of a TLR2 agonist for treating, preventing, or minimizing the progression of cancer in a subject who has received or is receiving an immunostimulant. Alternatively, the use of the TLR2 agonist is in any other method or use of the present invention described herein. In one aspect of the present invention, the TLR2 agonist is suitable for intraperitoneal, intratumoral, topical, oral, or respiratory tract administration, preferably by inhalation or intranasal, intravenous, subcutaneous, or intramuscular administration. Preferably, the TLR2 agonist is suitable for intravenous administration or by inhalation administration. In another aspect, the TLR2 agonist can be formulated as a nasal spray or nasal drops.

[0052] In any embodiment of the present invention, the immunostimulant is - cellular immunotherapy (cytotoxic cellular immunotherapy or adoptive cellular immunotherapy); -Oncolytic viruses; -Cancer vaccines; -T cell engager; bispecific T cell engagers; and -Checkpoint inhibitors is selected from the group consisting of:

[0053] In any embodiment of the present invention, the method comprises: antigen; peptide antigens; or T helper antigens This does not include administration of

[0054] In any embodiment of the invention, the TLR2 agonist is typically not administered as part of a vaccine formulation when administered via subcutaneous, inhaled, intranasal, intradermal or intramuscular routes.

[0055] In one embodiment of the present invention, the TLR2 agonist is not administered with an antigen, hi another embodiment, the TLR2 agonist is not administered with a cell-penetrating peptide.

[0056] In any embodiment of the invention, the TLR2 agonist is not Pam3Cys.

[0057] In any embodiment of the present invention, the effect of any TLR2 agonist and immunostimulant described herein may be significant compared to the effect of the TLR2 agonist alone or the immunostimulant alone. In one aspect, the effect may be additive or synergistic.

[0058] In another aspect, when any of the TLR2 agonists and immunostimulatory agents described herein are administered to a subject, the TLR2 agonist can improve the efficacy of any of the immunostimulatory agents described herein. Preferably, the improved efficacy of any of the immunostimulatory agents described herein is against tumors that are partially or completely resistant to checkpoint inhibitors, e.g., PD-1-resistant tumors.

[0059] In one embodiment, the effect of any of the TLR2 agonists and immunostimulatory agents described herein on the survival rate of a subject may be significantly greater than the effect of the TLR2 agonist and immunostimulatory agent when administered alone. In a further embodiment, the effect of any of the TLR2 agonists and immunostimulatory agents described herein on tumor growth or metastasis in a subject may be significantly greater than the effect of the TLR2 agonist and immunostimulatory agent when administered alone.

[0060] In some aspects of the invention, the TLR2 agonist and / or immunostimulant is administered once, hi other embodiments, the TLR2 agonist and / or immunostimulant is administered two, three, four or more times to the subject.

[0061] In any embodiment of the present invention, the TLR2 agonist and / or immunostimulatory agent may be administered in the same composition or in separate compositions. Thus, in another embodiment, the TLR2 agonist and / or immunostimulatory agent may be administered together or sequentially. Alternatively, the administration may be staggered. The TLR2 agonist and / or immunostimulatory agent may also be administered at the same frequency or at different frequencies.

[0062] In any embodiment of the present invention, the TLR2 agonist and immunostimulant may be administered by any route of administration known in the art, including intraperitoneally, intratumorally, topically, orally, via inhalation or intranasally to the respiratory tract, intravenously, subcutaneously, or intramuscularly. Preferably, the TLR2 agonist and / or immunostimulant is administered intravenously or by inhalation.

[0063] In any aspect of the invention, the amount of TLR2 agonist administered can be in the range of about 250 nmoles / kg body weight / dose to 0.005 nmoles / kg body weight / dose. Preferably, the range is about 250 nmoles / kg body weight / dose to 0.05 nmoles / kg body weight / dose. In certain embodiments, the body weight / dose range is about 250 nmoles / kg to 0.1 nmoles / kg, about 50 nmoles / kg to 0.1 nmoles / kg, about 5 nmoles / kg to 0.1 nmoles / kg, about 2.5 nmoles / kg to 0.25 nmoles / kg, or about 0.5 nmoles / kg to 0.1 nmoles / kg body weight / dose. In certain embodiments, the amount is 250 nmoles, 50 nmoles, 5 nmoles, 2.5 nmoles, 0.5 nmoles, 0.25 nmoles, 0.1 nmoles, or 0.05 nmoles / kg body weight / dose, or approximately such amounts of compound.

[0064] In any embodiment of the invention, the amount of TLR2 agonist administered can be in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 μg / kg or more.

[0065] In any embodiment of the present invention, the amount of immunostimulant, particularly the amount of checkpoint inhibitor, administered can be within the range of about 0.01 to about 20 mg / kg, about 0.1 to about 10 mg / kg, about 0.1 to about 5 mg / kg, about 1 to about 5 mg / kg, about 2 to about 5 mg / g, about 7.5 to about 12.5 mg / kg, or about 0.1 to about 30 mg / kg of the subject's body weight. For example, the dosage can be about 0.1, about 0.3, about 1, about 2, about 3, about 5, or about 10 mg / kg of body weight, or about 0.3, about 1, about 2, about 3, or about 5 mg / kg of body weight.

[0066] In any aspect of the invention, the cancer is selected from the group consisting of breast cancer, colon cancer, adenocarcinoma, mesothelioma, bladder cancer, prostate cancer, germ cell carcinoma, hepatocellular / bile duct carcinoma, neuroendocrine carcinoma, pituitary tumor, small round cell tumor, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, nonseminoma, stromal Leydig cell tumor, Sertoli cell tumor, skin tumor, kidney tumor, testicular tumor, brain tumor, ovarian tumor, stomach tumor, oral tumor, bladder tumor, bone tumor, neck tumor, esophageal tumor, laryngeal tumor, liver tumor, lung tumor, fibrosarcoma, vaginal tumor, or Wilms' tumor. In preferred embodiments, the cancer is melanoma, breast cancer, or colon cancer.

[0067] In any embodiment of the invention, the checkpoint inhibitor may be a PD-1, PD-L1, or CTLA-4 checkpoint inhibitor. In one embodiment, the checkpoint inhibitor is an antibody. Preferably, the checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4 in the form of an antibody.

[0068] Preferably, the TLR2 agonist is any one of those described herein, even more preferably Pam2Cys-Ser-PEG. As used herein, Pam2Cys-Ser-PEG refers to a compound of the following formula: [ka] It could be.

[0069] In another preferred embodiment, the TLR2 agonist is a compound of the formula: [ka] is.

[0070] In any embodiment of the invention, the method does not include administering an agonist of a TLR other than a TLR2 homodimer or heterodimer.

[0071] In one embodiment of the present invention, administering to a subject a therapeutically effective amount of a TLR2 agonist and a therapeutically effective amount of an immunostimulatory agent comprises administering Pam2Cys-Ser-PEG and the immunostimulatory agent according to the methods described herein. Preferably, the immunostimulatory agent is a checkpoint inhibitor that is an inhibitor of PD-1, PD-L1, or CTLA-4. More preferably, the checkpoint inhibitor is in the form of an antibody.

[0072] In any embodiment of the invention, administering to a subject a therapeutically effective amount of a TLR2 agonist and a therapeutically effective amount of a checkpoint inhibitor comprises administering compound A108 and an immunostimulant according to the methods described herein. Preferably, the immunostimulant is a checkpoint inhibitor that is an inhibitor of PD-1, PD-L1, or CTLA-4. More preferably, the checkpoint inhibitor is in the form of an antibody. In this or any other embodiment, the TLR2 agonist and / or checkpoint inhibitor may be administered to the respiratory tract, preferably by inhalation.

[0073] In any aspect of the present invention, the TLR2 agonist comprises a lipid, peptidoglycan, lipoprotein, or lipopolysaccharide. Preferably, the TLR2 agonist comprises palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl. The TLR2 agonist may be selected from the group consisting of Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys, and Oct2Cys. In a preferred embodiment, the TLR2 agonist comprises Pam2Cys.

[0074] In any embodiment of the present invention, the TLR2 agonist may be conjugated to another compound or functional group. The other compound or functional group may be any of those described herein. Preferred compounds are selected based on their ability to aid in dissolving the TLR2 agonist in a carrier, diluent, excipient, or solvent.

[0075] Depending on the polarity of the solvent, the solubility of the TLR2 agonist can be increased by the solubilizing agent. Thus, the compound can include a TLR2 agonist and a solubilizing agent. Preferably, the TLR2 agonist and the solubilizing agent are combined. The TLR2 agonist is PEGylated. Preferably, the solubilizing agent is any molecule described herein.

[0076] The solubilizer may comprise, consist essentially of, or consist of a positively or negatively charged group. Preferably, the charged group is a branched or linear peptide. Preferably, the positively charged group comprises at least one positively charged amino acid, such as an arginine or lysine residue. Preferably, the negatively charged group comprises at least one negatively charged amino acid, such as glutamate or aspartate. The charged amino acid may be at the terminal, preferably the N-terminus.

[0077] Typically, the solubilizer comprises polyethylene glycol (PEG) or R4. In any embodiment of the invention, the solubilizer comprises polyethylene glycol (PEG) and R4.

[0078] In any embodiment of the present invention, the compound is PEG 11 In any embodiment of the present invention, the Pam2Cys and PEG are conjugated to 11 The molecules are separated by at least one serine.

[0079] In any aspect of the present invention, the TLR2 agonist has improved solubility when compared to the efficacy of other TLR2 agonists. In a preferred embodiment, the solubility of compound A108 is about 10 times better than that of PEG-Pam2Cys-R4.

[0080] In one embodiment, Compound 1 has an EC 50In a preferred embodiment, Compound 1 has a stimulatory effect on human TLR2 at EC values ​​of 0.5 pg / ml, 1.9 pg / ml, 7.8 pg / ml, 31.25 pg / ml, 125 pg / ml, and 500 pg / ml. 50 and has a stimulatory effect on human TLR2.

[0081] In another embodiment, Compound 1 has an EC 50 In other words, Compound 1 stimulates only TLR2 at an EC 50 and does not stimulate any other TLR2 agonists.

[0082] In one embodiment, Pam2CysSK4 has an EC value of about 0.05 pg / ml to 64 pg / ml or greater. 50 In another embodiment, Pam2CysSK4 has a stimulatory effect on human TLR2 at an EC of 0.5 pg / ml, 1.9 pg / ml, 7.8 pg / ml, 31.3 pg / ml, 125 pg / ml, and 500 pg / ml. 50 In another embodiment, Pam2CysSK4 has a stimulatory effect on human TLR2 at an EC of 0.0625 pg / ml, 0.25 pg / ml, 1.0 pg / ml, 4 pg / ml, 16 pg / ml, and 64 pg / ml. 50 and has a stimulatory effect on human TLR2.

[0083] In one embodiment, compound A108 has an EC 50 and has a stimulatory effect on human TLR2.

[0084] In any embodiment of the present invention, any TLR2 agonist described herein does not exhibit cytotoxicity. In one embodiment, any TLR2 agonist described herein does not inhibit human cytochrome p450 enzymes.

[0085] In a further aspect, any TLR2 agonist described herein does not significantly increase type I interferon (IFN-α) and type II interferon (IFN-γ). In another aspect, any TLR2 agonist described herein is capable of eliciting an immune response, preferably by increasing monocyte chemoattractant protein-1 (MCP-1).

[0086] In another aspect, any TLR2 agonist described herein has a half-life of about 1 to 10 hours, preferably about 3 to 6 hours. In a preferred embodiment, the TLR2 agonist is compound A108.

[0087] In one embodiment, any TLR2 agonist described herein can activate the TLR2 pathway by activating TLR2 homodimers or TLR2 / 6 heterodimers. In one embodiment, compound 1 has a stimulatory effect on human TLR2 homodimers and TLR2 / 6 heterodimers at concentrations greater than about 1 pg / ml, preferably greater than about 2 pg / ml. In one embodiment, compound A108 has a stimulatory effect on human TLR2 homodimers at concentrations greater than about 6 pg / ml, preferably greater than about 7 pg / ml. In one embodiment, compound A108 has a stimulatory effect on human TLR2 / 6 heterodimers at concentrations greater than about 0.3 pg / ml, preferably greater than about 0.4 pg / ml. In another embodiment, any TLR2 agonist described herein does not activate TLR2 / 1 heterodimers.

[0088] TLR2 agonists contemplated for use in any aspect of the present invention are any of the compounds described herein, including compounds of formulae (I)-(XIX).

[0089] In another aspect, the present invention provides a method for treating, preventing, or minimizing the progression of cancer in a subject, comprising administering to the subject a therapeutically effective amount of a compound described herein, e.g., a compound represented by any one of Formulas (I) to (XIX), and any other compound comprising moiety A), and an immunostimulant, thereby treating, preventing, or minimizing the progression of cancer in the subject.

[0090] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0091] [Figure 1A] Compound 1 / Compound A101 improves the efficacy of anti-PD1 immunotherapy in the MC38 model. Experimental setting: MC38-bearing WT mice were intratumorally treated with the indicated doses of Compound 1 together with anti-PD1 blocking antibodies. Tumor growth was measured and monitored over time. [Figure 1B] Compound 1 / Compound A101 improves the efficacy of anti-PD1 immunotherapy in the MC38 model. Kaplan-Meier survival curves (mean ± SEM; n = 10 for control, n = 9 for anti-PD1, anti-PD1 + 5 μg Compound 1, and anti-PD1 + 50 μg Compound 1, n = 8 for anti-PD1 + 25 μg Compound 1; statistics: log-rank test). [Figure 2A] Compound 1 / Compound A101 improves the efficacy of anti-PD1 immunotherapy in the B16F10 model. Experimental setting: B16F10-bearing WT mice were intratumorally treated with the indicated doses of Compound 1 or control, together with an anti-PD1 blocking antibody. Tumor growth was measured and monitored over time. [Figure 2B] Compound 1 / Compound A101 improves the efficacy of anti-PD1 immunotherapy in the B16F10 model. Kaplan-Meier survival curves (n=10 for control and anti-PD1, n=11 for anti-PD1 + 25 μg Compound 1, n=12 for anti-PD1 + 50 μg Compound 1; log-rank test). [Figure 3A] Systemic delivery of Compound 1 / Compound A101 improves the efficacy of anti-PD1 immunotherapy. Experimental setting: MC38-bearing WT mice were treated intravenously with the indicated doses of Compound 1 or control, and with intraperitoneal injections of anti-PD1 or control IgG. [Figure 3B] Systemic delivery of Compound 1 / Compound A101 improves the efficacy of anti-PD1 immunotherapy. Tumor growth was measured and monitored over time and assessed in response to Compound 1, anti-PD-1, Compound 1 and anti-PD-1, or control IgG. [Figure 4A] Combination immunotherapy reduces metastatic spread of 4T1.2 breast cancer cells. Experimental setting: Balb / c mice were injected with 4T1.2 breast cancer cells. Primary tumors were surgically removed and mice were treated as indicated. The number of metastases on the lung surface was counted. [Figure 4B] Combination immunotherapy reduces metastatic spread of 4T1.2 breast cancer cells. Number of 4T1.2 lung metastases in the indicated cohorts (mean ± SEM; n = 8 for control and anti-PD1; n = 9 for anti-PD1 + 10 μg Compound 1; statistics, one-way ANOVA with Dunnett's test for multiple concentrations). [Figure 5A] Compound A108 in combination with checkpoint inhibitors significantly reduces the growth rate of EMT6.5 tumors in vivo. Longitudinal monitoring (caliper measurements) of EMT6.5 tumors demonstrates that treatment regimens containing compound A108 result in reduced EMT6.5 tumor growth and tumor size. (Vehicle vs. anti-PD1 monotherapy (200 μg), p=not significant (ns); vehicle vs. INNA-042 (10 μg), p=0.0001; vehicle vs. INNA-042 + anti-PD1, p<0.0001; anti-PD1 monotherapy (200 μg) vs. INNA-042 + anti-PD1, p<0.0001). [Figure 5B] Compound A108 in combination with checkpoint inhibitors significantly reduces the growth rate of EMT6.5 tumors in vivo. Kaplan-Meier survival plots show a better prognosis for animals treated with the combination of compound A108 and anti-PD1. Log-rank test. [Figure 5C]Compound A108 in combination with checkpoint inhibitors significantly reduces the growth rate of EMT6.5 tumors in vivo. Violin diagrams detail individual tumor sizes within each group (black dots). [Figure 6] Compound A108 in combination with checkpoint inhibitors has antitumor activity in the immune cell-rich MC38 model. MC38 melanoma-bearing WT mice were treated intratumorally with 25 μg of compound A108. Kaplan-Meier survival analysis highlights the significant improvement in survival in response to administration of compound A108 and anti-PD1 immunotherapy. [Figure 7] Compound A108 in combination with checkpoint inhibitors has antitumor activity against large tumors in the MC38 colon cancer model. Compound A108 was administered intratumorally every two days and anti-PD1 (10 mg / Kg (200 μg dose) intraperitoneally (ip)) every four days. Tumor growth was measured and monitored over time. Data show the antitumor effect of compound A108, anti-PD-1, or the combination over the treatment period. Two-way ANOVA with Tukey's multiple comparison test. [Figure 8A] Compound A108 in combination with anti-PD-1, anti-PDL1, or anti-CTL4 prevents tumor growth in the WEHI164 fibroblast tumor model. A. A single 2.5 μg dose of compound A108 via the intraperitoneal (ip) route was combined with three doses of anti-PD1 (200 μg ip) at 7 days post-inoculation. A. Anti-PD1 vs. anti-PD1+A108 p=0.034; anti-PD1+A108 vs. PBS p=0.0081. [Figure 8B] Compound A108 in combination with anti-PD-1, anti-PDL1, or anti-CTL4 prevents tumor growth in the WEHI164 fibroblast tumor model. B. A single 2.5 μg dose of compound A108 via the intraperitoneal (ip) route was administered in combination with anti-CTLA4 (100 μg i.p. -7 days) at 7 days post-inoculation. B. Anti-CTLA4 + A108 vs. anti-CTLA4 p=0.0237; anti-CTLA4 + A108 vs. A108 alone p=0.0001; anti-CTLA4 alone vs. PBS p=0.0453; anti-CTLA4 + A108 vs. PBS p=0.0001. [Figure 8C] Compound A108 in combination with anti-PD-1, anti-PDL1, or anti-CTL4 prevents tumor growth in the WEHI164 fibroblastoma tumor model. C. A single 2.5 μg dose of compound A108 administered intraperitoneally (ip) in combination with anti-PDL1 at 7 days post-inoculation. C. Anti-PDL1 + A108 vs. A108 alone p=0.0001; anti-PDL1 alone vs. PBS p=0.0414; anti-PDL1 + A108 vs. PBS p=0.0028. [Figure 9] Human TLR2 Activity. Human TLR2 activity of compounds A107, A108, A115, A116, A203, A204, A215 and A216 from the NK-κB luciferase assay described in Example 5.

[0092] Detailed Description of the Embodiments Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the invention is not limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0093] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described in or apparent from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0094] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0095] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0096] During tumor development, tumor cells are the patient's own cells (e.g., they are the patient) and are not effectively recognized by the patient's immune system, allowing them to grow and divide to varying degrees without proper regulatory control. Therefore, the patient's own immune system needs stimulation to attack cancer cells. Cancer immunotherapy involves harnessing the immune system of a cancer patient to reject cancer by stimulating the patient's immune system. The activated immune system then attacks the cancer cells without damaging the patient's normal cells. One so-called immunotherapy that has been shown to be useful in the treatment of cancer is the use of checkpoint inhibitors.

[0097] Although such checkpoint inhibitor cancer immunotherapy has demonstrated efficacy in some cancers, such therapies are ineffective in a significant proportion of patients, and some initial responders eventually develop resistance to these therapies and experience disease recurrence. A patient's ability to respond to immunotherapy depends on numerous factors, including the individual's genetic makeup, infection history, age, nutritional status, HLA type, and the use of specific drugs. Masking of tumor antigens, which prevents tumor cells from being detected by immune surveillance, is also particularly problematic, as is the generally impaired immune status of cancer patients.

[0098] For example, PD-1 blockade alone has been shown to be ineffective in a proportion of patients with certain cancers, such as melanoma and large B-cell lymphoma. Therefore, there is a need for more reliable and effective immunotherapy regimens that are useful in cancer treatment. The inventors unexpectedly discovered that when an immunostimulant (e.g., a checkpoint inhibitor) is administered together with a TLR2 agonist, a favorable response to immunotherapy is observed. This effect has been tested in many cancer models, including colon cancer, breast cancer, metastatic breast cancer, fibrosarcoma, and melanoma. Significantly, in some embodiments, administration of an immunostimulant (e.g., a checkpoint inhibitor) and a TLR2 agonist can significantly improve cancer in a subject.

[0099] Specifically, when a combination of an immunostimulant and a TLR2 agonist is administered to a subject, it results in significant suppression of tumor growth. This effect is observable even at low doses of a given TLR2 agonist. Significantly, when a combination of an immunostimulant and a TLR2 agonist is administered to a subject, it results in an increased survival rate compared to the effect of the immunostimulant alone. In some cases, the inventors have found that while administration of the immunostimulant alone did not increase the survival rate of a subject, when the immunostimulant was administered in the presence of a TLR2 agonist, there was a significant increase in survival rate. Specifically, administration of a checkpoint inhibitor alone was less effective or ineffective, but favorable responses were observed when the checkpoint inhibitor was administered together with a TLR2 agonist in melanoma and breast cancer models.

[0100] The inventors have demonstrated this remarkable effect in multiple models of cancers of different etiologies and pathogenesis.

[0101] In particular, the combination of a TLR agonist and an immunostimulant described herein: · Breast epithelial carcinoma with triple-negative breast cancer characteristics; Metastatic PD1-insensitive breast cancer; ·Colon cancer; PD1-insensitive melanoma; and Fibrosarcoma It has been tested in several cancer models, including

[0102] Thus, one of skill in the art will understand the applicability of the present invention to any of the other cancers described herein. The findings described herein are important because they demonstrate that tumors that were previously untreatable using checkpoint inhibitors alone become treatable when a combination of an immune stimulant and a TLR2 agonist is used.

[0103] The inventors also describe herein the utility of several different TLR2 agonists in treating cancer in combination with various immune stimulants, including: Compound 1 / Compound A101 in models of colon cancer, melanoma, and metastatic breast cancer was able to reduce tumor growth, prolong survival, and reduce metastasis when administered intraperitoneally, intratumorally, or systemically; and Compound A108 significantly reduced tumor growth, improved survival, and reduced metastasis when administered intraperitoneally, intratumorally, or systemically in models of colon cancer, breast cancer, melanoma, fibrosarcoma, and metastatic breast cancer.

[0104] Thus, one of skill in the art will understand the applicability of the present invention to any of the other TLR2 agonists described herein.

[0105] The inventors also ·PD1; PD-L1; and CTLA-4 Several checkpoint inhibitors have demonstrated utility in the treatment of cancer in combination with the TLR2 agonists listed above, including

[0106] Thus, one of skill in the art will understand the applicability of the present invention to any of the other immunostimulants described herein or known in the art.

[0107] Thus, this study identifies a novel immunotherapy treatment that may improve response rates in cancer patients, including those who have previously failed to respond to immune stimulation.

[0108] This effect was surprising to the present inventors, as the efficacy of TLR2 agonists in cancer treatment has been unclear. In particular, it has been reported that subcutaneous or intraperitoneal administration of synthetic TLR2 / 6 agonists (including Pam2cysSK4 and MALP2) has no antitumor activity but instead induces IL-10 and Tregs (Yamazaki et al. PLOS ONE 2011 6(4):e18833). The same group reported that intravenous administration of Pam2cysSK4, a TLR2 / 6 agonist, promotes bone marrow-derived immunosuppressive cells (Maruyama et al. Biochemical and Biophysical Research Communications 2015 457:445e450). Importantly, it has been suggested that endogenous TLR2 / 6 agonists derived from cancer cells may promote metastasis (Kim et al. Nature 2009 457:102-106). Another study also suggested that TLR2 stimulation could promote colon cancer cell proliferation through the PI3K / Akt and NFκB signaling pathways (Liu et al. International Immunopharmacology 2018 59:375-383).

[0109] [Toll-like receptors (TLRs)] Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) expressed by diverse cell types that play important roles in both innate and adaptive immunity. Cells of the innate immune system respond to TLR activation by producing proinflammatory cytokines and chemokines that signal the clearance of pathogens and damaged self. Upon binding to specific ligands, TLR activation leads to the activation of transcription factors, such as nuclear factor kappa B (NF)-kB, activator protein-1 (AP-1), and interferon regulatory factors (IRFs), via several adaptor molecules, including myeloid differentiation primary response gene 88 (MyD88), the Toll-interleukin 1 receptor (TIR) ​​domain-containing adaptor protein TIRAP, and the TIR-domain-containing adaptor that induces interferon-β (TRIF), to regulate cytokine expression.

[0110] There are several TLRs that belong to this membrane receptor protein family, including TLR1, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9.

[0111] As used herein, the term "TLR2" is intended to mean Toll-like receptor 2 protein. In humans, TLR2 is encoded by the TLR2 gene. TLR2 is expressed on the surface of certain cells and plays a fundamental role in pathogen recognition and innate immune activation.

[0112] A TLR2 agonist is an agent that binds to Toll-like receptor 2. A TLR2 agonist can bind to and activate TLR2 as a homodimer or heterodimer. Any TLR2 agonist known in the art is contemplated for use in the present invention.

[0113] In any embodiment of the present invention, the TLR2 agonist comprises a lipid, peptidoglycan, lipoprotein, or lipopolysaccharide. Preferably, the TLR2 agonist comprises palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl. The TLR2 agonist may be selected from the group consisting of Pam2Cys, Pam3Cys, Ste2Cys, Lau2Cys, and Oct2Cys. In a preferred embodiment, the TLR2 agonist comprises Pam2Cys.

[0114] An exemplary lipopeptide according to any embodiment of the present invention is the lipopeptide "Pam2Cys." Those skilled in the art will understand that the term "lipopeptide" refers to any composition comprising one or more conjugated lipid moieties and one or more amino acid sequences. "Pam2Cys" (also known as dipalmitoyl-S-glyceryl-cysteine ​​or S-[2,3-bis(palmitoyloxy)propyl]cysteine) corresponds to the lipid moiety of MALP-2, a macrophage-activating lipopeptide synthesized and isolated from Mycoplasma fermentans. Pam2Cys is known to be a ligand for TLR2.

[0115] Pam2Cys has the structure: [ka] It has.

[0116] As used herein, the reference to "S" shown in the above chemical structures defines a sulfur atom.

[0117] Another exemplary lipopeptide is the lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteine, also known as Pam3Cys, or Pam3Cys-OH, which is a synthetic form of the N-terminal portion of Brown's lipoprotein that spans the inner and outer membranes of Gram-negative bacteria, Pam3Cys, and has the following structure: [ka] It has.

[0118] U.S. Patent No. 5,700,910 describes several N-acyl-S-(2-hydroxyalkyl)cysteines for use as intermediates in the preparation of lipopeptides used as synthesis adjuvants, B lymphocyte stimulators, macrophage stimulators, or synthetic vaccines. U.S. Patent No. 5,700,910 also teaches the use of such compounds as intermediates in the synthesis of Pam3Cys-OH and lipopeptides containing this lipoamino acid or its analog at the N-terminus.

[0119] Other lipid moieties that can be used to target cell surface TLRs include palmitoyl, myristoyl, stearoyl, lauroyl, octanoyl, or decanoyl.

[0120] In addition to Pam2Cys and Pam3Cys, the present invention also contemplates the use of Ste2Cys, Lau2Cys, and Oct2Cys according to the present invention. Those skilled in the art will recognize that Ste2Cys is also known as S-[2,3-bis(stearoyloxy)propyl]cysteine ​​or distearoyl-S-glyceryl-cysteine; Lau2Cys is also known as S-[2,3-bis(lauroyloxy)propyl]cysteine ​​or dilauroyl-S-glyceryl-cysteine; and Oct2Cys is also known as S-[2,3-bis(octanoyloxy)propyl]cysteine ​​or dioctanoyl-S-glyceryl-cysteine.

[0121] Other suitable TLR2 agonists include, but are not limited to, synthetic triacylated and diacylated lipopeptides, FSL-1 (a synthetic lipoprotein derived from Mycoplasma salivarium 1), Pam3Cys (tripalmitoyl-S-glyceryl cysteine) and S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-cysteine, where "Pam3" is "tripalmitoyl-S-glyceryl." Derivatives of Pam3Cys are also suitable TLR2 agonists, including, but not limited to: S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-(Lys)4-hydroxy trihydrochloride; Pam3Cys-Ser-Ser-Asn-Ala; Pam3Cys-Ser-(Lys)4; Pam3Cys-Ala-Gly; Pam3Cys-Ser-Gly; Pam3Cys-Ser; Pam3Cys-OMe; Pam3Cys-OH; PamCAG, palmitoyl-Cys((RS)-2,3-di(palmitoyloxy)-propyl)-Ala-Gly-OH, and the like.

[0122] Another non-limiting example of a suitable TLR2 agonist is Pam2CSK4 (dipalmitoyl-S-glycerylcysteine-serine-(lysine)4; or Pam2Cys-Ser-(Lys)4), a synthetic diacylated lipopeptide. Other synthetic TLR agonists include those described, for example, in Kellner et al. (1992) Biol. Chem. 373:1:51-5; Seifer et al. (1990) Biochem. J, 26:795-802; and Lee et al. (2003) J. Lipid Res., 44:479-486.

[0123] The TLR2 agonist can be conjugated with one or more compounds or functional groups. Examples of specific compounds or functional groups are shown below. One type of compound or functional group can act to increase the solubility of the TLR2 agonist. As will be understood by those skilled in the art, TLR2 agonists are typically non-polar and therefore soluble in non-polar solvents, while only slightly soluble in polar and aqueous solvents. If it is desired to use the TLR2 agonist in a polar or aqueous solvent, the TLR2 agonist can be conjugated with a solubilizing agent.

[0124] The solubilizing agent may include one or more solubilizing agents that may be conjugated to the TLR2 agonist to enhance the solubility of the TLR2 moiety. The solubilizing agent will generally be a polar moiety that enhances the solubility of the TLR2 moiety in polar or aqueous solvents.

[0125] In any embodiment of the present invention, the solubilizing agent can be a positively charged group, including, but not limited to, penetratin, HIV Tat 48-60, HIV Rev 34-50, transportan, oligoarginine peptides (linear and branched), oligolysine peptides, pyrrochoricin, α-helical amphipathic model peptides, polylysine, protamine, FL17, Magnafloc 1697, and polycationic compounds described in U.S. Patent Nos. 6,689,478 and 4,035,558.

[0126] In yet another embodiment of the present invention, the solubilizing agent comprises, consists essentially of, or consists of a linear or branched peptide. Typically, the linear or branched peptide contains a positively or negatively charged amino acid. The positively charged amino acid may be lysine, arginine, histidine, ornithine, or a combination thereof. The branched or linear peptide may contain at least one lysine or arginine residue. Preferably, the charged amino acid is at the terminal, e.g., the N-terminus. The branched peptide may have one of the following structures: [ka]

[0127] In the above structure, X can independently be a charged residue, either a positively or negatively charged residue. Preferably, the positively charged amino acid is lysine, arginine, histidine, or ornithine. Preferably, the negatively charged amino acid is glutamate or aspartate.

[0128] As used herein, "PEG" refers to the polymeric compound polyethylene glycol. Unless otherwise defined, reference to "PEG" includes any length polymer of ethylene oxide. Reference to PEG also includes substituted PEGs.

[0129] Compounds or functional groups that can act as solubilizers can be one or more of the group consisting of "PEG" (or polyethylene glycol) and polar polypeptides, such as "R4", a hyperbranched tetra-arginine conjugate; "H4", a hyperbranched tetra-histidine conjugate; "H8", a linear peptide containing a histidine residue; and "E8", a linear peptide containing a glutamate residue. Other linear and branched lipid solubilizers are also contemplated, including hyperbranched peptides containing glutamate residues (see, for example, "Branched E8" below). In yet another embodiment of the present invention, the solubilizer comprises one or more of the group consisting of PEG and R4, H4, H8, and E8 (linear or branched). R4, H4, H8, and E8 have been previously described in PCT / AU2009 / 000469 (WO 2010 / 115230) and have the following structures: [ka] [ka] [ka] It has.

[0130] Below are schematic diagrams of several examples of branched (structures 1-5) and linear (structures 6-8) immunogenic compositions containing positively charged (arginine, R; lysine, K) or negatively charged (aspartic acid, D; glutamic acid, E) amino acids at the terminal positions, so that their respective electrostatic charges are exposed to the environment. Each immunogenic composition also contains dipalmitoyl-S-glycerylcysteine ​​(Pam2Cys), a ligand for Toll-like receptor 2. Two serine residues (Ser) are also incorporated. For construct 2, the peptide structure was assembled in an N→C direction; all other structures shown in the diagram were assembled C→N. Positive and negative electrostatic charges are indicated as 2-, 2+, 1-, 1+, etc., depending on the magnitude of the charge. Ac = acetyl group used to suppress the positive charge of the alpha-amino group in the case of N-terminally positioned glutamic acid. [ka]

[0131] Those skilled in the art will understand that the present invention is not limited to the particular exemplified compounds or functional groups that can act as solubilizing agents, and that other suitable compounds or functional groups, including those known in the art that can act as solubilizing agents, such as carbohydrates, can be used in accordance with the present invention.

[0132] Those skilled in the art will be familiar with methods by which one or more compounds or functional groups (such as solubilizing agents) can be conjugated to lipids according to the present invention. For example, conjugation via Fmoc chemistry, disulfide or thioether bridges, or oxime chemistry is envisioned. In a specific embodiment of the present invention, a soluble form of Pam2Cys was prepared by the addition of O-(N-Fmoc-2-aminoethyl)-O'-(2-carboxyethyl)-undecaethyleneglycol (Fmoc-PEOn-OH, Merck Ltd) to Pam2Cys. This then results in a PEGylated form of the lipid, Pam2Cys-PEG, suitable for administration to a subject. 11 This resulted in the formation of

[0133] In another aspect of the invention, the TLR2 moiety comprises a conjugate comprising a Pam2Cys conjugated to a pendant R4 form. In a preferred aspect, the pendant Pam2Cys has the following structure: [ka] It is conjugated to R4 according to the formula:

[0134] In a preferred form according to any embodiment of the invention, the TLR2 moiety comprises a conjugate comprising Pam2Cys conjugated to PEG. 11 or PEG 12 Preferably, the conjugate comprises Pam2Cys conjugated to PEG. 11 or PEG 12 The molecule contains at least two serine (PEG 11 -SS-Pam2Cys or PEG 12 -SS-Pam2Cys).

[0135] As used herein, a reference to a TLR2 agonist also includes a pharmaceutically acceptable salt, solvate, polymorph or prodrug thereof.

[0136] Further compounds, including TLR2 agonists, that are useful in any embodiment of the present invention are described below.

[0137] In any embodiment, the compound has formula (I): AYB (I) (Wherein A is A1 and A2: [ka] comprising or consisting of a moiety selected from wherein each z is independently selected from 1 or 2; each X is independently selected from -S-, -S(=O)-, and -S(=O)-; In part A1: each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; In part A2: b and w are each independently an integer of 0 to 7, and v is an integer of 0 to 5, for example, 2 to 5, with the proviso that: the sum of b, v, and w is at least 3; the sum of b and w is 0 to 7; Z1 and Z2 are each independently selected from the group consisting of -O-, -NR-, -S-, S(=O), -S(=O)2-, -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; R 11 , R 12 , R x , R y , R 14 , R 15 , R 16 , and R 17 are each independently H or a C1-C6 aliphatic; R, R 13 and R 18 are each independently H or a C1-C6 aliphatic; R 19 is H, a C1-C6 aliphatic group, an amino protecting group, L3-C(=O)-, or A2; L1 and L2 are each independently C5 to C 21 Aliphatic or C4~C 20 is heteroaliphatic; L3 is C1~C 21 Aliphatic or C2-C 20is heteroaliphatic; A2 is an amino acid or a peptide; where R, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R x , R y any aliphatic or heteroaliphatic group present in any of L1, L2, and L3 is optionally substituted; Y is, [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; B comprises or consists of polyethylene glycol (PEG). or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0138] In any embodiment, the compound has the formula (IA1): AYB (IA1) wherein A is a moiety A1: [ka] comprising or consisting of wherein each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; Y is, [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; B comprises or consists of polyethylene glycol (PEG). or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0139] In one embodiment, g is an integer of 12-16.

[0140] In some embodiments, g is 14.

[0141] In any embodiment, the compound has the formula (IA2): AYB (IA2) (Wherein A is [ka] comprising or consisting of In the formula, b and w are each independently an integer of 0 to 7, and v is an integer of 0 to 5, for example, 2 to 5, with the proviso that: the sum of b, v, and w is at least 3; the sum of b and w is 0 to 7; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; Z1 and Z2 are each independently selected from the group consisting of -O-, -NR-, -S-, S(=O), -S(=O)2-, -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; R 11 , R 12 , R x , R y , R 14 , R 15 , R 16 , and R 17 are each independently H or a C1-C6 aliphatic; R, R 13 and R 18 are each independently H or a C1-C6 aliphatic; R 19 is H, a C1-C6 aliphatic group, an amino protecting group, L3-C(=O)-, or A2; L1 and L2 are each independently C5 to C 21 Aliphatic or C4~C 20 is heteroaliphatic; L3 is C1~C 21 Aliphatic or C2-C 20 is heteroaliphatic; A2 is an amino acid or a peptide; where R, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R x , R y any aliphatic or heteroaliphatic group present in any of L1, L2, and L3 is optionally substituted; Y is, [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; B comprises or consists of polyethylene glycol (PEG). or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0142] In some embodiments, v is an integer selected from 2, 3, 4, or 5. In some embodiments, v is 2 or 3. In some embodiments, v is 2.

[0143] In one embodiment, R x , R y , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , and R 17 is H.

[0144] In some embodiments, R and R 13 are H respectively.

[0145] In certain embodiments, Z1 and Z2 are the same and are selected from the group consisting of -O-, -NR-, -S-, S(=O), S(=O)2-, -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, OC(=O)O-, NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-.

[0146] In certain embodiments, Z1 and Z2 are independently selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-.

[0147] In some embodiments, w is an integer selected from 1 to 7. In some embodiments, w is 1.

[0148] In some embodiments, b is 0.

[0149] In some embodiments, the sum of b and w is 1 to 7. In these embodiments, b can be an integer selected from 0 to 7, and w can be an integer selected from 1 to 7, preferably 1.

[0150] In some embodiments, b is 0, w is 1, and v is 2.

[0151] In one embodiment, R 18 is H.

[0152] In one embodiment, R 19 is H, C1-C6 alkyl, -C(=O)C1-C6 alkyl or -C(=O)C 11 ~C 19 alkyl.

[0153] In one embodiment, R 19 is selected from H, C1-C6 alkyl, —C(═O)C1-C6 alkyl, preferably H, C1-C4 alkyl, —C(═O)C1-C4 alkyl.

[0154] In one embodiment, R 19 is selected from H and —C(═O)CH 3 .

[0155] In one embodiment, L1 and L2 are independently C5 to C 21 Aliphatic or C4~C 20 In some embodiments, L and L are independently selected from C 10 ~C 18 Aliphatic or C 10 ~C 18 In some embodiments, L and L are independently selected from C 14 -alkyl and C 15 -alkyl.

[0156] In one embodiment, X is S.

[0157] In some embodiments, X is S(=O).

[0158] In certain embodiments, X is S(=O)2.

[0159] In some embodiments, R6 and R7 are each H.

[0160] In one embodiment, R 18 and R 19 are H respectively.

[0161] In certain embodiments, the present invention provides a compound of formula (I), wherein: v is an integer from 2 to 5; b is 0; R x , R y , R 13 , R 14 , R 15 , R 16 , and R 17 is H; Z1 and Z2 are independently selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; w is an integer from 1 to 7; R 19 is H, C1-C6 alkyl, -C(=O)C1-C6 alkyl or -C(=O)C 11 ~C 19 selected from the group consisting of alkyl; L1 and L2 are independently C 10 ~C 18 Aliphatic or C 10 ~C 18 Heteroaliphatic.

[0162] In certain embodiments, the present invention provides a compound wherein: v is 2; b is 0; w is 1; the sum of v, b, and w is 3; the sum of b and w is 1; z is 1; X is S, Z1 and Z2 are independently selected from the group consisting of -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; R in each case of b, v, w, and z 11 , R 12 , R x , R y , R 14 , R 15 , R 16 , and R 17 are H, respectively; R and R 13 are H, respectively; R 18 is H; R 19 is H, C1-C6 alkyl, -C(=O)C1-C6 alkyl or -C(=O)C 11 ~C 19 selected from the group consisting of alkyl; L1 and L2 are independently C 10 ~C 18 Aliphatic or C 10 ~C18 Heteroaliphatic.

[0163] Substituents R1, R2, R4, R5, R6, R7, R9, R 10 , z, X, g, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R x , R y It will be understood that any embodiment of the substituents described herein, including L1, L2, Z1, Z2, b, v, w, n, m, p, q, R3, L, t, k, and h, is intended to apply in any instance to the substituents of any compound described herein, including compounds of formulae (I) through (XIX).

[0164] In any embodiment, the compound has formula (II): A-Y'-B (II) wherein A comprises or consists of a moiety A1 or A2 as defined herein; Y' is [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; B comprises or consists of polyethylene glycol (PEG) or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0165] In one embodiment, the compound comprises a moiety A1, wherein: each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; z is 1; X is S; R6 and R7 are H; R9 and R 10 are both single bonds.

[0166] In one embodiment, moiety A1 is moiety A1′ [ka] is defined by wherein each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18.

[0167] In any aspect, any of the compounds described herein can be a compound comprising a moiety A selected from A1' and A2 as defined herein and PEG, wherein moiety A and PEG are linked by a glycine, serine, homoserine, threonine, phosphoserine, asparagine, or glutamine residue, or an ester of a glutamine residue.

[0168] In any embodiment, the compound has the moiety A1Y' or A2Y': [ka] wherein R and R are independently selected from the group consisting of H, -CHOH, -CHCHOH, -CH(CH)OH, -CHOPO(OH), -CHC(=O)NH, -CHCHC(=O)OH, and -CHCHC(=O)OR, wherein any one of the alkyl hydrogens may be replaced with a halogen; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; b and w are each independently an integer from 0 to 7, and v is an integer from 0 to 5, with the proviso that: the sum of b, v, and w is at least 3; The sum of b and w is 0 to 7; Z1 and Z2 are each independently selected from the group consisting of -O-, -NR-, -S-, -S(=O)-, -S(=O)2-, -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; R in each case of b, v, w, and z 11 , R 12 , R x , R y , R 14 , R 15 , R 16 , and R 17 are each independently H or a C1-C6 aliphatic; R, R 13 and R 18 are each independently H or a C1-C6 aliphatic; R 19 is H, a C1-C6 aliphatic group, an amino protecting group, L3-C(=O)-, or A2; L1 and L2 are each independently C5 to C 21 Aliphatic or C4~C 20 is heteroaliphatic; L3 is C1~C 21 Aliphatic or C2-C 20 is heteroaliphatic; A2 is an amino acid or a peptide; where R, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R17 , R 18 , R 19 , R x , R y Any aliphatic or heteroaliphatic group present in any of L1, L2, and L3 is optionally substituted; A1Y' or A2Y' is covalently linked to polyethylene glycol (PEG). may comprise or consist of or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0169] In certain embodiments, moiety A and PEG are linked through a serine, homoserine, threonine, or phosphoserine residue.

[0170] In one embodiment, moiety A and PEG are [ka] is covalently attached to a glycine, serine, homoserine, threonine, phosphoserine, asparagine, or glutamine residue, or an ester of a glutamine residue, via a bond represented by the formula:

[0171] In any embodiment, the compound is covalently linked to polyethylene glycol (PEG): [ka] wherein R and R are independently selected from the group consisting of H, -CHOH, -CHCHOH, -CH(CH)OH, -CHOPO(OH), -CHC(=O)NH, -CHCHC(=O)OH, and -CHCHC(=O)OR, wherein any one of the alkyl hydrogens may be replaced with a halogen; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0172] In one embodiment, the compound is covalently attached to polyethylene glycol (PEG): [ka] wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0173] In one embodiment, the compound is covalently attached to polyethylene glycol (PEG): [ka] wherein R and R are independently selected from the group consisting of H, -CHOH, -CHCHOH, -CH(CH)OH, -CHOPO(OH), -CHC(=O)NH, -CHCHC(=O)OH, and -CHCHC(=O)OR, wherein any one of the alkyl hydrogens may be replaced with a halogen; R6 and R7 are H; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R 10 are both single bonds; z is 1; X is S.) or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0174] In one embodiment, PEG is [ka] is covalently attached via a bond indicated by

[0175] In one embodiment, the compound is covalently attached to polyethylene glycol (PEG): [ka] wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; R6 and R7 are H; R9 and R 10 are both single bonds; z is 1; X is S. ); or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0176] In one embodiment, PEG is [ka] is covalently attached via a bond indicated by

[0177] In one embodiment, the compound is [ka] (wherein R1, R2, and g are as defined herein.) or a salt, solvate or prodrug thereof.

[0178] In one embodiment, PEG is [ka] is covalently attached via a bond indicated by

[0179] In one embodiment, the compound is covalently attached to polyethylene glycol (PEG): [ka] wherein R and R are independently selected from the group consisting of H, -CHOH, -CHCHOH, -CH(CH)OH, -CHOPO(OH), -CHC(=O)NH, -CHCHC(=O)OH, and -CHCHC(=O)OR, wherein any one of the alkyl hydrogens may be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; b and w are each independently an integer from 0 to 7, and v is an integer from 0 to 5, with the proviso that: the sum of b, v, and w is at least 3; The sum of b and w is 0 to 7; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; Z1 and Z2 are each independently selected from the group consisting of -O-, -NR-, -S-, -S(=O)-, -S(=O)2-, -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; R in each case of b, v, w, and z 11 , R 12 , R x , R y , R 14 , R 15 , R 16 , and R 17 are each independently H or a C1-C6 aliphatic; R, R 13 and R 18 are each independently H or a C1-C6 aliphatic; R 19 is H, a C1-C6 aliphatic group, an amino protecting group, L3-C(=O)-, or A2; L1 and L2 are each independently C5 to C 21 Aliphatic or C4~C 20 is heteroaliphatic; L3 is C1~C 21 Aliphatic or C2-C 20 is heteroaliphatic; A2 is an amino acid or a peptide; where R, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R x , R y Any aliphatic or heteroaliphatic group present in any of L1, L2, and L3 is optionally substituted. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0180] In one embodiment, PEG is [ka] is covalently attached via a bond indicated by

[0181] In any embodiment, the compound has formula (III): AY-B (III) (In the formula, AY is AY1 and AY2 [ka] comprising or consisting of a moiety selected from In the formula, R1, R2, R6, R7, R9, R 10 , z, X, g, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R x , R y , L1, L2, Z1, Z2, b, v and w are as defined for compounds of formula (I); B comprises or consists of polyethylene glycol (PEG). The compound may be:

[0182] In any embodiment, the compound has formula (IV): [ka] (In the formula, n is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; q is 0 or 1; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain of an amino acid or the second hydrogen atom or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0183] In any embodiment, the compound has the formula (V): [ka] (In the formula, n is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; q is 0 or 1; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain of an amino acid or the second hydrogen or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0184] In certain embodiments, the compound is a compound of formula (IV) or (V), wherein: R6 and R7 are H; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R 10 are both single bonds; z is 1; X is S.

[0185] In some embodiments, the compound of any one of Formulas (I)-(V) has Formula (VI): [ka] (In the formula, n is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; q is 0 or 1; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain of an amino acid or the second hydrogen or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0186] In any embodiment, the compound has formula (VII): [ka] During the ceremony, n is 3 to 100; m is 1, 2, 3 or 4; p is 2, 3 or 4; q is 0 or 1; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain of an amino acid or the second hydrogen; b and w are each independently an integer from 0 to 7, and v is an integer from 0 to 5, with the proviso that: the sum of b, v, and w is at least 3; the sum of b and w is 0 to 7; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; Z1 and Z2 are each independently selected from the group consisting of -O-, -NR-, -S-, -S(=O)-, -S(=O)2-, -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; R in each case of b, v, w, and z 11 , R 12 , R x , R y , R 14 , R15 , R 16 , and R 17 are each independently H or a C1-C6 aliphatic; R, R 13 and R 18 are each independently H or a C1-C6 aliphatic; R 19 is H, a C1-C6 aliphatic group, an amino protecting group, L3-C(=O)-, or A2; L1 and L2 are each independently C5 to C 21 Aliphatic or C4~C 20 is heteroaliphatic; L3 is C1~C 21 Aliphatic or C2-C 20 is heteroaliphatic; A2 is an amino acid or a peptide; where R, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R x , R y Any aliphatic or heteroaliphatic group present in any of L1, L2, and L3 is optionally substituted. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0187] In any embodiment, the compound has formula (VIII): AY-NH-(CH2) p -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3 (VIII) (In the formula, A is a moiety selected from A1 and A2 as defined herein; Y is, [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; n is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; q is 0 or 1; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0188] In any embodiment, the compound has formula (IX): A1-Y-NH-(CH2) p -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3 (IX) (In the formula, A1 is represented by the moiety A1 defined for formula (I), Y is, [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; n is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; q is 0 or 1; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0189] In certain embodiments, the compound is a compound of formula (VIII) or (IX), wherein: R6 and R7 are H; R9 and R 10 are both single bonds; z is 1; X is S.

[0190] In any embodiment, the compound has the formula (X): Pam2Cys-Y-NH-(CH2) p -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3 (X) (In the formula, Pam2Cys has the structure: [ka] having; Y is: [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; n is 3 to 100; m is 1, 2, 3 or 4; p is 2, 3 or 4; q is 0 or 1; When q=1, R3 is H, —NH2, or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0191] In any embodiment, the compound has formula (XI): Pam2Cys-Y-NH-(CH2) p -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3 (XI) (In the formula, Pam2Cys has the structure: [ka] having; Y is: [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH), wherein any one of the alkyl hydrogens may be replaced with a halogen, and wherein R1 and R2 are not both H; n is 3 to 100; m is 1, 2, 3 or 4; p is 2, 3 or 4; q is 0 or 1; When q=1, R3 is H, —NH2, or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0192] In any embodiment, the compound has formula (XII): Pam2Cys-Y-NH-(CH2) p -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3 (XII) (In the formula, Pam2Cys has the structure: [ka] having; Y is: [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; n is 3 to 100; m is 1, 2, 3 or 4; p is 2, 3 or 4; q is 0 or 1; When q=1, R3 is H, —NH2, or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0193] In any embodiment, the compound has formula (XIII): Pam2Cys-Ser-NH-(CH2) p -O-(CH2-CH2-O) n -[(CH2) m -CO-L-] q R3 (XIII) (In the formula, Pam2Cys-Ser has the structure: [ka] having; n is 3 to 100; m is 1, 2, 3 or 4; p is 2, 3 or 4; q is 0 or 1; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0194] In one embodiment, the compound has formula (XIV): [ka] (In the formula, n is 3 to 100; k is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; t is 2, 3 or 4; h is 1, 2, 3 or 4; q is 0 or 1; wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0195] In one embodiment, the compound has formula (XV): [ka] (In the formula, n is 3 to 100; k is 3 to 100; m is 1, 2, 3 or 4; p is 2, 3 or 4; t is 2, 3 or 4; h is 1, 2, 3 or 4; q is 0 or 1; wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is zero or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of an amino acid; b and w are each independently an integer from 0 to 7, and v is an integer from 0 to 5, with the proviso that: the sum of b, v, and w is at least 3; The sum of b and w is 0 to 7; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; Z1 and Z2 are each independently selected from the group consisting of -O-, -NR-, -S-, -S(=O)-, -S(=O)2-, -C(=O)O-, -OC(=O)-, -C(=O)NR-, -NRC(=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)O-, -NRC(=O)O-, -OC(=O)NR-, and -NRC(=O)NR-; R in each case of b, v, w, and z 11 , R 12 , R x , R y , R 14 , R 15 , R 16 , and R 17 are each independently H or a C1-C6 aliphatic; R, R 13 and R 18 are each independently H or a C1-C6 aliphatic; R 19 is H, a C1-C6 aliphatic group, an amino protecting group, L3-C(=O)-, or A2; L1 and L2 are each independently C5 to C 21 Aliphatic or C4~C 20 is heteroaliphatic; L3 is C1~C 21 Aliphatic or C2-C20 is heteroaliphatic; A2 is an amino acid or a peptide; where R, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R x , R y Any aliphatic or heteroaliphatic group present in any of L1, L2, and L3 is optionally substituted. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0196] In any embodiment, the compound has formula (XVI): [ka] (In the formula, n is 3 to 100; k is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; t is 2, 3 or 4; h is 1, 2, 3 or 4; q is 0 or 1; wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; R6 and R7 are independently selected from the group consisting of H, linear or branched C1-C4 alkyl, and —C(═O)CH3; R9 and R 10 is independently selected from the group consisting of -NH-, -O-, or a single bond; z is 1 or 2; X is selected from -S-, -S(=O)-, and -S(=O)2-; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain of an amino acid or the second hydrogen or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0197] In any embodiment, the compound has formula (XVII): [ka] (In the formula, n is 3 to 100; k is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; t is 2, 3 or 4; h is 1, 2, 3 or 4; q is 0 or 1; wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R6 and R7 are H; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; R9 and R 10 are both single bonds; z is 1; X is S; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0198] In any embodiment, the compound has formula (XVIII): [ka] (In the formula, n is 3 to 100; k is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; t is 2, 3 or 4; h is 1, 2, 3 or 4; q is 0 or 1; wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; R6 and R7 are H; R9 and R 10 are both single bonds; z is 1; X is S; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0199] In any embodiment, the compound has formula (XIX): [ka] (In the formula, n is 3 to 100; k is 3 to 100; m is 1, 2, 3 or 4; each g is independently 10, 11, 12, 13, 14, 15, 16, 17, or 18; p is 2, 3 or 4; t is 2, 3 or 4; h is 1, 2, 3 or 4; q is 0 or 1; R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, and -CH2OPO(OH)2, wherein any one of the alkyl hydrogens may be replaced with a halogen, and R1 and R2 are not both H; When q=1, R3 is —NH2 or —OH; When q=0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0200] In certain embodiments, any compound disclosed herein that includes polyethylene glycol (PEG) (including a compound of any one of Formulas (I)-(XIX)) may include PEG in the form of a substituted PEG.

[0201] In one embodiment, the substituted PEG has the subformula BI: [ka] (In the formula, n is 3 to 100; m is 1, 2, 3 or 4; p is 2, 3 or 4; q is 0 or 1; R3 is H, -NH2 or -OH, and when q is 0, R3 is H, and when q is 1, R3 is -NH2 or -OH; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain of the amino acid or the second hydrogen).

[0202] In one embodiment, the substituted PEG has the subformula B-II: [ka] (In the formula, p is 2, 3 or 4; n is 3 to 100; m is 1, 2, 3 or 4; t is 2, 3 or 4; k is 3 to 100; h is 1, 2, 3 or 4; q is 0 or 1; When q is 1, R3 is -NH2 or -OH; When q is 0, R3 is H; L is 0 or consists of 1 to 10 units, where each unit is or is derived from a naturally occurring alpha amino acid and has the formula: [ka] is expressed as wherein R4 is H; R5 is the side chain or the second hydrogen of the amino acid. is expressed by

[0203] In some embodiments of the substituted PEG of formula BI or B-II, q is 1.

[0204] In some embodiments of the substituted PEG of formula BI or B-II, n can be 10-14, such as 11, or 24-30, such as 27.

[0205] In some embodiments of the substituted PEG of formula BI or B-II, m is 1 to 3, for example, 2.

[0206] In some embodiments of the substituted PEG of formula BI or B-II, when q is 1, R3 is -NH2.

[0207] In some embodiments of the substituted PEG of formula BI or B-II, L is a naturally occurring alpha amino acid residue.

[0208] The compounds described herein can exist in and be isolated in optically active and racemic forms. As will be appreciated by those skilled in the art, the present invention is intended to encompass any racemic, optically active, or stereoisomer of the compounds of the present invention, or mixtures thereof, that possess the useful properties described herein. Methods for preparing such forms (e.g., by resolution of racemic mixtures by recrystallization, synthesis from optically active starting materials, chiral synthesis, or chiral chromatographic separation) are well known in the art. In certain embodiments, compositions can include compounds in enantiomerically or diastereomeric enriched forms. For example, compounds can have an enantiomeric excess (ee) or diastereomeric excess (de) of at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99%. In certain embodiments, the compound may be enriched by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% at any stereocenter in the compound.

[0209] In any embodiment, the compound has the following chiral center: * (indicated by): [ka] may include a chiral center around wherein the chiral center is in the R configuration. This form of the compound may also be referred to as an R-Pam2 analog diastereomer of the compound of the invention described herein. [ka] It can be shown as:

[0210] In any embodiment, the compound has a chiral center ( * (indicated by): [ka] may include wherein the chiral center is in the R configuration. This form of the compound is also referred to as the R-Pam2 diastereomer of the compounds of the invention described herein. [ka] It can be shown as:

[0211] In any embodiment, the compound has the following chiral center: * (indicated by): [ka] may include a chiral center around wherein the chiral center is in the S configuration. This form of the compound may also be referred to as the S-Pam2 analog diastereomer of the compound of the invention described herein. [ka] It can be shown as:

[0212] In any embodiment, the compound has a chiral center ( * (indicated by): [ka] Including, wherein the chiral center is in the S configuration. This form of the compound may also be referred to as the S-Pam2 diastereomer of the compounds of the invention described herein. [ka] It can be shown as:

[0213] In any embodiment, the compound has the following chiral center: * (indicated by): [ka] containing a chiral center around wherein the chiral center is in the L configuration. This form of the compound may also be referred to as the L-Cys analog diastereomer of the Pam2Cys of the compounds described herein. [ka] It can be shown as:

[0214] In any embodiment, the compound has a chiral center ( * (indicated by): [ka] Including, wherein the chiral center is in the L configuration. This form of the compound may also be referred to as the L-Cys diastereomer of the Pam2Cys of the compounds described herein. [ka] It can be shown as:

[0215] Other stereocenters in these compounds may be racemic or enriched in either the R or S configuration.

[0216] In any embodiment, the compound has the following chiral center: * (indicated by): [ka] containing a chiral center in moiety A1 around wherein the chiral center is in the D configuration. This form of the compound may also be referred to as the D-Cys analog diastereomer of the Pam2Cys of the compounds described herein. [ka] It can be shown as:

[0217] Other stereocenters in these compounds may be racemic or enriched in either the R or S configuration.

[0218] In any embodiment, the compound has a chiral center ( * (indicated by): [ka] Including, wherein the chiral center is in the D configuration. This form of the compound may also be referred to as the D-Cys diastereomer of the Pam2Cys of the compounds described herein. [ka] It can be shown as:

[0219] Other stereocenters in these compounds may be racemic or enriched in either the R or S configuration.

[0220] In any aspect or embodiment of the present invention, the compounds of the present invention have a chiral center at the carbon atom of the moiety A2: * (indicated by): [ka] and wherein the chiral center is in the R configuration. In one embodiment, this stereoisomer of the compound is [ka] where L1, L2, Z1, Z2, R x , R y , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , b, v and z are as defined for compounds of formula (I) and w is 1. Other stereocenters in these compounds may be racemic or enriched in either the R or S configuration.

[0221] In any aspect or embodiment of the present invention, the compounds of the present invention have a chiral center at the carbon atom of the moiety A2: * (indicated by): [ka] and wherein the chiral center is in the S configuration. In one embodiment, moiety A of this stereoisomer of the compound is [ka] where L1, L2, Z1, Z2, R x , R y , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19, b, v, w, and z are as defined for the compounds or formula (I). Other stereocenters in these compounds may be racemic or enriched in either the R or S configuration.

[0222] In any aspect or embodiment of the present invention, the compounds of the present invention have a chiral center at the carbon atom of the moiety A2: ** (indicated by): [ka] and wherein the chiral center is in the L configuration. This form of the compound may also be referred to as the L-Cys analog stereoisomer of the compound of the invention. In one embodiment, this stereoisomer of the compound is [ka] where L1, L2, Z1, Z2, R x , R y , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , b, v, w, and z are as defined for the compounds or formula (I). Other stereocenters in these compounds may be racemic or enriched in either the R or S configuration.

[0223] In any aspect or embodiment of the present invention, the compounds of the present invention have a chiral center at the carbon atom of the moiety A2: ** (indicated by): [ka] and wherein the chiral center is in the D configuration. This form of the compound may also be referred to as the D-Cys analog stereoisomer of the compound of the invention. In one embodiment, the moiety A of this stereoisomer of the compound is: [ka] where L1, L2, Z1, Z2, R x , R y , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , b, v, and z are as defined for the compounds or formula (I), and w is 1. Other stereocenters in these compounds may be racemic or enriched in either the R or S configuration.

[0224] In any embodiment, the compound has a chiral center ( * (indicated by): [ka] Including, wherein the chiral center is in the L configuration. This form of the compound may also be referred to as the LY diastereomer of the compounds of the invention described herein.

[0225] In any embodiment, the compound has a chiral center ( * (indicated by): [ka] Including, wherein the chiral center is in the D configuration. Compounds of this form can also be:

[0226] In any aspect, a composition comprising a compound of the present invention (including any one of the compounds of formulae (I) to (XIX)) or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier, diluent or excipient may be used in the method or use of the present invention.

[0227] In certain embodiments, the compounds described herein are R diastereomers about the chiral center of the 2,3-bis(palmitoyloxy)propyl portion of the compound.

[0228] In certain embodiments, the compounds described herein are S diastereomers about the chiral center of the 2,3-bis(palmitoyloxy)propyl portion of the compound.

[0229] In any embodiment, the compositions described herein include a compound that is an R diastereomer about the chiral center of the 2,3-bis(palmitoyloxy)propyl portion of the compound.

[0230] In either embodiment, the composition comprises a compound that is an S diastereomer about the chiral center of the 2,3-bis(palmitoyloxy)propyl portion of the compound.

[0231] In any embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the compound present in the composition is the R diastereomer about the chiral center of the 2,3-bis(palmitoyloxy)propyl portion of the compound.

[0232] In any embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the compound present in the composition is the S diastereomer about the chiral center of the 2,3-bis(palmitoyloxy)propyl moiety (e.g., moiety A1) of the compound.

[0233] In any embodiment, the compounds described herein are L diastereomers about the chiral center of the cysteine ​​analog residue of the Pam2Cys analog portion (eg, moiety Y) of the compound.

[0234] In any embodiment, the compounds described herein are L diastereomers about the chiral center of the cysteine ​​residue of the Pam2Cys portion (eg, moiety Y) of the compound.

[0235] In any embodiment, the compounds described herein are D diastereomers about the chiral center of the cysteine ​​analog residue of the Pam2Cys analog portion (eg, moiety Y) of the compound.

[0236] In any embodiment, the compounds described herein are D diastereomers about the chiral center of the cysteine ​​residue of the Pam2Cys portion (eg, moiety Y) of the compound.

[0237] In any embodiment, the compositions described herein include compounds that are L diastereomers about the chiral center of the cysteine ​​analog residue of the Pam2Cys analog portion (eg, moiety Y) of the compound.

[0238] In any embodiment, the compositions described herein include a compound that is an L diastereomer about the chiral center of the cysteine ​​residue of the Pam2Cys portion (eg, moiety Y) of the compound.

[0239] In any embodiment, the compositions described herein include compounds that are D diastereomers about the chiral center of the cysteine ​​analog residue of the Pam2Cys analog portion (eg, moiety Y) of the compound.

[0240] In any embodiment, the compositions described herein include compounds that are D diastereomers about the chiral center of the cysteine ​​residue of the Pam2Cys portion (eg, moiety Y) of the compound.

[0241] In any embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the compound present in the composition is the L diastereomer about the chiral center of the cysteine ​​analog residue of the Pam2Cys analog portion of the compound.

[0242] In any embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the compound present in the composition is the L diastereomer about the chiral center of the cysteine ​​residue of the Pam2Cys portion of the compound.

[0243] In any embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the compound present in the composition is a D diastereomer about the chiral center of the cysteine ​​analog residue of the Pam2Cys analog portion of the compound.

[0244] In any embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the compound present in the composition is a D diastereomer about the chiral center of the cysteine ​​residue of the Pam2Cys portion of the compound.

[0245] In either embodiment, the compounds of the present invention are L diastereomers about the chiral center of the Y moiety.

[0246] In either embodiment, the compounds described herein are D diastereomers about the chiral center of the Y moiety.

[0247] In any embodiment, the compositions described herein include compounds that are L diastereomers about the chiral center of the Y moiety.

[0248] In any embodiment, the compositions described herein include compounds that are D diastereomers about the chiral center of the Y moiety.

[0249] In any embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the compounds present in the composition are L diastereomers about the chiral center of the Y moiety.

[0250] In any embodiment, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater than 99% of the compounds present in the composition are D diastereomers about the chiral center of the Y moiety.

[0251] The compounds of Formulas (I)-(XIX) described herein may demonstrate considerable stability in solution, which may be observed by storing solutions of the compounds under ambient storage conditions (e.g., at 25°C) or accelerated decomposition conditions (e.g., at 40°C) for at least about 14 days.

[0252] In any embodiment, any of the compounds described herein may be administered in the form of a pharmaceutically acceptable salt.

[0253] The term "pharmaceutically acceptable" may be used to refer to any pharmaceutically acceptable salt, hydrate or prodrug, or any other compound that, when administered to a subject, is capable of providing (directly or indirectly) a compound of the invention described herein, or a pharmaceutically acceptable salt, prodrug or ester thereof, or an active metabolite or residue thereof.

[0254] Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid, or salts of pharmaceutically acceptable organic acids such as acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, malic acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid, and valeric acid.

[0255] Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, zinc, ammonium, alkylammonium salts such as those formed with triethylamine, alkoxyammonium salts such as those formed with ethanolamine, and salts formed with ethylenediamine, choline, or amino acids such as arginine, lysine, or histidine. General information about the types of pharmaceutically acceptable salts and their formation is known to those skilled in the art and can be found in general texts such as "Handbook of Pharmaceutical Salts" by P.H. Stahl and C.G. Wermuth, 1st edition, 2002, Wiley-VCH.

[0256] For compounds that are solids, it will be understood by those skilled in the art that the compounds, agents and salts of the invention may exist in different crystalline or polymorphic forms, all of which are intended to be included within the scope of the invention and defined formula.

[0257] The term "polymorph" includes any crystalline form of a compound of the invention described herein, including anhydrous, hydrated, solvated and mixed solvate forms.

[0258] The compounds of the invention described herein are intended to include solvated as well as unsolvated forms of the compounds, where applicable. Thus, the compounds of the invention described herein include compounds having the depicted structure, including hydrated or solvated forms as well as non-hydrated and non-solvated forms.

[0259] As used herein, the term "solvate" refers to a complex of variable stoichiometry formed by a solute (in the present invention, the compound of the present invention described herein, or a pharmaceutically acceptable salt, prodrug, or ester thereof) and a solvent. Such a solvent for the present invention must not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol, and acetic acid. Preferably, the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include, but are not limited to, water, ethanol, and acetic acid. Most preferably, the solvent used is water.

[0260] Basic nitrogen-containing groups may be quaternized with such materials as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dialkyl sulfates, such as dimethyl sulfate and diethyl sulfate;

[0261] The compounds described herein also include isotopic forms such as the replacement of deuterium with hydrogen.

[0262] A "prodrug" is a compound that may not fully meet the structural requirements of the compounds provided herein, but is modified in vivo after administration to a subject or patient to produce a compound of the invention described herein. For example, a prodrug can be an acylated derivative of a compound provided herein. Prodrugs include compounds in which a hydroxy, carboxy, amine, or sulfhydryl group is bonded to any group that, when administered to a mammalian subject, cleaves to form the free hydroxy, carboxy, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, phosphate, and benzoate derivatives of alcohol and amine functional groups in the compounds provided herein. Prodrugs of the compounds provided herein can be prepared by modifying functional groups present in the compound such that the modifications are cleaved in vivo to produce the parent compound.

[0263] Prodrugs include compounds in which an amino acid residue, or a polypeptide chain of two or more (e.g., 2, 3, or 4) amino acid residues, is covalently bonded to any of the free amino and amide groups of the compounds of Formulas (I) through (XIX). The amino acid residues include the 20 naturally occurring amino acids, commonly represented by their three-letter symbols, and also include 4-hydroxyproline, hydroxylysine, demosin, isodesin, 3-methylhistidine, norbrine, β-alanine, γ-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine, and methionine sulfone. Prodrugs also include compounds in which carbonates, carbamates, amides, and alkyl esters are covalently bonded to the above-mentioned substituents of the compounds described herein, including the compounds of Formulas (I) through (XIX), or to other structures shown herein.

[0264] General chemical terms used in the formulae herein have their usual meanings.

[0265] The term "aliphatic" is intended to include saturated and unsaturated, non-aromatic, straight-chain, branched-chain, acyclic, and cyclic hydrocarbons. One of ordinary skill in the art will understand that aliphatic groups include, for example, alkyl, alkenyl, alkynyl, cycloalkyl, and cycloalkenyl groups, as well as hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, and (cycloalkyl)alkenyl groups. In various embodiments, aliphatic groups contain 1 to 12, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In some embodiments, aliphatic groups contain 5 to 21, 9 to 21, or 11 to 21 carbon atoms, e.g., 11, 13, 15, 17, or 19 carbon atoms. In some embodiments, aliphatic groups are saturated.

[0266] The term "heteroaliphatic" is intended to include aliphatic groups in which one or more chain and / or ring carbon atoms are independently replaced with a heteroatom, preferably a heteroatom selected from oxygen, nitrogen, and sulfur. In certain embodiments, heteroaliphatic groups are saturated. Examples of heteroaliphatic groups include linear or branched heteroalkyl, heteroalkenyl, and heteroalkynyl groups.

[0267] The term "alkyl" is intended to include saturated straight-chain and branched-chain hydrocarbon groups. In certain embodiments, an alkyl group has 1 to 12, 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms. In certain embodiments, an alkyl group has 5 to 21, 9 to 21, or 11 to 21 carbon atoms, e.g., 11, 13, 15, 17, or 19 carbon atoms. Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl. Examples of branched-chain alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl.

[0268] The term "alkenyl" is intended to include straight-chain and branched-chain alkyl groups having at least one double bond between two carbon atoms. In certain embodiments, an alkenyl group has 2 to 12, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In certain embodiments, an alkenyl group has 5 to 21, 9 to 21, or 11 to 21 carbon atoms, e.g., 11, 13, 15, 17, or 19 carbon atoms. In certain embodiments, an alkenyl group has 1, 2, or 3 carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, -CH=CH(CH), -CH=C(CH), -C(CH)=CH, and -C(CH)=CH(CH).

[0269] The term "alkynyl" is intended to include straight-chain and branched-chain alkyl groups having at least one triple bond between two carbon atoms. In certain embodiments, an alkynyl group has 2 to 12, 2 to 10, 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In certain embodiments, an alkynyl group has 1, 2, or 3 carbon-carbon triple bonds. Examples include, but are not limited to, -C=CH, -C=CH3, -CH2C=CH3, and -C=CH2CH(CH2CH3)2.

[0270] The term "heteroalkyl" is intended to include alkyl groups in which one or more chain carbon atoms are replaced with a heteroatom, preferably a heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur. In some embodiments, the heteroalkyl is saturated. Examples of heteroalkyl groups include polyethylene glycol groups and polyethylene glycol ether groups.

[0271] The term "cycloalkyl" is intended to include monocyclic, bicyclic, and tricyclic alkyl groups. In certain embodiments, cycloalkyl groups have 3 to 12, 3 to 10, 3 to 8, 3 to 6, or 3 to 5 carbon atoms in the ring. In certain embodiments, cycloalkyl groups have 5 or 6 ring carbon atoms. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In certain embodiments, cycloalkyl groups have 3 to 8, 3 to 7, 3 to 6, 4 to 6, 3 to 5, or 4 to 5 ring carbon atoms. Bicyclic and tricyclic ring systems include bridged, spiro, and fused cycloalkyl ring systems. Examples of bicyclic and tricyclic cycloalkyl systems include, but are not limited to, bicyclo[2.1.1]hexanyl, bicyclo[2.2.1]heptanyl, adamantyl, and decalinyl.

[0272] The term "cycloalkenyl" is intended to include non-aromatic cycloalkyl groups having at least one double bond between two carbon atoms. In certain embodiments, cycloalkenyl groups have 1, 2, or 3 double bonds. In certain embodiments, cycloalkenyl groups have 4 to 14, 5 to 14, 5 to 10, 5 to 8, or 5 to 6 carbon atoms in the ring. In certain embodiments, cycloalkenyl groups have 5, 6, 7, or 8 ring carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl.

[0273] The term "aryl" is intended to include cyclic aromatic hydrocarbon groups that do not contain any ring heteroatoms. Aryl groups include monocyclic, bicyclic, and tricyclic ring systems. Examples of aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl. In certain embodiments, aryl groups have 6 to 14, 6 to 12, or 6 to 10 carbon atoms in the ring. In certain embodiments, aryl groups are phenyl or naphthyl. Aryl groups include aromatic-aliphatic fused ring systems. Examples include, but are not limited to, indanyl and tetrahydronaphthyl.

[0274] The term "heterocyclyl" is intended to include non-aromatic ring systems containing three or more ring atoms, one or more of which is a heteroatom. In certain embodiments, the heteroatom is nitrogen, oxygen, or sulfur. In certain embodiments, heterocyclyl groups contain 1, 2, 3, or 4 heteroatoms. In certain embodiments, heterocyclyl groups include monocyclic, bicyclic, and tricyclic rings having 3 to 16, 3 to 14, 3 to 12, 3 to 10, 3 to 8, or 3 to 6 ring atoms. Heterocyclyl groups include partially unsaturated and saturated ring systems, such as imidazolinyl and imidazolidinyl. Heterocyclyl groups include fused and bridged ring systems containing heteroatoms, such as quinuclidyl. Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, azepanyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, isoxazolidinyl, morpholinyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolidinyl, and trithianyl.

[0275] The term "heteroaryl" is intended to include aromatic ring systems containing five or more ring atoms, one or more of which is a heteroatom. In certain embodiments, the heteroatom is nitrogen, oxygen, or sulfur. In certain embodiments, heteroaryl groups include monocyclic, bicyclic, and tricyclic ring systems having 5 to 16, 5 to 14, 5 to 12, 5 to 10, 5 to 8, or 5 to 6 ring atoms. Heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl, benzothiazolyl, imidazopyridinyl, isoxazolopyridinylxanthinyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl. Heteroaryl groups include fused ring systems in which all of the rings are aromatic, e.g., indolyl, and fused ring systems in which only one of the rings is aromatic, e.g., 2,3-dihydroindolyl.

[0276] The term "halo" or "halogen" is intended to include F, Cl, Br, and I.

[0277] The term "heteroatom" is intended to include oxygen, nitrogen, sulfur, or phosphorus. In certain embodiments, heteroatoms are selected from the group consisting of oxygen, nitrogen, and sulfur.

[0278] As used herein, the term "substituted" is intended to mean that one or more hydrogen atoms in the indicated group are replaced with one or more independently selected suitable substituents, provided that the substituent does not exceed the normal valence of each atom to which it is attached and that the substitution results in a stable compound. In some embodiments, optional substituents in the compounds described herein include, but are not limited to, halo, CN, NO, OH, NH, NHR. 100 , N.R. 100 R 200 , C 1~6 Haloalkyl, C 1~6 Haloalkoxy, C(O)NH2, C(O)NHR 100 , C(O)NR 100 R 200 , SO2R 100 , OR 100 , S.R. 100 , S(O)R 100 , C(O)R 100 , and C 1~6 aliphatic groups; where R 100 and R 200 are each independently, C 1~6 Aliphatic, e.g., C 1~6 It is alkyl.

[0279] Where a protecting group (PG) is shown, one skilled in the art will readily understand what type of protecting group is suitable.

[0280] The term "amine protecting group" as used herein is intended to mean a group that can be easily removed to provide the NH group of the amine group and that protects the amine group from undesired reactions during synthetic procedures. Such protecting groups are described in Protective Groups in Organic Synthesis, edited by T.W. Greene et al. (John Wiley & Sons, 1999) and in 'Amino Acid-Protecting Groups' by Fernando Albericio (with Albert Isidro-Llobet and Mercedes Alvarez), Chemical Reviews 2009(109)2455-2504. Examples include, but are not limited to, acyl and acyloxy groups such as acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, picolinoyl, aminocaproyl, benzoyl, methoxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,2,2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl, tert-butyloxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, etc. Further examples include Cbz (carboxybenzyl), Nosyl (o- or p-nitrophenylsulfonyl), Bpoc (2-(4-biphenyl)isopropoxycarbonyl), and Dde (1-(4,4-dimethyl-2,6-dioxohexylidene)ethyl). In certain embodiments, amine protecting groups for the purposes described herein include (but are not limited to) tert-butyloxycarbonyl (t-Boc) and 9H-fluoren-9-ylmethoxycarbonyl (Fmoc).

[0281] As used herein, the term "carboxyl-protecting group" is intended to mean a group that can be easily removed to provide an OH group of the carboxyl group and that protects the carboxyl group from undesired reactions during synthetic procedures. Such protecting groups are described in Protective Groups in Organic Synthesis, edited by T.W. Greene et al. (John Wiley & Sons, 1999) and in "Amino Acid-Protecting Groups" by Fernando Albericio (with Albert Isidro-Llobet and Mercedes Alvarez), Chemical Reviews 2009 (109) 2455-2504. Examples include, but are not limited to, alkyl and silyl groups, such as methyl, ethyl, tert-butyl, methoxymethyl, 2,2,2-trichloroethyl, benzyl, diphenylmethyl, trimethylsilyl, and tert-butyldimethylsilyl.

[0282] As used herein, the term "carboxamide protecting group" is intended to mean a group that can be easily removed to provide the NH group of the carboxamide group and that protects the carboxamide group from undesired reactions during synthetic procedures. Such protecting groups are described in Protective Groups in Organic Synthesis, edited by T.W. Greene et al. (John Wiley & Sons, 1999) and 'Amino Acid-Protecting Groups' by Fernando Albericio (with Albert Isidro-Llobet and Mercedes Alvarez), Chemical Reviews 2009(109)2455-2504. Examples include, but are not limited to, 9-xanthenyl (Xan), trityl (Trt), methyltrityl (Mtt), cyclopropyldimethylcarbinyl (Cpd), and dimethylcyclopropylmethyl (Dmcp).

[0283] The term "ester" refers to a carboxylic acid group in which the hydrogen of the hydroxyl group has been replaced with a saturated, straight-chain (i.e., linear) or branched hydrocarbon group. Specific examples of alkyl groups are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, and 2,2-dimethylbutyl. The alkyl group can be a C1-C6 alkyl group. As used herein, a term specifying an end value of a length range, such as "1-5," means any integer between 1 and 5, i.e., 1, 2, 3, 4, and 5. In other words, any range specified by two explicitly stated integers is meant to include and disclose any integers specifying the end values ​​and any integers within the range. The alkyl group can be a branched alkyl group.

[0284] As used herein, "Ser" refers to the amino acid serine and "Cys" refers to the amino acid cysteine.

[0285] As used herein, "PEG" refers to the polymeric compound polyethylene glycol. Unless otherwise defined, reference to "PEG" includes any length polymer of ethylene oxide. Reference to PEG also includes substituted PEG. In some embodiments, the substituted PEG can be defined by formula B-I or B-II, as described herein.

[0286] As used herein, the term "and / or" means "and," or "or," or both.

[0287] The term "(s)" after a noun contemplates the singular and / or plural.

[0288] Reference to a range of numerical values ​​disclosed herein (e.g., 1 to 10) also incorporates a reference to every rational number within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10), and further to any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus all subranges of every range explicitly disclosed herein are intended to be expressly disclosed herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the minimum and maximum values ​​recited are likewise considered to be expressly stated in this application.

[0289] [Immune system stimulant] In one aspect of the invention, there is provided a method for treating, preventing, or minimizing the progression of cancer, comprising administering a TLR2 agonist and an immune stimulant.

[0290] Those skilled in the art will understand that the terms "immunostimulant," "immunostimulatory," or "stimulating / inducing an immune response," and grammatical equivalents, as used herein, refer to inducing, increasing, promoting, or otherwise providing a beneficial effect with respect to the immune response.

[0291] "Immunostimulatory" or "immunostimulatory" or "stimulating / inducing an immune response" refers to the direct or indirect response of immune system cells or components to any of the treatments described herein. Such a response can be measured by any means known in the art, including activation, proliferation, or differentiation of immune system cells (B cells, T cells, dendritic cells, APCs, macrophages, NK cells, NKT cells, etc.), up-regulated or down-regulated expression of markers, cytokines, interferons, IgM and IgG release in serum and mixed cellular infiltrates in various organs.

[0292] Immunostimulatory agents can include specific immunostimulatory agents and non-specific immunostimulatory agents. Specific immunostimulatory agents provide antigen specificity in the immune response, such as a vaccine or any antigen. Non-specific immunostimulatory agents act regardless of antigen specificity to enhance immune responses to other antigens, or stimulate components of the immune system without antigen specificity, such as adjuvants and non-specific immunostimulatory agents.

[0293] Such immunostimulatory agents can help the immune system in one or more different ways, such as by enhancing or strengthening the immune system by stimulating antigen-presenting cells, T cells, or innate immune cells; by reducing immunosuppression in the tumor environment by modulating inhibitory pathways; and / or by promoting adaptive or innate immune responses. Thus, stimulation of the immune system can be of innate immune system cells or adaptive immune system cells. Immunostimulatory agents can inhibit the immunosuppressive effects induced by cancer cells or antigen-presenting cells.

[0294] As used herein, an immunostimulatory agent may be a molecule that does not directly stimulate the immune system but can repolarize immune cells or inhibit immunosuppression, or may directly stimulate or activate the immune system.

[0295] Innate immunity refers to immune responses that occur rapidly after infection or the onset of cancer. They are initiated without prior sensitization to pathogens or malignant cells, are not antigen-specific, and are mediated directly by phagocytes such as macrophages, cytotoxic cells such as natural killer (NK) cells, and antigen-presenting cells such as dendritic cells (DCs), as well as indirectly by cytokines produced by these cells. Adaptive immunity, or cellular immunity, refers to responses that must occur after initial infection or cancer onset, as the case may be, and involves the education of immune cells, resulting in the development of highly specific, highly potent, and long-lasting responses. This is mediated by cytotoxic T-lymphocytes (CTLs), helper T-lymphocytes, and antibody-producing B-lymphocytes. Along these lines, adaptive immune responses are classified as either cellular (those mediated by CTLs) or humoral (antibody-mediated responses), with helper T-lymphocytes promoting both responses. Taken together, a rapid innate immune response functions to control early metastasis of disease and promotes the development of an adaptive immune response, while a highly potent, highly specific, and long-lasting adaptive response functions to eliminate disease and prevent recurrence.

[0296] The present invention contemplates the use of immunostimulants in combination with any of the TLR2 agonists described herein for the treatment, prevention, or minimization of the progression of cancer. Suitable immunostimulants that may be used in accordance with the methods described herein include: - cellular immunotherapy (cytotoxic cellular immunotherapy or adoptive cellular immunotherapy); -Oncolytic viruses; -Cancer vaccines; -T cell engagers; and -Bispecific T cell engagers Examples include:

[0297] Specific examples of suitable immunostimulants include Blincyto (blinatumomab), Oncotice (Bacille Calmette-Guérin [BCG] [Tice strain] vaccine), BCG (BCG [Rivm strain] vaccine), ImmuCyst (ImmuCyst), Pacis (BCG [Montreal strain] vaccine), Provenge (sipuleucel-T), DCVax-L (DCVax-L), Oncorine (human adenovirus type 5 [recombinant]), and Imlygic (talimogene laherparepvec).

[0298] Immune stimulatory agents suitable for use in the methods of the present invention also include activators of costimulatory molecules or inhibitors of immune checkpoint molecules. Thus, the immune stimulatory agent can be an immune checkpoint inhibitor, a costimulatory molecule agonist, or an immune activator. Examples of checkpoint inhibitors useful in the present invention are described herein. Inhibition of inhibitory molecules can be achieved by inhibition at the DNA, RNA, or protein level. In embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA, or shRNA) can be used to inhibit the expression of inhibitory molecules. In other embodiments, the inhibitor of an inhibitory signal is a polypeptide that binds to the inhibitory molecule, such as a soluble ligand, or an antibody or antigen-binding fragment thereof.

[0299] The costimulatory molecule can be any one of the following costimulatory molecules selected from agonists of one or more of OX40, CD2, CD27, CDS, ICAM-1, LFA-1, (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand. Agonists of any one of these costimulatory molecules are also contemplated as immunostimulatory agents.

[0300] [Checkpoint inhibitors] "Checkpoint inhibitors" inactivate proteins in the inhibitory checkpoint pathways of the immune response.

[0301] With respect to cancer, checkpoint inhibitors modulate the immune system by blocking proteins that stop the immune system from attacking cancer cells. In particular, they control how detection-evading cancer cells and T cells interact so that T cells can recognize tumor cells and mount an appropriate immune response against them. Non-limiting examples of checkpoint inhibitors that can be used in accordance with the methods described herein include inhibitors that target PD-1 (programmed cell death protein 1), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), and PD-L1 (programmed death ligand 1). Those skilled in the art will understand that CTLA-4 and PD-1 are found on T cells, and PD-L1 is expressed on cancer cells. Other non-limiting examples include PD-L2, TIM3, LAG3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD107), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF-β.

[0302] Other immune checkpoints include indoleamine 2,3-dioxygenase (IDO) and CSF-R1. Inhibitors of these proteins are also contemplated as immune checkpoint inhibitors for use in the present invention.

[0303] "Programmed death-1 (PD-1)" refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is primarily expressed on activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence can be found at GenBank accession number U64863.

[0304] When PD-1 binds to programmed death-ligand 1 (PD-L1), the immune response is shut down to prevent T cells from damaging or killing the cells. In the context of cancer, cancer cells can evade the immune response by being coated with PD-L1 protein to make them appear healthy. Programmed death-ligand 1 (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (HPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found in GenBank accession number Q9NZQ7.

[0305] Cytotoxic T-lymphocyte antigen-4 (CTLA-4) refers to an immunoinhibitory receptor belonging to the CD28 family. CTLA-4 is expressed exclusively on T cells in vivo and binds to two ligands, CD80 and CD86 (also known as B7-1 and B7-2, respectively). As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found in GenBank accession number AAB59385.

[0306] Any of the checkpoint inhibitors described herein may be administered in the form of an antibody. "Antibody" (Ab) is intended to include, but is not limited to, a glycoprotein immunoglobulin, or an antigen-binding portion thereof, that specifically binds to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further divided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigen. The constant regions of Abs can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0307] The term "antibody" includes, by way of example, monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless otherwise specified, and unless the context requires otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the above immunoglobulins, including monovalent and bivalent fragments or portions, and single-chain Abs.

[0308] An "isolated antibody" refers to an Ab that is substantially free of other Abs with different antigen specificities (e.g., an isolated Ab that specifically binds to PD-1 is substantially free of Abs that specifically bind to antigens other than PD-1). However, an isolated Ab that specifically binds to PD-1 may have cross-reactivity with other antigens, such as PD-1 molecules from different species. Furthermore, an isolated Ab may be substantially free of other cellular material and / or chemicals. The term "monoclonal antibody" (mAb) refers to a non-naturally occurring preparation of Ab molecules of single molecular composition, i.e., Ab molecules that are essentially identical in nucleotide sequence and display a single binding specificity and affinity for a particular epitope. mAbs are an example of isolated Abs. mAbs may be produced by hybridoma, recombinant, transgenic, or other techniques known to those of skill in the art.

[0309] A "human" antibody (huMAb) refers to an Ab having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the Ab contains a constant region, the constant region also is derived from a human germline immunoglobulin sequence. The human Abs of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include Abs in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms "human" Ab and "fully human" Ab are used interchangeably.

[0310] A "humanized antibody" refers to an Ab in which some, most, or all of the amino acids outside the CDR domains of a non-human Ab have been replaced with corresponding amino acids from a human immunoglobulin. In one embodiment of a humanized form of an Ab, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not abolish the Ab's ability to bind to a particular antigen. A "humanized" Ab retains antigen specificity similar to that of the original Ab.

[0311] A "chimeric antibody" refers to an Ab whose variable region is derived from one species and whose constant region is derived from another species, e.g., the variable region is derived from a murine Ab and the constant region is derived from a human Ab.

[0312] An "anti-antigen" Ab refers to an Ab that specifically binds to an antigen. For example, an anti-PD-1 Ab specifically binds to PD-1, and an anti-CTLA-4 Ab specifically binds to CTLA-4.

[0313] An "antigen-binding portion" (also called an "antigen-binding fragment") of an Ab refers to one or more fragments of an Ab that retain the ability to specifically bind to the antigen bound by the whole Ab.

[0314] Examples of immune checkpoint and antibody inhibitors that target these checkpoints include anti-CTLA-4 (e.g., ipilimumab, tremelimumab, KAHR-102), anti-TIM3 (e.g., F38-2E2.ENUM005), anti-LAG3 (e.g., BMS-986016, IMP701.IMP321, C9B7W), anti-KIR (e.g., lirilumab, IPH2101, IPH4102), anti-PD-1 (e.g., nivolumab, pidilizumab, pembrolizumab, BMS-936559, atezolizumab, lambrolizumab, MK-3475), and anti-PD-1 (e.g., nivolumab, pidilizumab, pembrolizumab, BMS-936559, atezolizumab, lambrolizumab, MK-3475). AMP-224, AMP-514, STI-A1110, TSR-042), anti-PD-L1 (e.g., KY-1003 (EP20120194977), MCLA-145, atezolizumab, BMS-936559, MEDI-4736, MSB0010718C, AUR-012, STI-A1010, PCT / US2001 / 020964, MPDL3280A, AMP-224, dapirolizumab pegol (CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42 (OSU-HDAC42), HDAC-42, AR42, AR 42, OSU-HDAC 42, OSU-HDAC-42, NSC D736012, HDAC-42, HDAC 42, HDAC42, NSCD736012, NSC-D736012, MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1, anti-B7-H4, anti-CD52 (such as alemtuzumab), anti-IL-10, anti-IL-35, anti-TGF-β (such as fresolimumab), anti-CSF1R (e.g., FPA008), anti-NKG2A (e.g., monalizumab), anti-MICA (e.g., IPH43), and anti-CD39.

[0315] [Anti-PD-1 antibody and anti-PD-L1 antibody] Examples of suitable PD-1 inhibitors that may be used in accordance with the present invention include Keytruda (pembrolizumab), Opdivo (nivolumab), AGEN 2034, BGB-A317, BI-754091, CBT-501 (genolimzumab), MEDI0680, MGA012, PDR001, PF-06801591, REGN2810 (SAR439684), and TSR-042 or those disclosed in U.S. Pat. No. 8,008,449. Other anti-PD-1 mAbs are described, for example, in U.S. Pat. Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, and PCT Publication No. WO 2012 / 145493.

[0316] Nivolumab (also known as "Opdivo®"; formerly designated 5C4, BMS-936558, MDX-1106, or ONO4538) is a fully human IgG4(S228P) PD-1 immune checkpoint inhibitor Ab that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of antitumor T cell function (U.S. Pat. No. 8,008,449).

[0317] Pembrolizumab (also known as "Keytruda®," lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Pat. Nos. 8,354,509 and 8,900,587. Pembrolizumab is approved by the FDA for the treatment of recurrent or refractory melanoma.

[0318] Other suitable PD-1 inhibitors include Libtayo (cemiplimab), Blincyto (blinatumomab), dostallimab, spartalizumab, cetrelimab, pidilizumab, and BI-754091.

[0319] Anti-PD-1 Abs suitable for use in the disclosed methods or compositions are Abs that bind to PD-1 with high specificity and affinity, block PD-L1 and / or PD-L2 binding, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, the anti-PD-1 antibodies include antigen-binding portions or fragments that bind to the PD-1 receptor, inhibit ligand binding, and exhibit functional properties similar to those of the whole Ab in downregulating the immune system.

[0320] In certain embodiments, the anti-PD-1 antibody used in the methods may be replaced with another PD-1 or anti-PD-L1 antagonist. For example, an anti-PD-L1 antibody may replace the use of an anti-PD-1 antibody in the methods disclosed herein, as the latter prevents the interaction between PD-1 and PD-L1, thereby exerting a similar effect on the PD-1 signaling pathway. In any embodiment, suitable PD-L1 inhibitors include Imfinzi (durvalumab or MEDI4736), Tecentriq (atezolizumab or MPDL3280A), Bavencio (avelumab; MSB0010718C), MS-936559 (12A4 or MDX-1105), and CX-072.

[0321] [Anti-CTLA-4 antibody] The anti-CTLA-4 antibodies of the present invention bind to human CTLA-4 in a manner that prevents its interaction with the human B7 receptor. Because the interaction of CTLA-4 with B7 transmits a signal that leads to the inactivation of T-cells that have the CTLA-4 receptor, it will be understood that inhibition of the interaction effectively induces, enhances, or prolongs the activation of such cells, thereby inducing, enhancing, or prolonging an immune response.

[0322] Suitable CTLA-4 inhibitors that can be used in accordance with the present invention include Yervoy (ipilimumab), tremelimumab, and AGEN 1884, or those disclosed in U.S. Patent Nos. 6,984,720 and 7,605,238. Ipilimumab is a fully human, IgG1 monoclonal Ab that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation. Tremelimumab is a human IgG2 monoclonal anti-CTLA-4 antibody. Another is Blincyto (blinatumomab), a bispecific CD19-directed CD3 T cell engager.

[0323] [Administration and dosage] In one embodiment of the invention, therapeutically effective amounts of a TLR2 agonist and a checkpoint inhibitor are administered to a subject.

[0324] Administration refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including those described herein. Pharmaceutical compositions can be formulated from the compounds of the present invention described herein for any suitable route of administration. Typically, in addition to a therapeutic agent (e.g., a TLR2 agonist and / or an immunostimulant), a pharmaceutical composition contains a pharmaceutically acceptable excipient, carrier, and / or diluent. Examples of suitable components for inclusion in pharmaceutical compositions are described in Martindale-The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences.

[0325] Suitable routes of administration for practicing the defined methods include oral, intravenous, intramuscular, topical, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to methods of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and in vivo electroporation. Administration can also be, for example, single, multiple, and / or over one or more extended periods of time.

[0326] Any composition described herein can be formulated for administration to the respiratory tract, or in other words, by the respiratory route. Where administration to all or part of the respiratory tract is envisaged, those skilled in the art will understand that this includes administration into the nasal cavity or by inhalation, particularly for administration to the lungs. The compositions described herein can be formulated for intranasal administration, including as a dry powder, spray, mist, or aerosol.

[0327] Suitable formulations wherein the carrier is a liquid, for administration as, for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient. Alternatively, the composition may be a dry powder and, as defined herein, may be administered only to the respiratory tract.

[0328] The selection of an appropriate carrier will depend on the particular type of administration envisioned. For administration via the respiratory tract, e.g., the nasal mucosal surface, the compounds can be formulated in solutions, e.g., water or buffered or unbuffered isotonic saline, or as suspensions for intranasal administration as drops or sprays. Preferably, such solutions or suspensions are isotonic with respect to nasal secretions and have approximately the same pH, e.g., from about pH 4.0 to about pH 7.4, or from pH 6.0 to pH 7.0. Buffers should be physiologically compatible, and examples include, by way of example only, phosphate buffers. For example, a typical nasal decongestant is described as being buffered to a pH of about 6.2 (Remington's, Id., page 1445). Of course, one of ordinary skill in the art can readily determine the appropriate saline content and pH for a non-toxic aqueous carrier for nasal and / or respiratory administration.

[0329] Other ingredients, such as preservatives, colorants, lubricants, or viscous mineral or vegetable oils, flavorings, natural or synthetic plant extracts, such as aromatic oils, as well as humectants and viscosity enhancers, such as glycerol, may also be included to provide additional viscosity, moisture retention, and a favorable texture and aroma of the formulation. For nasal administration of the solutions or suspensions of the present invention, various devices for producing drops, droplets, and sprays are available in the art. For example, the TLR2 agonists and / or immunostimulants or compositions described herein may be administered intranasally using a manual pump, such as a simple dropper (or pipette) containing a glass, plastic, or metal dispensing tube that expels its contents dropwise by air pressure provided by a flexible rubber bulb attached to one end.

[0330] Because tear secretions in the eye drain from the orbit into the nasal cavity, if desired, suitable pharmaceutically acceptable ophthalmic solutions can be readily provided by those skilled in the art as carriers for the compounds or compositions described herein to be delivered and administered into the orbit in the form of eye drops to provide for both intraocular and intranasal administration.

[0331] In one embodiment, a pre-measured unit dose dispenser, including a dropper or spray device containing a solution or suspension for delivery as drops or a spray, is provided containing one or more doses of the drug to be administered. The invention also includes kits containing one or more unit dry doses of the compound, along with any necessary salts and / or buffers, preservatives, colorants, etc., ready to be prepared into a solution or suspension by the addition of a suitable amount of water. The water can be sterile or non-sterile, although sterile water is generally preferred.

[0332] The phrase "therapeutically effective amount" or "effective amount" generally refers to an amount of a TLR2 agonist and / or checkpoint inhibitor of the present invention, a pharmaceutically acceptable salt, polymorph, or prodrug thereof, that (i) treats a particular disease, condition, or disorder; (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder; or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. Undesirable effects, e.g., side effects, can sometimes occur along with the desired therapeutic effect; therefore, a physician balances the potential benefits against the potential risks when determining what is an appropriate "effective amount."

[0333] For example, for the treatment of tumors, a therapeutically effective amount of a compound or composition described herein may inhibit tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to untreated controls. Alternatively, the treatments described herein may cause a complete reduction in tumor burden. In other embodiments of the invention, tumor reduction may be observed and sustained for a period of at least about 10 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days, at least about 70 days, at least about 80 days, at least about 90 days, at least about 100 days, or more.

[0334] A therapeutically effective amount of an agent also includes a "prophylactic" or "prophylactically effective amount," which is any amount of a TLR2 agonist and / or checkpoint inhibitor administered to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or a subject who has relapsed from cancer, that inhibits the onset or recurrence of cancer. In certain embodiments, a prophylactically effective amount completely prevents the onset or recurrence of cancer. "Inhibiting" or "preventing" the onset or recurrence of cancer means either reducing the likelihood of the onset or recurrence of cancer, or completely preventing the onset or recurrence of cancer.

[0335] The exact therapeutically effective amount required may vary from subject to subject, depending on the species, age, and general condition of the subject, the method of administration, etc. Therefore, it may be impossible to define an exact therapeutically effective amount. However, the appropriate therapeutically effective amount in any individual case can be determined by one of ordinary skill in the art using only routine experimentation. In one embodiment, the dose administered to the subject is any therapeutically effective amount that reduces symptoms associated with cancer, resulting in any one of the following: a reduction in the number of cancer cells; a reduction in tumor size; inhibition (i.e., slowing to some extent, preferably stopping) of cancer cell invasion into peripheral organs; inhibition (i.e., slowing to some extent, preferably stopping) of tumor metastasis; inhibition (i.e., slowing to some extent, preferably stopping) of tumor growth; or alleviation of one or more symptoms associated with cancer. Additionally or alternatively, a therapeutically effective amount may result in an increase in the subject's survival time.

[0336] In certain embodiments, a therapeutically effective amount of a TLR2 agonist for a human subject is in the range of about 250 nmoles / kg body weight / dose to 0.005 nmoles / kg body weight / dose. Preferably, the range is about 250 nmoles / kg body weight / dose to 0.05 nmoles / kg body weight / dose. In certain embodiments, the body weight / dose range is about 250 nmoles / kg to 0.1 nmoles / kg, about 50 nmoles / kg to 0.1 nmoles / kg, about 5 nmoles / kg to 0.1 nmoles / kg, about 2.5 nmoles / kg to 0.25 nmoles / kg, or about 0.5 nmoles / kg to 0.1 nmoles / kg body weight / dose. In certain embodiments, the amount is 250 nmoles, 50 nmoles, 5 nmoles, 2.5 nmoles, 0.5 nmoles, 0.25 nmoles, 0.1 nmoles, or 0.05 nmoles / kg body weight / dose, or approximately such an amount of compound. Dosage regimens may be adjusted to suit the needs of the situation and to provide the optimum therapeutic dosage.

[0337] Typically, a therapeutically effective dose is formulated to contain a concentration (by weight) of at least about 0.1% up to about 50% or more, and all combinations and subcombinations of ranges therein. Compositions may be formulated to contain a concentration of one or more compounds, or pharmaceutically acceptable salts, polymorphs, or prodrugs thereof, of about 0.1 to less than about 50%, e.g., about 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40%, with concentrations ranging from greater than about 0.1%, e.g., about 0.2, 0.3, 0.4, or 0.5%, to less than about 40%, e.g., about 39, 38, 37, 36, 35, 34, 33, 32, 31, or 30%. Exemplary compositions may contain from about 0.5% to about 30%, e.g., less than about 29, 28, 27, 26, 25, 25, 24, 23, 22, 21, or 20%, with concentrations from greater than about 0.5%, e.g., greater than about 0.6, 0.7, 0.8, 0.9, or 1%, to less than about 20%, e.g., about 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10%. Compositions may contain from about 1%, e.g., greater than about 2%, to less than about 10%, e.g., less than about 9 or 8%, including concentrations from greater than about 2%, e.g., greater than about 3 or 4%, to less than about 8%, e.g., less than about 7 or 6%. The active agent may be present, for example, at a concentration of about 5%. In all cases, the amount may be adjusted to account for differences in the amount of active ingredient actually delivered to the treated cells or tissue.

[0338] For administration of checkpoint inhibitors, including PD-1, PD-L1, or CTLA-4 inhibitors, dosages can range from about 0.01 to about 20 mg / kg, about 0.1 to about 10 mg / kg, about 0.1 to about 5 mg / kg, about 1 to about 5 mg / kg, about 2 to about 5 mg / kg, about 7.5 to about 12.5 mg / kg, or about 0.1 to about 30 mg / kg of the subject's body weight. For example, dosages can be about 0.1, about 0.3, about 1, about 2, about 3, about 5, or about 10 mg / kg of body weight, or about 0.3, about 1, about 2, about 3, or about 5 mg / kg of body weight. Dosing schedules are typically designed to achieve an exposure that results in sustained receptor occupancy (RO) based on the typical pharmacokinetic properties of the Ab. Exemplary treatment regimens involve administration about once per week, about once per 2 weeks, about once per 3 weeks, about once per 4 weeks, about once per month, or about once every 3 to 6 months or more. In certain embodiments, the checkpoint inhibitor is administered to a subject about once per 2 weeks. In other embodiments, the Ab is administered about once per 3 weeks. Dosages and schedules can vary over the course of treatment. For example, dosing schedules for anti-PD-1 therapy can include administering Ab (i) about every 2 weeks in about a 6-week cycle; (ii) about every 4 weeks for about 6 doses, then about every 3 months; (iii) about every 3 weeks; or (iv) about 3 to about 10 mg / kg once, followed by about 1 mg / kg about every 2 to 3 weeks. Given that IgG4 Abs typically have a half-life of 2-3 weeks, dosing regimens for the anti-PD-1 Abs of the invention include about 0.3 to about 10 mg / kg body weight, 1 to 5 mg / kg body weight, or about 1 to about 3 mg / kg body weight by intravenous administration, with the Ab administered about every 14-21 days in cycles of up to about 6 weeks or about 12 weeks until a complete response or progressive disease is confirmed.

[0339] In certain embodiments, the checkpoint inhibitor and / or TLR2 agonist treatment disclosed herein continues for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 18 months, at least about 24 months, at least about 3 years, at least about 5 years, or at least about 10 years.

[0340] It will be understood that the specific dose level for any particular patient may vary depending on a variety of factors, including the activity of the specific compound used, age, body weight, general health, sex, dietary habits, time of administration, route of administration, and rate of excretion, concomitant medications (i.e., other drugs used to treat the patient), and the severity of the specific disorder being treated.

[0341] The term "treatment" or "treating" a subject includes the application or administration of a compound of the invention to a subject for the purpose of delaying, slowing, stabilizing, curing, curing, palliating, alleviating, altering, treating, arresting, alleviating, improving, or affecting a disease or condition, symptoms of a disease or condition, or risk for (or susceptibility to) a disease or condition. The term "treating" refers to any indicator of successful treatment or amelioration of an injury, condition, or condition, including any objective or subjective parameter, such as relief; remission; a reduction in the rate of deterioration; a reduction in the severity of the disease; a stabilization, reduction in symptoms, or making the injury, condition, or condition more tolerable to the subject; a reduction in the rate of deterioration or decline; a reduction in decline at the endpoint of deterioration; or an improvement in the physical or mental health of the subject.

[0342] As used herein, minimizing or preventing the progression of cancer means treating a subject to prevent or slow the recurrence or metastasis of a tumor, or to prevent an existing tumor. Minimizing or preventing the progression of cancer includes preventing or slowing the recurrence of cancer after treatment of the cancer, or preventing the growth of an existing tumor. Prevented recurrence includes, for example, recurrence in a tumor bed after surgical resection. Alternatively, recurrence includes metastasis of cancer to another part of the body. As used herein, the terms "preventing recurrence" and "preventing relapse" are synonymous.

[0343] The present invention also includes a method for preventing the onset of cancer in an individual. For example, an individual in need of cancer prevention may be considered to be at risk of developing cancer, but does not yet have detectable cancer. An individual at risk of developing cancer may be an individual with a family history of cancer, and / or an individual for whom a genetic or other test indicates a high risk or high likelihood of developing cancer. An individual may have cancer stem cells, but does not yet have any detectable tumors. It will be understood that a method for preventing the onset of cancer includes a method for delaying the onset of cancer in a subject.

[0344] It will be understood that the terms "subject" and "patient" are synonymous. While the present invention applies to humans, it is also useful for therapeutic veterinary purposes. It is useful for livestock or farm animals such as cattle, sheep, horses, and poultry; companion animals such as cats and dogs; and zoo animals.

[0345] [cancer] It will be understood that the term "cancer" includes benign, pre-cancerous, pre-cancerous or non-metastatic tumors or metastatic tumors.

[0346] In certain embodiments, the types of cancer to be treated include cancers having benign, precancerous, precancerous, or non-metastatic tumors. It will be understood that a benign tumor is not a malignant tumor and does not invade adjacent tissues or spread to other parts of the body. Similarly, it will be understood that a non-metastatic cancer does not invade adjacent tissues or spread to other parts of the body. "Precancerous" or "precancerous" generally refers to a condition or growth that typically precedes or develops into cancer. A "precancerous" growth may have cells characterized by aberrant cell cycle regulation, proliferation, or differentiation, which may be determined by cell cycle markers.

[0347] In one embodiment, the cancer is a secondary cancer or metastasis. The secondary cancer may be located in any organ or tissue, particularly an organ or tissue with relatively high hemodynamic pressure, such as the lung, liver, kidney, pancreas, intestine, and brain. The secondary cancer may be detected in ascites and / or lymph nodes.

[0348] Precancerous, neoplastic, and metastatic cancers are specific examples to which the methods of the present invention may be applied. Broad examples include breast tumors, colon tumors, adenocarcinomas, mesotheliomas, bladder tumors, prostate tumors, germ cell tumors, hepatocellular carcinoma / cholangiocarcinoma, carcinomas, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinomas, melanomas, atypical fibroxanthomas, seminomas, nonseminomas, stromal Leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral tumors, bladder tumors, bone tumors, cervical masses, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilms' tumors.

[0349] Examples of specific cancers include, but are not limited to, adenocarcinoma, adenoma, adenofibroma, adenolymphoma, odontoma, AIDS-related cancer, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adrenocortical carcinoma, idiopathic myeloid metaplasia, alopecia, alveolar soft part sarcoma, ameloblastoma, angiokeratoma, angiolymphocytosis with eosinophilia, sclerosing hemangioma, hemangiomatosis, apdoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancer, intestinal cancer, brain stem glioma, brain and CNS tumors, breast cancer, branchiomas, CNS tumors, carcinoid tumors, cervical Cancer, childhood brain tumor, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancer, cutaneous T-cell lymphoma, carcinoma (e.g., Walker, basal cell, basosquamous cell, Brown-Pierce, ductal, Ehrlich tumor, Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell, papillary, sclerotic, bronchiolar, bronchogenic, squamous cell, and transitional cell), carcinosarcoma, cervical dysplasia, cystosarcoma phyllodes, cementoma, chordoma, choristoma, chondrosarcoma, chondroblastoma, craniopharyngioma, cholangioma, cholesteatoma, cystadenocarcinoma, cystadenoma, cystocarcinoma, cystadenoma, cyst ... Dermatofibrosarcoma, desmoplastic small round cell tumor, ductal carcinoma, dysgerminoma, endocrine carcinoma, endometrial carcinoma, ependymoma, esophageal cancer, Ewing's sarcoma, extrahepatic bile duct cancer, eye cancer, ocular melanoma, retinoblastoma, fallopian tube cancer, Fanconi anemia, fibroma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, genitourinary cancer, germ cell tumor, gestational trophoblastic disease, glioma, gynecological cancer, giant cell tumor, ganglioneuroma, glioma, glomus angiomas, granulosa cell tumor, hemispheric ovarian tumor, hematologic malignancies, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papilloma Pillomavirus, hydatidiform mole, hypercalcemia, hypopharyngeal carcinoma, hamartoma, hemangioendothelioma, hemangiopericytoma, hemangioendothelioma, histiocytic disorders, malignant histiocytosis, histiocytoma, hepatocellular carcinoma, hidradenoma, chondrosarcoma, immunoproliferative disorders, opoma, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leiomyosarcoma, leukemia (e.g., B cell, mixed cell, null cell,T cell, T cell chronic, HTLV-II associated, lymphangiosarcoma, lymphocytic acute, lymphocytic chronic, mast cell and myeloid), leukemia, Leydig cell tumor, liposarcoma, leiomyoma, leiomyosarcoma, lymphangioma, lymphangiocytoma, lymphangiomyoblastoma, male breast cancer, malignant rhabdoid tumor of the kidney, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic carcinoma, oral cancer, multiple endocrine neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloma, myeloproliferative disorder, malignant carcinoid syndrome, carcinoid Cardiac disease, medulloblastoma, meningioma, melanoma, mesenchymal cell carcinoma, mesothelioma, myoblastic leiomyoma, fibroid, myosarcoma, myxoma, myxosarcoma, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen chromosomal instability syndrome, non-melanoma skin cancer, non-small cell lung cancer (NSCLC), schwannoma, neuroblastoma, neuroepithelioma, neurofibromatosis, neurofibroma, neuroma, neoplasms (e.g., bone, breast, digestive system, colorectal, liver), eye cancer, esophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ostomy ovarian cancer cancer), pancreatic cancer, sinus cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral neuroectodermal tumors, pituitary cancer, polycythemia vera, prostate cancer, osteoma, osteosarcoma, ovarian cancer, papilloma, paraganglioma, nonchromaffin paraganglioma, pinealoma, plasmacytoma, proto-oncogenes, rare cancers and related disorders, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson syndrome, reticuloendotheliosis, rhabdomyoma, salivary gland cancer, sarcoma, schwannoma, Sézary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine Cancer, soft tissue sarcoma, spinal tumor, squamous cell carcinoma (skin), gastric cancer, synovial sarcoma, sarcoma (e.g., Ewing's experimental sarcoma, Kaposi's and mast cell sarcoma), Sertoli cell tumor, synovioma, testicular cancer, thymic cancer, thyroid cancer, transitional cell carcinoma (bladder), transitional cell carcinoma (renal pelvis / ureter), trophoblastic carcinoma, teratoma, theca cell tumor, thymoma, trophoblastic tumor, urethral cancer, urinary system cancer, uroplakin, uterine sarcoma, uterine cancer, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0350] The presence, amelioration, treatment, or minimization of cancer progression may be determined by any clinically or biochemically relevant method described herein or known in the art. A favorable response to cancer treatment or minimization of cancer progression may be determined by any method known in the art and may include determining: - A decrease in the number of cancer cells; - Reduction in tumor size; - inhibiting (i.e., slowing to some extent and preferably stopping) cancer cell invasion into peripheral organs; - inhibiting (i.e., slowing to some extent and preferably stopping) tumor metastasis; - Reduction or complete prevention of tumor metastasis after removal of the primary tumor; - Some inhibition of tumor growth; - some relief of one or more of the symptoms associated with cancer; and / or -Increased subject survival time.

[0351] Any of the above determinations may be considered a favorable response to the TLR agonists and / or checkpoint inhibitors described herein.

[0352] In contrast, a negative response or lack of response of a cancer to a treatment comprising any of the TLR agonists and / or checkpoint inhibitors described herein may be determined by any method known in the art, and may include determining: - No change or increase in the number of cancer cells; - No change or increase in tumor size; - No change, persistent or increased cancer cell infiltration into peripheral organs; - No change, persistent or increased tumor metastasis; - No change or increased tumor metastasis despite removal of the primary tumor; - No change or increase in tumor growth; - One or more cancer-related symptoms remain unchanged or increase; and / or - No change or decrease in subject survival time.

[0353] In one embodiment of the invention, the subject may have received a previous treatment. In one embodiment, the previous treatment is a checkpoint inhibitor, which may be in the form of an inhibitor of PD-1, PD-L1, or CTLA-4. In a preferred embodiment, the checkpoint inhibitor is in the form of an antibody.

[0354] A subject treated for cancer may be in partial or complete remission. In other words, a subject treated for cancer as described above may have a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in measurable parameters of tumor growth, such as those seen in physical examinations, radiological examinations, or biomarker levels from blood or urine tests. Alternatively, when a subject is in complete remission, there is a complete disappearance of all detectable signs of the disease, such that the subject does not have any detectable signs of cancer. A subject may have substantially undetectable signs of cancer. A "substantially undetectable" cancer generally refers to a situation in which the treatment reduces the size, volume, or other physical measures of the cancer so that the cancer is not clearly detectable as a result of the treatment using relevant standard detection techniques, such as in vivo imaging.

[0355] The purpose or outcome of treatment with a TLR2 agonist and / or checkpoint inhibitor may be to reduce the number of cancer cells; reduce primary tumor size; inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder.

[0356] The efficacy of treatment may be measured by assessing survival, time to disease progression, response rate (RR), duration of response, and / or quality of life.

[0357] In one embodiment, the method is particularly useful for delaying the progression of cancer. In one embodiment, the method is particularly useful for extending the survival of a subject, including overall survival and progression-free survival. Overall survival will be understood to be the length of time from either the date of diagnosis or the start of cancer treatment that a patient diagnosed with cancer survives. Progression-free survival will be understood to be the length of time during or after cancer treatment that a patient survives with the disease but without it worsening.

[0358] Survival analysis can be performed using techniques well known in the art, including the Kaplan-Meier method. The Kaplan-Meier method estimates a survival function from lifespan data. In medical research, it can be used to measure the proportion of patients surviving a certain period of time after treatment. A Kaplan-Meier plot of a survival function is a series of horizontal steps of decreasing magnitude, which, if a sufficiently large sample is taken, approaches the true survival function for the population. The value of the survival function between successive distinct sample observations ("clicks") is assumed to be constant.

[0359] A key advantage of the Kaplan-Meier curve is that it can account for data loss from "censored" samples (e.g., when a patient drops out of the study) before the final outcome is observed. In this plot, small vertical tick marks indicate loss while patient data are censored. If no truncation or censoring occurs, the Kaplan-Meier curve is equivalent to the empirical distribution.

[0360] In one embodiment, the method is particularly useful for providing a complete response to treatment, whereby all signs of cancer disappear in response to treatment. This does not necessarily mean that the cancer has been cured. In one embodiment, the method is particularly useful for providing a partial response to treatment, whereby there is a decrease in the size of one or more tumors or lesions, or the extent of cancer in the body, in response to treatment.

[0361] [kit] In another embodiment, a kit or article of manufacture is provided comprising a TLR2 agonist and / or checkpoint inhibitor described herein, a pharmaceutically acceptable salt, a diluent or excipient, and / or a pharmaceutical composition as described above. Additionally, the kit may include instructions for use in any of the methods or uses of the invention described herein.

[0362] In another embodiment, there is provided a kit for use in the above therapeutic and / or prophylactic applications, comprising: - a container holding a therapeutic composition in the form of a TLR2 agonist and / or checkpoint inhibitor as described herein, or a pharmaceutically acceptable salt, diluent or excipient or pharmaceutical composition; -Label or package insert containing instructions for use A kit is provided comprising:

[0363] In certain embodiments, the kit may contain one or more additional active ingredients or materials for the treatment of cancer.

[0364] The kit or "article of manufacture" may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a therapeutic composition that is effective for treating a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used to treat the condition of choice. In one embodiment, the label or package insert includes instructions for use indicating that the therapeutic or prophylactic composition can be used to treat a cancer described herein.

[0365] The kit may include (a) the therapeutic or prophylactic composition; and (b) a second container having a second active ingredient or material contained therein. The kit in this embodiment of the invention may further include a package insert indicating that the composition and other active ingredients can be used to treat or prevent the progression of a cancer described herein.

[0366] As used herein, the following compounds are depicted in the specific structures shown in the tables below and elsewhere herein and are contemplated in any method or use of the invention.

[0367] [Table 1]

[0368] [Table 2]

[0369] [Table 3]

[0370] [Table 4]

[0371] [Table 5]

[0372] [Table 6]

[0373] [Table 7]

[0374] [Table 8]

[0375] [Table 9]

[0376] [Table 10]

[0377] [Table 11]

[0378] [Table 12]

[0379] [Table 13]

[0380] [Table 14]

[0381] The compounds of the present invention can be prepared by techniques known in the art. For example, the compounds of the present invention comprising any one of formulas (I) to (XIX) containing the A1 moiety can be prepared by the techniques described in International Publication No. 2019 / 119067, the entire contents of which are incorporated herein by reference.

[0382] Compounds of the present invention comprising any one of formulas (I)-(XIX) containing the A2 moiety include compounds of formula A2-I: [ka] (In the formula, L1, L2, Z1, Z2, v, b, w, z, R x , R y , R 11 , R12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and X has the meaning defined for any compound of the invention as defined herein, R 19 is an amino protecting group), Compounds of formula (YB-I): [ka] (In the formula, Y' is [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; B' is polyethylene glycol (PEG); [ka] is a solid support resin) It can be provided by coupling with

[0383] In some embodiments, B' comprises a substituted PEG of formula B1. In these embodiments, the following solid-phase reaction sequence may be used: a) optionally coupling, using Fmoc chemistry, 1 to 10 alpha amino acids or compounds derived from natural alpha amino acids that constitute L to a solid phase resin; b) PG-NH-(CH2) p -O-(CH2CH2O) n-(CH2) m coupling of -COOH to a solid phase resin or a substituted resin if L is present (where PG represents an amino protecting group compatible with Fmoc chemistry); c) removing PGs; d) PG-NH-CR 13 R 14 coupling -COOH (where PG' represents an amino protecting group compatible with Fmoc chemistry); e) removing PG'; f) coupling an acid of formula (AI); g) optionally, R 19 and optionally acylation and / or alkylation to form R 18 and / or R 19 Introducing; and h) Removing the compound from the solid support.

[0384] In some embodiments, where B' comprises a substituted PEG represented by formula (B-II), the following solid-phase reaction sequence may be used: a) optionally coupling, using Fmoc chemistry, 1 to 10 alpha amino acids or compounds derived from natural alpha amino acids that constitute L to a solid phase resin; b) PG-NH-(CH2) t -O-(CH2CH2O) k -(CH2) h coupling of -COOH to a solid phase resin or a substituted resin if L is present (where PG represents an amino protecting group compatible with Fmoc chemistry); c) removing PGs; d) PG'-NH-(CH2) p -O-(CH2CH2O) n -(CH2) m coupling -COOH (where PG' represents an amino protecting group compatible with Fmoc chemistry); e) removing PG'; f) PG''-NH-CR 13 R 14coupling -COOH (where PG'' represents an amino protecting group compatible with Fmoc chemistry); g) removing PG''; h) coupling an acid of formula (AI); i) optionally, R 19 and optionally acylation and / or alkylation to form R 18 and / or R 19 Incorporating; and j) Removing the compound from the solid phase resin.

[0385] It will be understood that the exact order of events may differ from that outlined and further steps, for example oxidation of the cysteine ​​sulfur to a sulfoxide or sulfone, may be added if necessary and synthetically convenient.

[0386] The compound of formula A2-I may be a compound of formula A2-II: [ka] (Wherein L1, L2, X, v, w and R 18 is as defined above for compounds of formula AI, and Z1 and Z2 are independently selected from -NHC(O)-, -C(O)NH-, -OC(O)-, -C(O)O-, -NHC(O)O-, and -OC(O)O-. It is provided in the form of

[0387] Compounds of formula A2-II can be provided by the synthesis shown in Scheme 1.

[0388] Scheme 1 depicts the synthesis of an embodiment of a compound of formula A2-II, wherein: X is S, L1-Z1 is -OC(O)E-(CH2) g -CH3, where E is -O- or -NH-, and g is 10, 11, 12, 13, 14, 15, 16, 17, or 18; L2-Z2 is -OC(O)E-(CH2) g-CH3, where E is -O- or -NH-, and g is 10, 11, 12, 13, 14, 15, 16, 17, or 18; R 19 is PG3, which is an amino protecting group.

[0389] [Scheme 1] [ka] Reaction of a protected alkene alcohol of formula (V') where PG is a suitable protecting group, e.g., a silyl group such as TBDMS, forms an epoxide of formula (VI'). It will be understood that the epoxide formation can be carried out to obtain the product racemically or to obtain an enantiomerically enriched material. If a racemic or scalemic mixture of enantiomers is produced, preparative chiral chromatography can be used to separate the enantiomers, if necessary.

[0390] The epoxide of formula (VI') is reacted with a suitably protected cystine analogue, such as tert-butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-S-(((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(tert-butoxy)-3-oxopropyl)thio)-D-cysteinate (wherein PG2 is the tert-butyl ester and PG3 is Fmoc) under reducing conditions to give an alcohol of formula (VII'). It will be appreciated that the alcohol of formula (VII') may consist of two or more stereoisomers, which, when present, may be separated by chiral preparative chromatography, if necessary.

[0391] The alcohol of formula (VII') is acylated using a suitable reagent to give a carbonyl-containing adduct of formula (VIII'). If an ester is required, an acid chloride can be reacted in the presence of a suitable base and solvent; if a carbamate is required, an isocyanate can be reacted in the presence of a suitable base and solvent; if a carbonate is required, a chloroformate can be reacted in the presence of a suitable base and solvent. The carbonyl-containing adduct of formula (VIII') can then be deprotected using a suitable reagent to expose the carboxylic acid of formula (IX'); for example, if PG2 is tert-butyl, trifluoroacetic acid can be used to preferentially remove the tert-butyl group.

[0392] The acid of formula (IX') can then be used as a reagent in solid phase synthesis to add groups of formula Y and B.

[0393] Compounds of the invention comprising any one of formulas (I) to (XIX) containing moiety A2 (wherein z is 1, w is 1 and b is 0) can be prepared by reacting a resin-bound peptide of the formula: [ka] (In the formula, Y' is [ka] and wherein R1 and R2 are independently selected from the group consisting of H, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2OPO(OH)2, -CH2C(=O)NH2, -CH2CH2C(=O)OH and -CH2CH2C(=O)OR8, wherein any one of the alkyl hydrogens can be replaced with a halogen; R8 is selected from the group consisting of H and linear or branched C1-C6 alkyl; B' is polyethylene glycol (PEG); PG s is H or a sulfur protecting group, such as tert-butyl; [ka] is a solid support resin) The compound can be provided by preparing

[0394] After optional sulfur deprotection, the resin-bound peptide is converted to a 1,2-epoxy-alkanol of the formula: [ka] (In the formula, R x , R y and v has the meaning given for moiety A2 in formula (I). to produce an alkylated thiol of formula S-1: [ka] where Y' and B' have the meanings given above and v has the meaning given for moiety A2), or a sulfone or sulfoxide thereof, is obtained.

[0395] The diol portion of resin-bound compound S-1 can be further reacted, such as by diol functionalization with palmitic acid or lauryl carbamate groups, to provide compounds of the present invention.

[0396] [Example] The following Examples 1-6 illustrate various properties of compounds of the present invention. The compounds tested in these Examples were prepared as described in WO 2019 / 119067 or in Synthesis Example 5.

[0397] Example 1: Effect of Combination Therapy on Tumor Growth, Survival, and Metastasis Blocking inhibitory immune cell receptors, also known as "immune checkpoints," has revolutionized the treatment of cancer. Despite impressive response rates, the majority of patients do not respond to or develop resistance to immune checkpoint blockade (ICB). The underlying mechanisms remain poorly understood, and there is an unmet need to develop new or improve existing immunotherapies. Here, we set out to investigate the effects of combining a synthetic TLR2 agonist, designated compound A101 (also referred to as compound 1 in these examples and figures), with anti-PD1 immunotherapy in multiple models of cancer and cancer metastasis.

[0398] Materials and Methods [mouse] C57BL / 6 or Balb / c wild-type (WT) mice were purchased from the Walter and Eliza Hall Institute for Medical Research, bred in-house, and maintained at the QIMR Berghofer Medical Research Institute. Mice older than 8 weeks were sex-matched to the appropriate model. The number of mice in each treatment group or strain for each experiment is indicated in the figure legends. In all studies, mice were not excluded based on predetermined criteria, and randomization was applied immediately before treatment in therapeutic experiments. Experiments were performed as approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee.

[0399] [Cell culture] Murine B16F10 (melanoma), MC38 (colon adenocarcinoma), and 4T1.2 (breast carcinoma) cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Bovogen), 1% glutamine (Gibco), and 1% penicillin / streptomycin (Gibco). B16F10 and 4T1.2 cells were maintained at 37°C and 5% CO. MC38 cells were maintained at 37°C and 10% CO. All cell lines were routinely tested negative for Mycoplasma, but cell line authentication was not routinely performed.

[0400] [Subcutaneous tumor model] For primary tumor growth experiments, MC38 (1 × 10 6 pcs) or B16F10(1×10 5 Mice were injected subcutaneously (sc) with 100 μl of cells in a final volume (day 0). Treatment of mice began as indicated in the figure or legend. The perpendicular diameter of each individual tumor was measured using a digital caliper. Tumor size was calculated and presented as the mean ± SEM. When tumors reached 150 mm 2 Mice were sacrificed when they reached a size of 100 mm.

[0401] [4T1.2 breast cancer metastasis model] 5×10 4 4T1.2 cells were injected into the fourth mammary fat pad in a volume of 50 μl (day 0). 12 days after injection, the primary tumor was surgically resected under isoflurane anesthesia. Mice were injected and treated as indicated in the figure legends on days 15, 18, and 21 after tumor cell injection. All mice were sacrificed on day 26 after tumor cell injection for evaluation of metastatic burden. For this purpose, macroscopic lung metastases were counted.

[0402] [Immune checkpoint blockade experiment] Immunotherapy was initiated when the implanted tumors reached a size of 3-5 mm in diameter. Therapeutic blockade of PD-1 was performed by intraperitoneal (ip) injection of 250 μg of rat anti-mouse PD-1 IgG2a (clone RMP1-14; BioXcell) or control rat IgG2a mAb (clone 2A3; BioXcell) in 100 μl of PBS. The amount of administration and the exact treatment schedule are indicated in the figures and / or legends.

[0403] [Intratumoral injection of Compound 1 / Compound A101] When mice received the first injection of anti-PD1 or control IgG, they were further treated with Compound 1. For this, 100 μl of a solution of Compound 1 in saline was injected intratumorally. The doses and treatment schedules are indicated in the figures and / or legends.

[0404] [Intravenous injection of Compound 1 / Compound A101] When mice received the first injection of anti-PD1 or control IgG, they were further treated with Compound 1. For this, 10 μg of Compound 1 in 200 μl of saline was injected into the lateral tail vein. The treatment schedule is shown in the figures and / or legends.

[0405] [result] C57BL / 6 WT mice were subcutaneously injected with MC38 cells and treated as indicated once tumors became palpable (Figure 1A). Anti-PD1 immunotherapy in combination with Compound 1 significantly improved survival of MC38 tumor-bearing mice (Figure 1B).

[0406] In the next experiment, the low-immunogenic and immunotherapy-resistant B16F10 melanoma cell line was injected into C57BL / 6 WT mice. Similarly, once tumors became palpable, mice were randomly divided into four groups and treated as indicated (Figure 2A). Surprisingly, there was an improved survival time in B16F10-bearing mice treated with Compound 1 and anti-PD1 (Figure 2B). Importantly, anti-PD1 monotherapy did not affect survival.

[0407] Taken together, we demonstrate that compound 1 effectively combines with anti-PD1 immunotherapy when injected locally into the tumor microenvironment in both highly and poorly immunogenic preclinical models of cancer.

[0408] While local injection is possible, it presents challenges in routine clinical practice. Furthermore, many cancer entities are inaccessible for intratumoral injection. Therefore, we investigated the therapeutic efficacy of systemically administered compound 1 in combination with anti-PD1 immunotherapy. To this end, C57BL / 6 WT mice were subcutaneously (sc) injected with MC38 cells. Once tumors became palpable, mice were intravenously (iv) injected with 10 μg of compound 1 or control, and intraperitoneally (ip) injected with anti-PD1 or control IgG (Figure 3A). Combination immunotherapy of compound 1 with anti-PD1 was superior to anti-PD1 monotherapy (Figure 3B).

[0409] These data demonstrate that compound 1 improves the efficacy of anti-PD1 immunotherapy in MC38 and B16F10 tumor models. Because metastatic dissemination of tumor cells is a major cause of death in cancer patients, we next evaluated the efficacy of combination immunotherapy in a spontaneous metastasis model. To this end, the highly aggressive 4T1.2 breast cancer cell line was injected into the fourth mammary fat pad of Balb / c WT mice. Similar to typical clinical procedures, the primary tumors of interest were surgically resected, and mice were treated with compound 1 alone or in combination with anti-PD1 immunotherapy (Figure 4A). All mice were sacrificed 14 days after surgery, and the number of lung metastases was assessed. Primary 4T1.2 tumors as well as disseminated cancer cells exhibited strong resistance to anti-PD1 immunotherapy. Systemic administration of 10 μg of compound 1 resulted in a lower number of lung metastases. Importantly, combination immunotherapy significantly reduced the metastatic burden in mice (Figure 4B) without affecting body weight loss (data not shown).

[0410] In summary, we demonstrate herein that Compound 1, a highly specific TLR2 agonist, enhances the efficacy of anti-PD1 immunotherapy, providing a rationale for such combination therapy in the treatment of cancers of diverse etiologies and pathogenesis. These results also demonstrate the utility of TLR2 agonist and checkpoint inhibitor combination therapy in cancers previously resistant to checkpoint inhibitor therapy.

[0411] Example 2. Effect of combination therapy on tumor growth and survival in the EMT6.5 model. Balb / c mice (n=48) were implanted with 100,000 EMT6.5 cells into the fourth inguinal mammary gland, and established tumors were monitored by caliper measurement. 3 Once the mice reached the target tumor size, they were randomly assigned to one of six treatment arms (n=8 per arm). To test additional TLR2 agonists, compound A108 was administered intravenously to tumor-bearing mice every third day for three doses. Checkpoint inhibitors were administered intraperitoneally on the same day. Treatment regimens including compound A108 induced a mild but reversible decrease in body weight (data not shown). The data show that compound A108 combined with anti-PDL-1 enhances the effect on tumor growth (Figure 5A, C). Compound A108 combined with anti-PL-1 also shows improved survival (Figure 5B).

[0412] These data demonstrate that repeated doses of systemically administered compound A108 were well tolerated in Balb / c mice bearing EMT6.5 tumors, and that repeated doses of compound A108 and anti-PDL-1 can statistically enhance the efficacy of various types of checkpoint inhibitors.

[0413] Example 3. Effect of combination therapy on tumor growth and survival in MC38-bearing mice. Compound A108 was also tested in the MC-38 model in the presence of checkpoint inhibitors. C57BL / 6 WT mice (n=10-14) were subcutaneously injected with highly immunogenic MC38 colon cancer cells. Once tumors became palpable (approximately 3-5 mm in diameter), mice were randomly divided into four groups and received three intratumoral injections of vehicle, anti-PD1 (200 μg, intraperitoneal (ip)), 25 μg of compound A108 in 100 μl of saline, or a combination of compound A108 and anti-PD-1. This study demonstrates that combination therapy improves mouse survival (Figure 6).

[0414] The above studies were further investigated by examining the effect of the combination therapy on large tumors in the MC38 mouse model. These studies aimed to understand whether repeated dosing over a two-week period could slow tumor growth. Mice were administered repeated doses via the intraperitoneal (ip) (10 μg dose) route every other day for 13 days. Mice were sacrificed when they reached a humane endpoint or one week after the last dose (up to the 25th day), whichever came first. Humane endpoints were a weight loss of more than 20% compared to the weight on the first day of treatment (or a weight loss of more than 15% on three consecutive days), a weight loss of 3000 mm 3 Individual mouse tumor volumes greater than 2,000 mm 3 The mean tumor volume of the group was >100% (all mice in the group were sacrificed). This study demonstrates that the combination therapy can slow tumor growth during the treatment phase via the intraperitoneal (ip) route (Figure 7).

[0415] These data demonstrate that the combination of compound A108 and anti-PD1 immunotherapy can result in reduced tumor growth and prolonged survival in mice bearing MC38 colon cancer.

[0416] Example 4. Compound A108 has antitumor activity in the WEHI164 model. This study was conducted to determine whether a low dose of compound A108 (2.5 μg) in combination with anti-CTLA4, anti-PDL1, or anti-PD-1 could inhibit tumor growth. The data from this experiment demonstrate that anti-PDL1 (Figure 8A), anti-CTLA-4 (Figure 8B), and anti-PD-1 (Figure 8C) have greater efficacy when administered in the presence of compound A108.

[0417] These data demonstrate that compound A108 increases response rates to anti-CTLA4, anti-PDL1 and anti-PD-1 checkpoint therapy in the WEHI-164 sarcoma model of cancer.

[0418] Example 5. Representative synthesis, characterization and TLR2 activity of selected compounds of the present invention: A107, A108, A115, A116, A117, A118, A203, A204, A215, A216, A220 and A224. [Synthesis of Compounds A107 (x=11) and A108 (x=27)] Fmoc S-2,3-di(palmitoyloxypropyl)-cysteine ​​(S-Fmoc-Dpc-OH) was purchased from Bachem Inc.

[0419] [Coupling of S-Fmoc-Dpc-OH to resin-bound peptide:] Fmoc-Dpc-OH (100 mg, 0.24 mmol) was activated with HOBt (36 mg, 0.24 mmol) and N,N'-diisopropylcarbodiimide (DICI; 37 µL, 0.24 mmol) in DCM and DMF (1:1, v / v, 3 mL) at 0 °C for 5 min. The mixture was then converted to Boc-Cys-Ser(tBu)CHCHO-(PEG). 11 -CH2CH2C(O)Gly resin or Boc-Cys-Ser(tBu)-CH2CH2O-(PEG) 27The resulting solution is added to a vessel containing -CH2CH2C(O)Gly resin (0.25 mmol / g, 0.25 g = 0.0625 mmol). After shaking for 2 hours, the solution is removed by filtration on a glass sinter funnel (porosity 3) and the resin is washed with DCM and DMF (3 x 30 mL each). The reaction is monitored for completion using the trinitrobenzenesulfonic acid (TNBSA) test. If necessary, a double coupling is performed.

[0420] Cleavage of peptide from solid support: Reagent B (93% TFA, 5% water, and 2% triisopropylsilane) for 2 hours. The peptide did not precipitate in cold ether. Most of the TFA had to be removed, and the residue was then dissolved in 50% acetonitrile and purified immediately or lyophilized.

[0421] [Synthesis of Compounds A115 and A116] The synthesis of compounds A115 (x=11) and A116 (x=27) was carried out as shown in Scheme 2. (R)-Glycidol was coupled to the thiol group of a cysteine ​​residue attached to a peptide resin by alkylation; 250 mg of Boc-Cys-Ser(tBu)CHCHO-(PEG) saturated in DMF was used. 11 -CH2CH2C(O)Gly resin or Boc-Cys-Ser(tBu)CH2CH2O-PEG 27To --CH2CH2C(O)Gly resin (0.25 mmol / g, 0.25 g = 0.0625 mmol) was added 250 μl of R-(+)-glycidol (molecular weight = 74.08, d = 1.1, 250 μl = 3.71 mmol, 60-fold excess over the free sulfhydryl groups on the peptide resin) and 25 μl of diisopropylethylamine (DIPEA, molecular weight = 129.2, d = 0.74, 25 μl = 0.14 mmol). The reaction mixture was kept at 50°C in a water bath for 2 hours, and then the solid support was washed extensively with DMF. To 250 mg of peptide resin washed with toluene after glycidolization, 100 μl of ethyl methyl sulfide (W = 76.16, d = 0.842, 100 μl = 1.10 mmol) was added, followed by 105 μl of tetradecyl isocyanate (molecular weight = 239, d = 0.869, 105 μl = 0.38 mmol, i.e., a three-fold excess over each hydroxyl group present on the solid support), and finally, 210 μl of dibutyltin dilaurate (molecular weight = 631.6, d = 1.053, 210 μl = 0.35 mmol). The reaction mixture was sparged with nitrogen gas for approximately 5 minutes and mixed overnight at room temperature (using an Intelli-Mixer RM-2, program F26). The reaction mixture was transferred to a 50 ml tube, and chloroform was added to 50 ml. After approximately 5 minutes of sonication, the white precipitate formed during the reaction dissolved. The solid support was washed with DMF and acetonitrile and the final product obtained after cleavage from the support was purified by HPLC.

[0422] [Scheme 2. Synthesis of Compounds A115 and A116] [ka] [Synthesis of Compounds A117 and A118] Compounds A117 (X = S( = O)) and A118 (X = S( = O)) were prepared following a similar synthetic route as described above for compound A115, except for the omission of the ethyl methyl sulfide scavenger and the optional omission of nitrogen sparging from the carbamate-forming step. Omission of the ethyl methyl sulfide scavenger gave a mixture of compounds A115, A117, and A118, which were separated and purified by HPLC.

[0423] Alternatively, sulfone or sulfoxide derivatives (e.g., A117 and A118) can be prepared by oxidation of the corresponding sulfides (e.g., A115) with an oxidizing agent such as meta-chloroperbenzoic acid (MCPBA) or tert-butyl hydroperoxide (t-BuOOH) under appropriate conditions.

[0424] [Synthesis of Compounds A203 and A204] The synthesis of compounds A203 and A204 is shown in Scheme 3 below.

[0425] After Fmoc-Gly was added as the first amino acid to the solid support, a two-fold molar excess of Fmoc-NHCHCHO-(PEG) was added in the presence of a two-fold excess of hexafluorophosphate benzotriazole tetramethyluronium (HBTU), hydroxybenzotriazole (HOBT), and a four-fold excess of diisopropylethylamine (DIPEA) in 2 ml of dimethylformamide (DMF) for 2 hours. 11 -CH2CH2COOH or Fmoc-NHCH2CH2O-(PEG) 27 -CH2CH2COOH was coupled. Next, Fmoc-Ser(tBu)-OH was coupled to give intermediate A2, followed by Boc-Cys(StBu)A1. The thiol-tert-butyl group on the cysteine ​​residue was removed by incubating the peptide resin in 0.5 M dithiothreitol in DMF for 1 hour at room temperature. 250 mg of Boc-Cys-Ser(tBu)-NHCH2CHO-(PEG) saturated in DMF was added. 11-CH2CH2C(O)Gly resin or Boc-Cys-Ser(tBu) CH2CH2O-(PEG) 27 To —CH2CH2C(O)-Gly resin (0.25 mmol / g, 0.25 g = 0.0625 mmol), 250 μl of R-(+)-1,2-epoxy-butan-4-ol [(R)-2-(oxiran-2-yl)ethan-1-ol] (M W = 88.11, d = 1.1, 250 μl = 3.125 mmol, corresponding to a 50-fold excess of free sulfhydryl groups present on the peptide resin) and 25 μl of diisopropylethylamine (DIPEA, M W = 129.2, d = 0.74, 25 μl = 0.14 mmol) was added. The reaction mixture was placed in a water bath at 50°C for 2 hours and then washed thoroughly with DMF to give intermediate A3.

[0426] Palmitic acid (320 mg, 1.25 mmol), DIPCDI (225 μL, 1.5 mmol), and 4-dimethylaminopyridine (DMAP; 15.25 mg, 0.125 mmol) were dissolved in 2 mL of dichloromethane (DCM) and then added to resin-bound BOC-Dhc-peptide resin A3 (0.0625 mmol, 0.25 g) and shaken at room temperature for 16 h. The supernatant was removed by filtration, and the solid support was thoroughly washed with DCM and dimethylformamide (DMF) to remove any residual urea before being subjected to the cleavage process described below.

[0427] The solid support with assembled lipopeptide was exposed to Reagent B (93% TFA, 5% water, and 2% triisopropylsilane) for 2 hours. To isolate the product, most of the TFA was removed, and the residue was then dissolved in 50% acetonitrile and either immediately purified using the purification protocol described below, or the material was lyophilized and stored for later purification.

[0428] [Scheme 3. Synthesis of Compound A203 (x=11) and Compound A204 (x=27)] [ka] [Synthesis of Compound A215 and Compound A216] The synthesis of compounds A215 and A216 was carried out as shown in Scheme 4. Intermediate A3 was prepared as described for compounds 3 and 4 above.

[0429] Next, after glycidolization, 250 mg of peptide resin, washed with toluene, was added with 100 μl of ethyl methyl sulfide (M W = 76.16, d = 0.842, 100 μl = 1.10 mmol), followed by 105 μl of tetradecyl isocyanate (MW = 239, d = 0.869, 105 μl = 0.38 mmol, i.e., a three-fold excess of each of the hydroxyl groups present on the solid support) and finally 210 μl of dibutyltin dilaurate (M W = 631.6, d = 1.053, 210 μl = 0.35 mmol) was added. The reaction mixture was sparged with nitrogen gas for approximately 5 minutes and mixed overnight at room temperature (Intelli-Mixer, RM-2, program F26 used). The reaction mixture was transferred to a 50 ml tube and chloroform was added to a volume of 50 ml. After approximately 5 minutes of sonication, the white precipitate formed during the reaction was dissolved. The solid support was washed with DMF and acetonitrile, and the final product obtained after cleavage from the support (as described above) was purified by HPLC.

[0430] [Scheme 4. Synthesis of Compounds A215 (x=11) and A216 (x=27)] [ka] [Synthesis of A220] Compound A220 was synthesized by standard Fmoc solid-phase peptide synthesis starting from Fmoc-RINK MBHA PS resin. Removal of the Fmoc group after each coupling was carried out using 20% ​​piperidine in DMF. Fmoc-Gly-OH (2-fold excess), Fmoc-NH-PEG 28Coupling of -CH2CH2COOH (1.4-fold excess), Fmoc-Ser(tBu)-OH (2-fold excess), and N-(Boc)-S-((R)-2,3-dihydroxybutyl)-L-cysteine ​​(1.5-fold excess) was carried out in DMF using an equivalent excess of ethyl cyano(hydroxyimino)acetate (Oxyma Pure) and diisopropylcarbodiimide (DIC) as coupling agents. Myristyl chloroformate coupling was carried out using myristyl chloroformate (12 equivalents relative to resin moles) and DIEA (24 equivalents relative to resin moles) in dry DCM at room temperature for 18 hours. This coupling was repeated three times ("recouplings"). The first recoupling was carried out using myristyl chloroformate (12 equivalents relative to resin moles) and NMM (24 equivalents relative to resin moles) in dry DCM / THF (85 / 15) at room temperature for 18 hours. The second recoupling was carried out with myristyl chloroformate (6 equivalents per mole of resin), NMM (12 equivalents per mole of resin) in dry DCM / THF (85 / 15) for 41 hours at room temperature. Finally, the third recoupling was carried out with myristyl chloroformate (6 equivalents per mole of resin), NMM (12 equivalents per mole of resin) in dry DCM / THF / toluene (85 / 15 / 5) for 21.5 hours at room temperature.

[0431] Cleavage of the peptide from the resin, removal of the N-terminal Boc group, and serine side chain deprotection were achieved by exposing the resin to a solution of 93% trifluoroacetic acid (TFA), 5% HO, and 3% triisopropylsilane (TIPS) for 1.5 h. After the cleavage reaction, the mixture was evaporated, and the resulting residue was redissolved in 30% acetonitrile / water and lyophilized.

[0432] [Synthesis of A224] Compound A224 was synthesized by standard Fmoc solid-phase peptide synthesis starting from chlorotrityl chloride resin with an initial substitution of 1.6 meq / g. The first amino acid, Fmoc-Gly-OH, was first loaded onto the resin using a 0.5-fold molar excess of Fmoc-Gly-OH and DIEA (1.5-fold excess), followed by capping with DMF / MeOH / DIEA (80 / 10 / 10) and Fmoc deprotection to give dry-loaded H-Gly-CT resin with a final substitution of 0.67 meq / g. Removal of the Fmoc group after each coupling was carried out using 20% ​​piperidine in DMF. Fmoc-NH-PEG 28 Coupling of -CH2CH2COOH (1.4 equiv.) was carried out using (7-azabenzotriazol-1-yloxy)trispirrolizinophosphonium hexafluorophosphate (PyAOp; 1.4 equiv.) and diisopropylethylamine (DIEA; 3.2 equiv.) in DMF, while coupling of Fmoc-Ser(tBu)-OH (2 equiv.) and N-(Boc)-S-((R)-2,4-dihydroxybutyl)-L-cysteine ​​(1.5 equiv.) was carried out in DMF using an equivalent excess of Oxyma Pure and DIC as coupling agents. Palmitic acid coupling was carried out at room temperature for 24 h using palmitic acid (20 equiv. relative to resin mole), DIC (20 equiv.), and DMAP (2 equiv.) in DCM / THF (85 / 15) (v / v).

[0433] Cleavage of the peptide from the resin, removal of the N-terminal Boc group, and serine side chain deprotection were achieved by exposing the resin to a solution of 93% TFA, 5% HO, 3% TIPS for 1.5 h. After the cleavage reaction, the mixture was evaporated and the resulting residue was redissolved in 30% acetonitrile / water and lyophilized.

[0434] Purification and characterization Purification and Characterization: After cleavage from the solid support, each of the analogs was purified by reverse-phase HPLC according to either protocol A or B described below.

[0435] Protocol A: Reverse-phase HPLC was performed using an Agilent Zorbax 300SB-C3, 5 μm column (9.4 mm × 250 mm; Agilent Technology, Australia) attached to an Agilent HPLC 1260 Infinity system (Agilent Technologies, Santa Clara, California, USA) with a chromatogram developed using buffer A (0.1% trifluoroacetic acid in water) and buffer B (0.1% trifluoroacetic acid in acetonitrile).

[0436] Protocol B: Reverse-phase chromatography was performed using a Novasep Axial Compression Column (5 cm diameter) packed with cyano medium (Daisogel SP-120-CN-P) using a gradient of acetonitrile in 0.1% TFA / water. After lyophilization of the intermediate, ion exchange was performed on a Dowex ion exchange resin to obtain the peptide as the acetate salt.

[0437] Identification and purity determination of the target material was performed using an in-line HPLC-MS system using the following conditions: Condition A: The following gradient conditions were used: 0-5 min, 20% B; 5-32 min, 20% B to 100% B; 32-40 min, 100% B to 20% B. HPLC column: Agilent Zorbax 300-SB C3 (150 x 0.5 mm; 5 μm). The flow rate was 20 μl / min. LC-MS: Agilent 1100 series LC / MSD ion trap mass spectrometer inline with an Agilent 1100 series capillary LC system. The mass spectrometer was operated with electrospray ionization configured in positive ion mode. Data analysis software from Agilent Technologies was used to deconvolute the set of charged ions for identification of the peptide material, which was then characterized by LC-MS.

[0438] Condition B: Analytical reversed-phase HPLC using a cyano column (Daiso Fine Chem, SP-120-3-CN-P, 150 × 4.6 mm, 3 μm, 120 Å). Peptides were also analyzed by ESI LC-MS using a Finnigan LCQ Deca XPMax in positive ion mode.

[0439] Compounds A107, A108, A115, A116, A203, A204, A215 and A216 were prepared and purified according to protocol A and conditions A, and compounds A220 and A224 were prepared and purified according to protocol B and conditions B, as described above, and were each found to be greater than 95% pure.

[0440] [Peptide quantification] Quantification of compounds A107, A108, A115, A116, A203, A204, A215, and A216 was performed by hydrolysis of samples in sealed glass vials under reduced pressure at 110°C in the presence of 6N HCl containing 0.1% phenol. Amino acids were then derivatized using Waters AccQTag reagent according to the manufacturer's instructions, followed by analysis on a Waters Acquity UPLC System (Waters Millipore) using an AccQTag Ultra column (2.1 mm x 100 mm; Waters Millipore). Quantification of other compounds can be performed using similar protocols.

[0441] Synthesis of sulfone and sulfoxide analogues of compounds A215 and A216. The sulfone and sulfoxide derivatives of compounds A215 and A216 can be obtained by a similar synthetic route described above, with the omission of the ethyl methyl sulfide scavenger from the carbamate-forming step and the optional omission of nitrogen sparging. This reaction can yield a mixture of thiol, sulfone, and sulfoxide derivatives, which can be separated and purified by HPLC.

[0442] Alternatively, sulfone or sulfoxide derivatives can be prepared by oxidation of the corresponding sulfides with an oxidizing agent, such as meta-chloroperbenzoic acid (MCPBA) or tert-butyl hydroperoxide (t-BuOOH), under appropriate conditions.

[0443] [Activation of human TLR2] The efficacy of compounds as activators of human and mouse TLR-2s is tested in an in vitro assay that assesses NF-kB activation in the HEKBlue-mTLR-2 cell line. These cells are stably transfected with mouse TLR-2 and endogenously express TLR-1 and TLR-6 at levels sufficient to allow fully functional TLR-1 / 2 and TLR-2 / 6 activation.

[0444] Toll-like receptor 2 (TLR2) stimulation is tested by assessing NF-kB activation in the HEKBlue-hTLR2 cell line. These cells are stably transfected with human TLR2 and endogenously express TLR1 and TLR6 at levels sufficient to allow fully functional TLR1 / 2 and TLR2 / 6 activation. Test substance activity is tested as a potential agonist against human TLR2. Test substances are evaluated at seven concentrations and compared to a control ligand. These steps are performed in triplicate.

[0445] NF-kB reporter gene assay protocol: This assay was performed as previously described (Jackson et al. 2004; Lau et al. 2006; Sandor et al. 2003; Zeng et al. 2010). HEK293T cells were cultured at 4 × 10 4Cells were cultured in 96-well plates at 100 ng / well and transfected with 100 ng of the NF-kB luciferase reporter gene (50 ng of TK-Renilla-luciferase expression plasmid (Promega Corporation, Madison, USA)) 24 hours later, with or without 5 ng of TLR2 expression plasmid, in the presence of 0.8 μl of Fugene 6 (Roche Diagnostic). Compounds were added to the wells at the concentrations shown in the histogram 24 hours later. Cell lysates were prepared 5 hours after stimulation using reporter lysis buffer (Promega Corporation, Madison, USA). Luciferase activity in the cell lysates was determined using a reagent kit (Promega Corporation, Madison, USA) and a FLUOstar microplate reader (BMG Labtech, Ortenberg, Germany). NF-kB-dependent firefly luciferase activity was normalized with NF-kB-dependent Renilla luciferase activity. Relative stimulation was calculated as the ratio of stimulated samples to unstimulated samples.

[0446] The results of this assay for compounds A107, A108, A115, A116, A203, A204, A215 and A216 are shown in Figure 9. These data indicate that these compounds exhibit significant activity at TLR2.

Claims

[Claim 1] 1. A method for treating, preventing, or minimizing the progression of cancer in a subject, comprising administering to the subject therapeutically effective amounts of a TLR2 agonist and an immune stimulant, thereby treating, preventing, or minimizing the progression of cancer in the subject.

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