Granzyme b directed imaging and therapy

Abstract

To provide compounds useful for imaging granzyme B by using medical imaging including positron tomography.SOLUTION: A method for imaging granzyme B in a cell or a tissue includes: i) a step of bringing a sample of the cell or tissue in contact with a compound of formula I or a pharmaceutically acceptable salt thereof; and ii) a step of imaging the cell or tissue by an appropriate imaging method and thus imaging granzyme B in the cell or tissue. In the formula, A includes one or a plurality of imaging agents, B denotes an arbitrary selected linking group, and C denotes a group binding to granzyme B.SELECTED DRAWING: None

Description

Technical Field

[0001] Research or development supported by the federal government This invention was made with government support under grant numbers P50-CA127003 and 5R01CA166582-0 3, awarded by the National Institutes of Health. The United States federal government has certain rights in this invention.

[0002] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 357,845, filed July 1, 2016, and U.S. Provisional Application No. 62 / 435,541, filed December 16, 2016, each of which is hereby incorporated by reference in its entirety herein. and U.S. Provisional Application No. 62 / 435,541, filed December 16, 2016.

[0003] Technical Field The present invention relates to compounds useful in imaging methods, and more particularly, to compounds useful for imaging granzyme B using medical imaging, including positron emission tomography

Background Art

[0004] Granzyme B is a serine protease released via exocytosis by cytotoxic lymphocytes (CTLs) during the cellular immune response, and represents one of two major mechanisms by which T cells mediate cancer cell death, along with the FAS / FASL pathway. Granzyme B is released together with perforin, a pore-forming protein, at the immune synapse formed between T cells and their targets. A portion of the released granzyme B then enters the cancer cells mainly through perforin pores, where granzyme B ​​​​​​​​Activates the substrate, resulting in the activation of the caspase cascade.

Summary of the Invention

Means for Solving the Problems

[0005] This application relates, inter alia, to a method for imaging granzyme B in cells or tissues comprising: i) contacting a sample of cells or tissue with a compound of formula I:

[0006]

Chemical Formula

[0007] or a pharmaceutically acceptable salt thereof; and ii) imaging the cells or tissue by an appropriate imaging method, thereby imaging granzyme B within the cells or within the tissue wherein: A comprises one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B. This application presents a method.

[0008] This application relates to a method for imaging granzyme B in a subject, comprising: i) administering to the subject a compound of formula I:

[0009]

Chemical Formula

[0010] or a pharmaceutically acceptable salt thereof; and ii) imaging the subject by an appropriate imaging method, thereby imaging granzyme B in the subject wherein: A comprises one or more imaging agents, A comprises one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B, and further provides a method.

[0011] This application is a method for imaging an immune response in a sample of cells or tissues, i) contacting a sample of cells or tissues with a compound of formula I:

[0012]

Chemical formula

[0013] or a pharmaceutically acceptable salt thereof; and ii) imaging the sample of cells or tissues by an appropriate imaging method, thereby imaging the immune response in the sample of cells or tissues and wherein, A comprises one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B, and further provides a method.

[0014] This application is a method for imaging an immune response in a subject, i) administering to the subject a compound of formula I:

[0015]

Chemical formula

[0016] or a pharmaceutically acceptable salt thereof; and ii) imaging the subject by an appropriate imaging method, thereby imaging the immune response in the subject and wherein, A comprises one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B, and further provides a method.

[0017] This application is a method for monitoring the treatment of a disease in a subject, i) administering to the subject a compound of formula I:

[0018]

Chemical formula

[0019] or a pharmaceutically acceptable salt thereof; and ii) imaging the subject by an appropriate imaging method and in the formula, A comprises one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B, and further provides a method.

[0020] This application is a method for monitoring the immune response in the treatment of a disease in a subject , i) administering to the subject a compound of formula I:

[0021]

Chemical formula

[0022] or a pharmaceutically acceptable salt thereof; and ii) imaging the subject by an appropriate imaging method and in the formula, A comprises one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B, and further provides a method.

[0023] In some embodiments, A comprises one or more imaging agents selected from the group consisting of paramagnetic ions, x-ray imaging agents, fluorophores, and radioisotopes.

[0024] In some embodiments, the paramagnetic ion is selected from the group consisting of chromium(III), manganese(II), iron(II), iron(II), cobalt(II), nickel(II), copper(II), neodymium(II), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and erbium(III).

[0025] In some embodiments, the x-ray imaging agent is selected from the group consisting of lanthanum(III), gold(III), lead(II), bismuth(III), and x-ray imaging agents iodinated .

[0026] In some embodiments, the radioisotope is 3 H, 11 C, 14 C, 18 F, 32 P, 3 5 S, 36 Cl, 51 Cr, 52 Fe, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Se, 76 Br, 77 Br, 89 Zr, 90 Y, 99m Tc, 111 In,​​123 I、 124 I、 125 I、 131 I、 152 Eu、 153 Sm 、 166 Ho、 177 Lu、 186 Re、 188 Re、 201 Tl、 203 Pb、 210 At、 211 At、 212 Bi、 213 Bi, and 225 selected from the group consisting of Ac .

[0027] In some embodiments, A is an imaging agent selected from the group consisting of a PET imaging agent, a SPECT imaging agent, and a computed tomography imaging agent. In some embodiments, A is a PET imaging agent or a SPECT imaging agent. In some embodiments, A is 3 H, 11 C, 14 C, 18 F, 35 S, 52 Fe, 58 Co, 64 Cu, 68 Ga, 76 Br, 77 Br, 89 Zr, 111 In, 123 I , 124 I, 125 I, 131 I, 186 Re, 188 Re, 201 Tl selected from the group consisting of a PET imaging agent or a SPECT imaging agent containing a radioisotope. In some embodiments, A is 68 a PET imaging agent containing Ga.

[0028] In some embodiments, A further comprises a chelating agent. In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetra azacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triaza cyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), ethylenediamine tetra acetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), cyclohexyl-1,2 -diamine tetraacetic acid (CDTA), ethylene glycol-O,O'-bis(2-aminoeth yl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl) -ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetramine hexaacetic acid( TTHA), hydroxyethyldiamine triacetic acid (HEDTA), and 1,4,8,11 -tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl )-cyclododecane (TCMC), and desferrioxamine B (DFO), and is selected from the group consisting of . In some embodiments, the chelating agent is selected from the group consisting of 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid -4,7-diacetic acid (NODAGA). In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA).

[0029] In some embodiments, A is a fluorophore. In some embodiments, A is Al exa 350, Alexa 430, AMCA, BODIPY 630 / 650, BO DIPY 650 / 665, BODIPY-FL, BODPY-R6G, 13BODLP Y-TMR, BODLPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Ore gon Green 500, Oregon Green 514, quantum dots, Pacific Blue, RE G, rhodamine green, rhodamine red, renografin, ROX, TAMRA, T ET, tetramethylrhodamine, Texas Red, AF350, AF405, AF532 , AF488, AF647, AF680, AF750, Cy5, Cy5.5, Cy7, I ndocyanine green (ICG), green fluorescent protein (GFP), red fluorescent protein (RFP), and a fluorophore selected from the group consisting of dsRED.

[0030] This application is a method for treating a disease in a subject, comprising: i) administering to the subject a compound of formula I:

[0031]

Chemical formula

[0032] [wherein, A contains a non-toxic radioisotope, B is an optional linking group, C is a group that binds to granzyme B] or a pharmaceutically acceptable salt thereof; and ii) imaging the subject by an appropriate imaging method; and iii) administering to the subject a compound of formula I:

[0033] [Chemical formula]

[0034] [wherein, A contains a toxic radioisotope, B is an optional linking group, and C is a group that binds to granzyme B] administering a compound of formula (I) or a pharmaceutically acceptable salt thereof, thereby treating a disease in a subject and further provides a method comprising

[0035] In some embodiments, the method further comprises determining whether a compound of the first formula (I) or a pharmaceutically acceptable salt thereof binds to the cells or tissues of the subject to be treated, prior to the administration of step (iii).

[0036] In some embodiments, the method further comprises determining whether a compound of the first formula (I) or a pharmaceutically acceptable salt thereof binds to granzyme B, prior to the administration of step (iii).

[0037] In some embodiments, the group A of the compound of the first formula (I) or a pharmaceutically acceptable salt thereof is , 3 H, 11 C, 14 C, 18 F, 35 S, 52 Fe, 58 Co, 64 Cu, 68 Ga, 76 Br, 77 Br, 89 Zr, 111 In, 123 I, 124 I, 125 I, 131 I , 186 Re,188 Re, 201 A non-toxic radioisotope selected from the group consisting of Tl is included.

[0038] In some embodiments, the group A of the compound of Formula I or a pharmaceutically acceptable salt thereof further comprises a chelating agent. In some embodiments, the chelating agent is 1,4,7-triaza cyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1-glutar ic acid-4,7-diacetic acid (NODAGA), ethylenediaminetetraacetic acid (EDTA), diethyl enetriaminepentaacetic acid (DTPA), cyclohexyl-1,2-diaminetetraacetic acid (CDTA ), ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N',N'- tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N '-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), hydroxyethyl diamine triacetic acid (HEDTA), and 1,4,8,11-tetraazacyclotetrade cane-N,N',N'',N'''-tetraacetic acid (TETA), 1,4,7,10-tetraaza -1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane (TCM C), and desferrioxamine B (DFO), and is selected from the group consisting of.

[0039] In some embodiments, the group A of the compound of Formula II or a pharmaceutically acceptable salt thereof comprises a toxic radioisotope selected from the group consisting of an alpha emitter and a beta emitter. In some embodiments, the group A of the compound of Formula II or a pharmaceutically acceptable salt thereof 211 At,212 Pb, 212 Bi, 213 Bi, 225 Ac, 227 Th, 9 0 Y, 177 Lu, and 131 contains a radioactive isotope of toxicity selected from the group consisting of I .

[0040] In some embodiments, B is one or more amino acid residues, one or more carbohydrates, one or more alkylene groups, one or more amine groups, one or more amide groups, one or more alkyleneoxy groups, one or more thiol groups, or an optional linking group comprising any combination thereof. In some embodiments, B is one or more C . alkyl groups, one or more amine groups, one or more amide groups, one or more C 1~30 alkyl groups, one or more amine groups, one or more amide groups, one or more C alkyl groups, one or more C 1~30 alkyl groups, one or more C 1~30 thio groups, or an optional linking group comprising any combination thereof. In some embodiments, B is an optional linking group comprising one or more -(OCH 2 CH 2 )- groups. In some embodiments, B is an optional linking group of the formula -(OCH CH 2 CH 2 ) p -, where p is , an integer from 1 to 40. In some embodiments, p is an integer from 10 to 40. In some embodiments, p is an integer from 20 to 40. In some embodiments, p is an integer from 25 to 35 .

[0041] In some embodiments, C is a polypeptide that binds to granzyme B, granzyme B An antibody that binds to, an antibody fragment that binds to granzyme B, and a selected from the group consisting of small organic molecules.

[0042] In some embodiments, C is an antibody that binds to granzyme B. In some embodiments C is an antibody that binds to granzyme B and is selected from the group consisting of clone GB11, clone GrB- 7, and NCL-L-Gran-B. In some embodiments, C is an antibody fragment that binds to granzyme B.

[0043] In some embodiments, C is a polypeptide that binds to granzyme B. In some embodiments, the polypeptide that binds to granzyme B is about 4 to about 100 amino acid residues in length. In some embodiments, the polypeptide that binds to granzyme B is about 4 to about 50 amino acid residues in length. In some embodiments, the polypeptide that binds to granzyme B is about 4 to about 25 amino acid residues in length. In some embodiments, the granzyme B-binding polypeptide is about 4 to about 15 amino acid residues in length.

[0044] In some embodiments, C is a polypeptide that binds to granzyme B, and the polypeptide is X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -D (SEQ ID NO: 1) and contains an amino acid sequence having at least 90% sequence identity thereto, wherein X 1 X 2 and X 3 are each independently selected from the group consisting of beta A, G, Q, N, S, T, Y, C, R, D, and E, X 4 is selected from the group consisting of I and V, X 5 is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D is made.

[0045] In some embodiments, X 4 is I. In some embodiments, X 5 is E. One In some embodiments, X 6 is F or P.

[0046] In some embodiments, the polypeptide is beta A - G - G - I - E - F - D (SEQ ID NO: 2); G - G - G - I - E - F - D (SEQ ID NO: 3); and beta A - G - G - I - E - P - D (SEQ ID NO: 4) has at least 90% sequence identity to an amino acid sequence selected from the group consisting of contains.

[0047] In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. One In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[0048] In some embodiments, C is a polypeptide that binds to granzyme B, and the polypeptide is X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -D - X 7 (SEQ ID NO: 8) comprises an amino acid sequence having at least 90% sequence identity, wherein, X 1 , X 2 , and X 3 are each independently selected from the group consisting of beta A, G, Q, N, S, T, Y, C, R, D, and E, X 4 is selected from the group consisting of I and V, X 5 is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D is selected, X 7 is an electrophilic group containing the C-terminus of the amino acid sequence of SEQ ID NO: 8.

[0049] In some embodiments, X 4 is I. In some embodiments, X 5 is E. In one In some embodiments, X 6 is F or P.

[0050] In some embodiments, X 7 is -C(O)H, -C(O)C 1~6 alkyl, -C(O )C 1~6 haloalkyl, -C(O)C 1~6 alkoxy, -C(O)C 1~6 haloalk oxy, -C(O)-(C 1~6 alkyl)-(5- to 10-membered heteroaryl), -C( O)-(C 1~6 haloalkyl)-(5- to 10-membered heteroaryl), -C(O)-(C 1~6 alkoxy)-(5- to 10-membered heteroaryl), and -C(O)-(C 1~6 haloalkoxy)-(5- to 10-membered heteroaryl) and is an electrophilic group selected from the group consisting of is. In some embodiments, X7 is -C(O)H.

[0051] In some embodiments, C is a small organic molecule that binds to granzyme B. In some embodiments the small organic molecule is a small peptide mimic.

[0052] In some embodiments, C is of formula III:

[0053]

Chemical formula

[0054] a small organic molecule of or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, or 2; m is 0, 1, or 2; R 1 and R 2 are each, independently, hydrogen, C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl, C 6~10 aryl, HET, and -N(R 10 ) 2 selected from the group consisting of wherein each C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl is independently optionally substituted with one, two, or three substituents selected from the group consisting of halo and hydroxy, and each C 6~10 aryl and HET are independently optionally substituted with one, two, or three 1~4 substituents selected from the group consisting of oxo, halo, hydroxy, C 1~4 alkyl, and C haloalkyl; or R 1 and R 2 together with the carbon atom to which they are attached form, each of which is 、optionally substituted by one, two, or three R 10 groups, a 5- to 6-membered cycloalkyl group or a 5- to 6-membered heterocycloalkyl group can be formed ; each R 3 and R 7 is independently selected from the group consisting of hydrogen, C 1~4 alkyl, and C 1~4 haloalkyl ; each R 4 , R 5 , R 6 , and R 8 is independently selected from the group consisting of hydrogen, halo, hydroxy, C 1~4 alkyl, and C alkyl 1~4 ; R 9 is HET optionally substituted by one, two, or three substituents independently selected from the group consisting of oxo, halo, hydroxy, C 1~4 alkyl, and C 1~4 halo alkyl; R 10 is selected from the group consisting of hydrogen, C 1~4 alkyl, and -C(O)C 1~4 alkyl, wherein -C(O)C alkyl is optionally substituted by -N(R 1~4 ) 11 , H 2 , H ET, and C 6~10 aryl, wherein C 6~10 aryl is optionally substituted by one, two, or three halo groups; each HET is an independently selected monocyclic or bicyclic 5- to 10-membered heteroaryl group or a monocyclic or bicyclic 5- to 10-membered heterocycloalkyl group, where ​​Combined, each HET contains one, two, three, or four heteroatoms selected from O, S, and N, and is optionally substituted by one or two oxo groups; and is optionally substituted by one or two oxo groups; R 11 is selected from hydrogen, C 1~4 alkyl, and C 1~4 haloalkyl, provided that if m is 0, then n is 0, and if n is 0, then m is 0. Provided that if m is 0, then n is 0, and if n is 0, then m is 0.

[0055] In some embodiments, C is selected from the group consisting of

[0056] [Table 1]

[0057] or a pharmaceutically acceptable salt thereof.

[0058] In some embodiments, A is an imaging agent comprising one or more of a paramagnetic ion, an x-ray imaging agent, a fluorophore, and a radioisotope, and B is an optional linking group comprising one or more alkylene groups, one or more amine groups, one or more amide groups, one or more alkyleneoxy groups, one or more thiol groups, or any combination thereof, and C is selected from the group consisting of a polypeptide that binds to granzyme B, an antibody that binds to granzyme B, an antibody fragment that binds to granzyme B, and a small organic molecule that binds to granzyme B. and C is selected from the group consisting of a polypeptide that binds to granzyme B, an antibody that binds to granzyme B, an antibody fragment that binds to granzyme B, and a small organic molecule that binds to granzyme B. In some embodiments, A contains a radioisotope, and B is one or more C selected from the group consisting of a polypeptide that binds to granzyme B, an antibody that binds to granzyme B, an antibody fragment that binds to granzyme B, and a small organic molecule that binds to granzyme B.

[0059] In some embodiments, A contains a radioisotope, and B is one or more C 1~30 alkylene groups, one or more amine groups, one or more amide groups, one or more C 1~30An alkyleneoxy group, one or more of C 1~30 is an optional linking group containing a thiol group, or any combination thereof, where C is selected from the group consisting of a polypeptide that binds to granzyme B and an organic small molecule.

[0060] In some embodiments, A is 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 36 C l, 51 Cr, 52 Fe, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 G a, 68 Ga, 75 Se, 76 Br, 77 Br, 89 Zr, 90 Y, 99m Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 152 Eu, 153 Sm, 166 Ho , 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 210 At, 211 At, 212 Bi, 213 Bi, and 225 contains a radioisotope selected from the group consisting of Ac, B is one or more C 1 ~ 30An alkylene group, one or more amide groups, one or more C 1 ~ 30 alkyleneoxy groups, or an optional linking group containing any combination thereof, wherein, C is selected from the group consisting of a polypeptide that binds to granzyme B and an organic small molecule that binds to granzyme B.

[0061] In some embodiments, A is 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 36 C l, 51 Cr, 52 Fe, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 G a, 68 Ga, 75 Se, 76 Br, 77 Br, 89 Zr, 90 Y, 99m Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 152 Eu, 153 Sm, 166 Ho , 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 210 At, 211 At, 212 Bi, 213 Bi, and 225 Ac, and contains a radioisotope selected from the group consisting of wherein, B is an optional linking group containing one or more -(-OCH 2 CH 2 )- groups, C is selected from the group consisting of a polypeptide that binds to granzyme B and an organic small molecule that binds to granzyme B.

[0062] In some embodiments, A further comprises a chelating agent. In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetra azacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triaza cyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), ethylenediamine tetra acetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), cyclohexyl-1,2 -diamine tetraacetic acid (CDTA), ethylene glycol-O,O’-bis(2-aminoeth yl)-N,N,N’,N’-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl) -ethylenediamine-N,N’-diacetic acid (HBED), triethylenetetramine hexaacetic acid ( TTHA), hydroxyethyldiamine triacetic acid (HEDTA), and 1,4,8,11 -tetraazacyclotetradecane-N,N’,N’’,N’’’-tetraacetic acid (TETA), 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl )-cyclododecane (TCMC), and desferrioxamine B (DFO). In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7, 10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1-glutaric acid 4,7-diacetic acid (NODAGA). - It is selected from the group consisting of (NODAGA). In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid (NOTA).

[0063] In some embodiments, C is a polypeptide that binds to granzyme B. In some embodiments, the polypeptide that binds to granzyme B is about 4 to about 100 amino acid residues in length. In some embodiments, the polypeptide that binds to granzyme B is about 4 to about 50 amino acid residues in length. In some embodiments, the polypeptide that binds to granzyme B is about 4 to about 25 amino acid residues in length. In some embodiments, the polypeptide that binds

[0064] to granzyme B is about 4 to about 15 amino acid residues in length. In some embodiments, C is a polypeptide that binds to granzyme B, and the polypeptide is 1 -X 2 -X 3 -X 4 -X 5 -X 6 -D (SEQ ID NO: 1) and comprises an amino acid sequence having at least 90% sequence identity, wherein 1 X 2 , X 3 , and X are each independently selected from the group consisting of beta A, G, Q, N, S, T, Y, C, 4 X is selected from the group consisting of I and V, 5 X is selected from the group consisting of E, G, D, and S, 6 X is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D.

[0065] In some embodiments, X 4 is I. In some embodiments, X 5 is E. In one embodiment, X 6 is F or P.

[0066] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% sequence identity to an array selected from the group consisting of beta A-G-G-I-E-F-D (SEQ ID NO: 2); G-G-G-I-E-F-D (SEQ ID NO: 3); and beta A-G-G-I-E-P-D (SEQ ID NO: 4)

[0067] In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[0068] In some embodiments, C is a polypeptide that binds to granzyme B, and the polypeptide comprises X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -D-X 7 (SEQ ID NO: 8) and comprises an amino acid sequence having at least 90% sequence identity thereto, wherein X 1 , X 2 and X 3 are each independently selected from the group consisting of beta A, G, Q, N, S, T, Y, C, R, D, and E, X 4 is selected from the group consisting of I and V, X 5is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D and X 7 is an electrophilic group containing the C-terminus of the amino acid sequence of SEQ ID NO: 8.

[0069] In some embodiments, X 4 is I. In some embodiments, X 5 is E. In one embodiment, X 6 is F or P.

[0070] In some embodiments, X 7 is -C(O)H, -C(O)C 1~6 alkyl, -C(O )C 1~6 haloalkyl, -C(O)C 1~6 alkoxy, -C(O)C 1~6 haloal koxy, -C(O)-(C 1~6 alkyl)-(5- to 10-membered heteroaryl), -C( O)-(C 1~6 haloalkyl)-(5- to 10-membered heteroaryl), -C(O)-(C 1~6 alkoxy)-(5- to 10-membered heteroaryl), and -C(O)-(C 1~6 haloalkoxy)-(5- to 10-membered heteroaryl) and is an electrophilic group selected from the group consisting of In some embodiments, X 7 is -C(O)H.

[0071] In some embodiments, C is a small organic molecule that binds to granzyme B. In some embodiments the small organic molecule is a small peptide mimic. In some embodiments, C is of the formula III:

[0072]

Chemical formula

[0073] an organic low-molecular substance, or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, or 2; m is 0, 1, or 2; R 1 and R 2 are each independently selected from the group consisting of hydrogen, C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl, C 6~10 aryl, HET, and -N(R 10 ) 2 wherein each C alkyl, C 1~6 alkoxy, C 1~6 cycloalkyl is independently optionally substituted with one, two, or three 3~6 substituents selected from the group consisting of halo and hydroxy, and each C aryl and HET are independently optionally substituted with one, two, or three substituents selected from the group consisting of oxo, halo, hydroxy, C 6~10 alkyl, and C haloalkyl; 1~4 alkyl, and C 1~4 haloalkyl wherein R or R 1 and R 2 may together with the carbon atom to which they are attached form a 5- to 6-membered cycloalkyl group or a 5- to 6-membered heterocycloalkyl group, each of which is optionally substituted with one, two, or three R 10 groups; or R ; each R 3 and R 7 is independently selected from the group consisting of hydrogen, C 1~4 alkyl, and C 1~4 haloalkyl; ; Each R 4 、R 5 、R 6 、and R 8 is independently selected from the group consisting of hydrogen, halo, hydroxy, C 1~4 alkyl, and C alkyl; 1~4 selected from the group consisting of hydrogen, halo, hydroxy, C R 9 is optionally substituted by one, two, or three substituents independently selected from the group consisting of oxo, halo, hydroxy, C 1~4 alkyl, and C 1~4 halo alkyl; is a HET optionally substituted by one, two, or three substituents independently selected from the group consisting of oxo, halo, hydroxy, C R 10 is hydrogen, C 1~4 alkyl, and -C(O)C 1~4 alkyl, where -C(O)C alkyl is optionally substituted by -N(R 1~4 ) 11 , H 2 ET, and C aryl, where C 6~10 aryl is optionally substituted by one, two, or three halo groups; 6~10 each HET is independently selected from a monocyclic or bicyclic 5- to 10-membered heteroaryl group or a monocyclic or bicyclic 5- to 10-membered heterocycloalkyl group, where in this case each HET contains one, two, three, or four hetero atoms selected from O, S, and N and is optionally substituted by one or two oxo groups; each HET is independently selected from a monocyclic or bicyclic 5- to 10-membered heteroaryl group or a monocyclic or bicyclic 5- to 10-membered heterocycloalkyl group, where in this case each HET contains one, two, three, or four hetero 11 atoms selected from O, S, and N and is optionally substituted by one or two oxo groups; 1~4 R 1~4 is selected from hydrogen, C alkyl, and C

[0074] In some embodiments, C is

[0075]

Table 2

[0076] It is selected from the group consisting of or a pharmaceutically acceptable salt thereof.

[0077] In some embodiments, C is an irreversible binder of granzyme B. In some embodiments C is an inhibitor of granzyme B.

[0078] In some embodiments, the method further comprises administering a therapeutic agent prior to the administration of step i). In some embodiments, the administration of the therapeutic agent induces an immune response in a sample of cells or tissue or in a subject.

[0079] In some embodiments, the therapeutic agent is selected from the group consisting of an anti-inflammatory agent, a steroid, an immunotherapeutic agent, a chemotherapeutic agent, and a therapeutic antibody. In some embodiments, the therapeutic agent is a chemotherapeutic agent.

[0080] In some embodiments, the disease is selected from the group consisting of an autoimmune disorder, an inflammatory disorder, a skin disorder, cancer, and a cardiovascular disorder. In some embodiments, the disease is cancer. In some embodiments, the cancer is a brain tumor, breast cancer, cervical cancer, colorectal cancer, lung cancer, lymphoma melanoma, bladder cancer, renal cell carcinoma, multiple myeloma, pancreatic cancer, and prostate cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is melanoma.

[0081] In some embodiments, the disease is graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus S, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, rheumatic fever, post-infectious glomerulonephritis, psoriasis, atopic dermatitis, contact dermatitis, eczematous dermatitis, lipophilic dermatitis, lichen planus, pemphigus, vesicular pemphigoid, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, erythroderma, acne, alopecia areata, keratoconjunctivitis, vernal catarrh, uveitis associated with Behçet's disease, keratitis, herpes keratitis, keratoconus, corneal epithelial dysplasia, corneal leukoma, ocular pemphigus, Mooren ulcer, scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergy, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma, chronic (chronic or inveterate) asthma, late-onset asthma and airway hyperresponsiveness, bronchitis, gastric ulcer, vascular damage caused by ischemic disease and thrombosis, ischemic bowel disease, inflammatory bowel disease, necrotizing enteritis, intestinal lesions associated with burns, celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis, Goodpasture syndrome, hemolytic uremic syndrome, diabetic nephropathy, polymyositis, Guillain-Barré syndrome, Ménière's disease, polyneuritis (polyneuritis, multiple neuritis), mononeuritis, radiculopathy, hyperthyroidism, Graves' disease, erythroleukemia, aplastic anemia (aplastic anemia, hypoplastic anemia), idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, erythropoiesis imperfecta, osteoporosis, sarcoidosis, pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, posterior uveitis, allergic encephalomyelitis, glomerulonephritis, rheumatic fever, post-infectious glomerulonephritis, psoriasis, atopic dermatitis, contact dermatitis, eczematous dermatitis, lipophilic dermatitis, lichen planus, pemphigus, vesicular pemphigoid, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, erythroderma, acne, alopecia areata, keratoconjunctivitis, vernal catarrh, uveitis associated with Behçet's disease, keratitis, herpes keratitis, keratoconus, corneal epithelial dysplasia, corneal leukoma, ocular pemphigus, Mooren ulcer, scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergy, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma, chronic (chronic or inveterate) asthma, late-onset asthma and airway hyperresponsiveness, bronchitis, gastric ulcer, vascular damage caused by ischemic disease and thrombosis, ischemic bowel disease, inflammatory bowel disease, necrotizing enteritis, intestinal lesions associated with burns, celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis, Goodpasture syndrome, hemolytic uremic syndrome, diabetic nephropathy, polymyositis, Guillain-Barré syndrome, Ménière's disease, polyneuritis (polyneuritis, multiple neuritis), mononeuritis, radiculopathy, hyperthyroidism, Graves' disease, erythroleukemia, aplastic anemia (aplastic anemia, hypoplastic anemia), idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, erythropoiesis imperfecta, osteoporosis, sarcoidosis, pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, vascular damage caused by ischemic disease and thrombosis, ischemic bowel disease, inflammatory bowel disease, necrotizing enteritis, intestinal lesions associated with burns, celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis, migraine, rhinitis, eczema, interstitial nephritis, Goodpasture syndrome, hemolytic uremic syndrome, diabetic nephropathy, polymyositis, Guillain-Barré syndrome, Ménière's disease, polyneuritis (polyneuritis, multiple neuritis), mononeuritis, radiculopathy, hyperthyroidism, Graves' disease, erythroleukemia, aplastic anemia (aplastic anemia, hypoplastic anemia), idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, erythropoiesis imperfecta, osteoporosis, sarcoidosis, pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, polymyositis, Guillain-Barré syndrome, Ménière's disease, polyneuritis (polyneuritis, multiple neuritis), mononeuritis, radiculopathy, hyperthyroidism, Graves' disease, erythroleukemia, aplastic anemia (aplastic anemia, hypoplastic anemia), idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, erythropoiesis imperfecta, osteoporosis, sarcoidosis, pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, hyperthyroidism, Graves' disease, erythroleukemia, aplastic anemia (aplastic anemia, hypoplastic anemia), idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, erythropoiesis imperfecta, osteoporosis, sarcoidosis, pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, erythropoiesis imperfecta, osteoporosis, sarcoidosis, pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, Photoallergic hypersensitivity, cutaneous T-cell lymphoma, arteriosclerosis, atherosclerotic arteriosclerosis, large arterial inflammatory syndrome, polyarteritis nodosa, cardiomyopathy, scleroderma, Wegener's granulomatosis, Sjogren syndrome, hyperlipidemia, eosinophilic fasciitis, gingiva, periodontal tissue, alveolar bone, lesions of dental cementum, glomerulonephritis, male pattern alopecia, senile alopecia due to hair loss, lack of hair germination, and / or senile alopecia due to decreased hair generation and hair growth, muscular dystrophy, impetigo , Sezary syndrome, Addison's disease, organ damage due to ischemia-reperfusion, transplantation diseases, ischemic diseases, endotoxin shock, pseudomembranous colitis, colitis caused by drugs or radiation, acute ischemic renal failure, chronic renal failure, oxygen lung or poisoning caused by drugs, lung cancer , pulmonary emphysema, cataract, hemosiderosis, retinitis pigmentosa, age-related macular degeneration, vitreous scarring, corneal ar caliburn, polymorphic erythematous dermatitis, linear IgA bullous dermatitis and cement dermatitis, gingivitis , periodontitis, sepsis, pancreatitis, aging, carcinogenesis, metastasis of cancer tumors and altitude sickness, histamine or ro eicosanoid C4 release-related diseases, Behcet's disease, autoimmune hepatitis, primary biliary cirrhosis , sclerosing cholangitis, partial hepatectomy, acute liver necrosis, necrosis caused by toxins, viral hepatitis, shock, anoxia, hepatitis B virus, non-A / non-B hepatitis, cirrhosis, alcohol -induced cirrhosis, liver failure, fulminant liver failure, late-onset liver failure, acute exacerbation of chronic hepatitis (acute -on-chronic liver failure), cytomegalovirus infection, HCMV infection, AIDS, senile dementia, trauma, chronic bacterial infection, lymphoid-derived malignant tumors acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphocytic lymphoma, and chronic Selected from the group consisting of lymphocytic lymphoma. In some embodiments, the disease is systemic erythematosus rheumatodes, rheumatoid arthritis, type I diabetes, inflammatory bowel disease, biliary cirrhosis, grape meningitis, multiple sclerosis, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis, ichthyosis, Graves' ophthalmopathy, asthma, scleroderma, and Sjogren's syndrome. In some embodiments, the disease is bone marrow rejection, organ graft rejection, and graft-versus-host disease.

[0082] In some embodiments, the compound of formula I is 68 Ga-NOTA-betaA-G-G-I-E-F-D (Compound 1; SEQ ID NO: 9); 68 Ga-NOTA-(OCH 2 CH 2 ) 27 -G-G-G-I-E-F-D (Compound 2; SEQ ID NO: 10); and 68 Ga-NOTA-betaA-G-G-I-E-P-D (Compound 3; SEQ ID NO: 11) selected from the group consisting of.

[0083] This application relates to a compound of formula I:

[0084]

Chemical formula

[0085] or a pharmaceutically acceptable salt thereof, wherein A comprises one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B, a) X 1 -X 2 -X 3 -X4 -X 5 -X 6 -D (array number 1) [wherein, X 1 、X 2 、and X 3 are each independently selected from the group consisting of beta A, G, Q, N, S, T, Y, C, R, D, and E, X 4 is selected from the group consisting of I and V, X 5 is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D selected] having at least 90% sequence identity with an amino acid sequence, and b) Formula III:

[0086]

Chemical formula

[0087] is a group selected from the group consisting of a compound of, or a pharmaceutically acceptable salt thereof, wherein 、 n is 0, 1, or 2; m is 0, 1, or 2; R 1 and R 2 are each independently selected from the group consisting of hydrogen, C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl, C 6~10 aryl, HET, and -N(R 10 ) 2 consisting of group selected, wherein each C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl is independently selected from one, two, or three selected from the group consisting of halo and hydroxy optionally substituted by one substituent, each C 6~10 aryl and HET are, independently, oxo, halo, hydroxy, C 1~4 alkyl, and C 1~4 haloalkyl, from the group optionally substituted by one, two, or three substituents selected therefrom; or R 1 and R 2 together with the carbon atom to which they are attached form a 5- or 6-membered cycloalkyl group or 5- or 6-membered heterocycloalkyl group, optionally substituted by one, two, or three R 10 groups; it is possible to form; ; each R 3 and R 7 are, independently, hydrogen, C 1~4 alkyl, and C 1~4 haloalkyl selected from the group consisting of; each R 4 , R 5 , R 6 , and R 8 are, independently, hydrogen, halo, hydroxy, C 1~4 alkyl, and C alkyl selected from the group consisting of; 1~4 ; R 9 is HET, optionally substituted by one, two, or three substituents selected from the group consisting of oxo, halo, hydroxy, C 1~4 alkyl, and C 1~4 halo alkyl; ; R 10 is hydrogen, C 1~4 alkyl, and -C(O)C 1~4 alkyl selected from the group consisting of, wherein -C(O)C alkyl is optionally -N(R 1~4 ) 11 ; 2 , H ET, and C 6~10 substituted by aryl, wherein C 6~10 aryl is optionally substituted by one, two, or three halo groups; each HET is an independently selected monocyclic or bicyclic 5- to 10-membered heteroaryl group, or a monocyclic or bicyclic 5- to 10-membered heterocycloalkyl group, wherein in this case, each HET contains one, two, three, or four hetero atoms selected from O, S, and N, and is optionally substituted by one or two oxo groups; R 11 is hydrogen, C 1~4 alkyl, and C 1~4 haloalkyl, and provided that if m is 0, then n is 0, and if n is 0, then m is 0, a compound, or a pharmaceutically acceptable salt thereof is further provided.

[0088] In some embodiments, the compound of formula I is 68 Ga-NOTA-betaA-G-G-I-E-F-D (Compound 1; SEQ ID NO: 9); 68 Ga-NOTA-(OCH 2 CH 2 ) 27 -G-G-G-I-E-F-D (Compound 2; SEQ ID NO: 10); and 68 Ga-NOTA-betaA-G-G-I-E-P-D (Compound 3; SEQ ID NO: 11) selected from the group consisting of.

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This specification The book describes the methods and materials for use in the present invention, but other suitable methods and materials known in the art can also be used. The materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will prevail. and other suitable methods and materials known in the art can also be used. The materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will prevail. merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will prevail. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will prevail. and other references are hereby incorporated by reference in their entirety. In case of conflict, the present document, including definitions, will prevail. In case of conflict, the present document, including definitions, will prevail.

Brief Description of the Drawings

[0090]

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Modes for Carrying Out the Invention

[0091] Cancer immunotherapy has shown remarkable progress in cancer treatment in recent years. Programs Antibodies targeting immune checkpoints such as programmed death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (C TLA-4) have been approved for some patients with positive outcomes. Research in the field of immuno-oncology continues with strategies including developing CAR-T cells, vaccines, small molecules, and antibodies. Despite the promise of these therapies, may be connected, which is costly and the response rate is typically 20 - 50%, meaning that most patients do not respond to the treatment. Furthermore, the response often shows signs of growth in the responding tumors in anatomical imaging (e.g., CT, MRI), is associated with immune cell infiltrates, making it difficult to determine the individual patient's response to the treatment using conventional methods. The increased affinity for FDG-PET imaging due to a large number of metabolically active immune cells supports this. Given the limitations of current imaging techniques, clinical studies on cancer immunotherapy typically invoke overall survival rather than

[0092] progression-free survival as their primary endpoint. Granzyme B, a downstream marker of cytotoxic T cell activity, could be used as a novel biomarker to evaluate the effectiveness of cancer immunotherapy. The expression of granzyme B within the tumor can not only be evaluated for the presence or absence of CTLs but also as an effector protein released by activated T cells that also integrates as a measure of CTL activity, thus explaining the problem of T cell exhaustion that makes it difficult to achieve an assessment of the presence of CTLs. Accordingly, the present application presents a novel granzyme B-specific imaging agent. Representative granzyme B imaging agents were examined in a model of cancer immunotherapy.

[0093] Compound The present application particularly relates to Formula I:

[0094] [Chemical formula]

[0095] [In the formula, A contains one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B] of the compound, or a pharmaceutically acceptable salt thereof, is presented.

[0096] In some embodiments, C is an irreversible binder of granzyme B. In some embodiments C is an inhibitor of granzyme B.

[0097] In some embodiments, the compound of formula I is of formula I-a:

[0098] [Chemical formula]

[0099] [In the formula, A contains one or more imaging agents, C is a group that binds to granzyme B] of the compound, or a pharmaceutically acceptable salt thereof.

[0100] In some embodiments, C is a polypeptide that binds to granzyme B, granzyme B an antibody that binds to, an antibody fragment that binds to granzyme B, and a selected from the group consisting of small organic molecules that bind to granzyme B.

[0101] In some embodiments, C is a) a polypeptide that binds to granzyme B, and b) a small organic molecule that binds to granzyme B selected from the group consisting of.

[0102] In some embodiments, C is a) a polypeptide that binds to granzyme B and that X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -D (SEQ ID NO: 1) comprises an amino acid sequence having at least 90% sequence identity thereto, wherein X 1 , X 2 , and X 3 are each independently selected amino acids (e.g., natural amino acids or unnatural amino acids), X 4 is selected from the group consisting of I and V, X 5 is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of P, S, T, Q, N, A, H, V, and D, a poly peptide, and b) a group selected from the group consisting of a compound of formula III:

[0103]

Chemical formula

[0104] or a pharmaceutically acceptable salt thereof, wherein , n is 0, 1, or 2; m is 0, 1, or 2; R 1 and R 2 are each independently hydrogen, C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl, C 6~10 aryl, HET, and -N(R 10 ) 2 selected from the group consisting of Selected from the group, wherein each C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloal kyl is independently optionally substituted by one, two, or three substituents selected from the group consisting of halo and hydroxy, and each C aryl and HET are independently optionally substituted by one, two, or three substituents selected from the group consisting of 6~10 oxo, halo, hydroxy, C alkyl, and C 1~4 haloalkyl; 1~4 or R and R together with the carbon atom to which they are attached form a 5- to 6-membered cycloalkyl group or a 5- to 6-membered heterocycloalkyl group, each of which may optionally be substituted by one, two, or three R 1 groups; 2 each R and R 10 is independently selected from the group consisting of hydrogen, C alkyl, and C haloalkyl; each R 3 and R 7 is independently selected from the group consisting of hydrogen, C 1~4 alkyl, and C 1~4 haloalkyl; each R , R 4 , R 5 , R 6 and R 8 is independently selected from the group consisting of hydrogen, halo, hydroxy, C 1~4 al kyl, and C 1~4 haloalkyl; R 9 is HET which may optionally be substituted by one, two, or three substituents selected from the group consisting of oxo, halo, hydroxy, C 1~4 alkyl, and C 1~4 halo alkyl; R is hydrogen, C 10 alkyl, and C 1~4Alkyl, and -C(O)C 1~4 selected from the group consisting of alkyl wherein -C(O)C 1~4 alkyl is optionally -N(R 11 ) 2 H ET, and C 6~10 substituted by aryl, wherein C 6~10 aryl is optionally substituted by one, two, or three halo groups; each HET is an independently selected monocyclic or bicyclic 5- to 10-membered heteroaryl group, or a monocyclic or bicyclic 5- to 10-membered heterocycloalkyl group, where in this case each HET contains one, two, three, or four hetero atoms selected from O, S, and N and is optionally substituted by one or two oxo groups; R 11 is hydrogen, C 1~4 alkyl, and C 1~4 selected from haloalkyl, if m is 0, then n is 0, and if n is 0, then m is 0.

[0105] In some embodiments, A comprises one or more imaging agents selected from the group consisting of paramagnetic ions, x-ray imaging agents, fluorophores, and radioisotopes. In some embodiments, A comprises one, two, or three imaging agents selected from the group consisting of paramagnetic ions, x-ray imaging agents, fluorophores, and radioisotopes. In some embodiments, A comprises one imaging agent. In some embodiments A comprises two imaging agents. In some embodiments, A comprises three imaging agents.

[0106] In some embodiments, A can include one or more independently selected paramagnetic ions and can include one or more imaging agents. In some embodiments, each of the paramagnetic ions is independently selected from the group consisting of chromium(III), manganese(II), iron(III), iron(II), cobalt (II), nickel(II), copper(II), neodymium(III), samarium(III) , ytterbium(III), gadolinium(III), vanadium(II), terbium (III), dysprosium(III), holmium(III), and erbium( III). In some embodiments, A includes one, two, or three independently selected paramagnetic ions. In some embodiments, one or more independently selected paramagnetic ions are independently bound, directly or indirectly (e.g., via a chelating agent), to the compounds presented herein.

[0107] In some embodiments, A includes one or more imaging agents that are independently selected x-ray imaging agents. In some embodiments, each of the x-ray imaging agents is independently selected from the group consisting of lanthanum(III), gold(III), lead(II), bismuth(III), and x-ray imaging agents iodinated (e.g., diatrizoate, ioxaglate, metrizoate , iopamidol, iohexol, ioxilan, iopromide, iodixanol, and ioversol). In some embodiments, A includes one, two, or three independently selected x-ray imaging agents.

[0108] In some embodiments, A includes one or more independently selected radioisotopes It contains one or more imaging agents. In some embodiments, as presented herein, in one or more of the methods presented herein, radioactive isotopes useful as imaging agents are presented. Additionally, one or more of the radioactive isotopes presented herein may also be useful in one or more therapeutic applications, for example, when administered to a subject in a therapeutically effective amount. For example, I and Cu, when administered to a subject at a low concentration (e.g., , 5 mCi), may be useful as imaging agents (e.g., non-toxic radioactive isotopes and / or non-therapeutic radioactive isotopes), but when administered to a subject at a high concentration, may also be useful as therapeutic agents (e.g., toxic radioactive isotopes and / or therapeutic radioactive isotopes). In some embodiments, each of the radioactive isotopes is independently, 131 H, 64 C, C, F, P, S, Cl, 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 36 Cl, 51 Cr, 52 Fe, , 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Se, , 76 Br, 77 Br, 89 Zr, 90 Y, 99m Tc, 111 In, 123 I, 124 I, 125 I, 131 I, 152 Eu, 153 Sm,166 Ho, 177 Lu, 186 R e, 188 Re, 201 Tl, 203 Pb, 210 At, 211 At, 212 Bi, 21 3 Bi, and 225 is selected from the group consisting of Ac. In some embodiments, one or more independently selected radioisotopes are independently bound, directly or indirectly (e.g., via a chelating agent), to the compounds presented herein.

[0109] In some embodiments, A is an imaging agent selected from the group consisting of positron emission tomography (PET) imaging agents, single photon emission computed tomography (SPECT) imaging agents, and computed tomography imaging agents. In some embodiments, A is a PET imaging agent or a SPECT imaging agent. In some embodiments A is a PET imaging agent. In some embodiments, A is a SPECT imaging agent. In some embodiments, A is a computed tomography imaging agent In some embodiments, A is a computed tomography imaging agent with a radioisotope agent.

[0110] In some embodiments, A is 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 52 F e, 58 Co, 64 Cu, 68 Ga, 76 Br, 77Br 89 Zr 111 In 12 3 I 124 I 125 I 131 I 186 Re 188 Re, and 201 selected from the group consisting of Tl to a PET imaging agent or an SPECT imaging agent, comprising one or more radioisotopes. In some embodiments, A is H 3 H 11 C 14 C 18 F 32 P 35 S 52 Fe 58 Co 64 Cu 68 Ga 76 Br 7 7 Br 89 Zr 111 In 123 I 124 I 125 I 131 I 186 Re , 188 Re, and 201 selected from the group consisting of Tl, one, two, or three radioisotopes, a PET imaging agent or an SPECT imaging agent. In some embodiments, A is H H 3 H 11 C 14 C 18 F 32 P 35 S 52 Fe 58 Co 64 Cu 68 Ga 76 Br 77 Br 89 Zr 111 In 123 I , 124 I, 125 I, 131 I, 186 Re, 188 Re, and 201 consisting of Tl, a PET imaging agent or SPEC A PET imaging agent or SPECT imaging agent containing one radioisotope selected from the group. In some embodiments, A is 68 A PET imaging agent or SPECT imaging agent containing Ga. In some embodiments, A further comprises a chelating agent. Examples of chelating agents are 1,4,7

[0111] -triazacyclononane triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane -1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1 -glutaric acid-4,7-diacetic acid (NODAGA), ethylenediaminetetraacetic acid (EDTA) , diethylenetriaminepentaacetic acid (DTPA), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N ',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine -N,N'-diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), hydroxy ethyldiaminetriacetic acid (HEDTA), and 1,4,8,11-tetraazacyclo tetradecane-N,N',N'',N'''-tetraacetic acid (TETA), 1,4,7,10 -tetraaza-1,4,7,10-tetra-(2-carbamoylmethyl)-cyclododecane (TCMC), and including but not limited to desferrioxamine B (DFO). In some embodiments, the chelating agent is 1,4,7-triazacyclononane triacetic acid ( ), ​NOTE), 1,4,7,10 - tetraazacyclododecane - 1,4,7,10 - tetraacetic acid (DOTA), 1,4,7 - triazacyclononane - 1 - glutaric acid - 4,7 - diacetic acid( NODAGA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid( DTPA), cyclohexyl - 1,2 - diamine tetraacetic acid (CDTA), ethylene glycol O,O’ - bis(2 - aminoethyl) - N,N,N’,N’ - tetraacetic acid (EGTA), N,N - bis(hydroxybenzyl) - ethylenediamine - N,N’ - diacetic acid (HBED ), triethylenetetraminehexaacetic acid (TTHA), hydroxyethyldiaminetriacetic acid (H EDTA), and 1,4,8,11 - tetraazacyclotetradecane - N,N’,N’ ’,N’’’ - tetraacetic acid (TETA), 1,4,7,10 - tetraaza - 1,4,7,10 - tetra - (2 - carbamoylmethyl) - cyclododecane (TCMC), and desfer rioxamine B (DFO) and is selected from the group consisting of. In some embodiments, the chelating agent is 1,4,7 - triazacyclononane triacetic acid (NOTA), 1,4,7,10 - tetra azacyclododecane - 1,4,7,10 - tetraacetic acid (DOTA), and 1,4,7 - tri azacyclononane - 1 - glutaric acid - 4,7 - diacetic acid (NODAGA) and is selected from the group consisting of. In some embodiments, the chelating agent is 1,4,7 - triazacyclononane tri acetic acid (NOTA). In some embodiments, A further comprises one or more chelating agents . In some embodiments, A further comprises one, two, or three chelating agents . In some embodiments, A further comprises one chelating agent. In some embodiments, A further comprises two chelating agents. In some embodiments, A further comprises three chelating agents also includes.

[0112] In some embodiments, A is one or more imaging agents that are independently selected fluorophores. Examples of fluorophores include Alexa 350, Alexa 43 0, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BOD IPY-FL, BODPY-R6G, 13BODLPY-TMR, BODLPY-TRX , cascade blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, quantum dots, Pacific Blue, REG, rhodamine green, rhodamine red, renografin, ROX, TAMRA, TET, tetramethylrhodamine, te xasred, Alexafluor family, Cy5, Cy5.5, Cy7, indocyanine green (ICG), and fluorescent proteins (e.g., green fluorescent protein (G FP), red fluorescent protein (RFP), and dsRED), but are not limited to these.

[0113] In some embodiments, A is Alexa 350, Alexa 430, AMCA, B ODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, B ODPY-R6G, 13BODLPY-TMR, BODLPY-TRX, cascade blue , Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JO E, Oregon Green 488, Oregon Green 500, Oregon Green 514, quantum dots, Pacific Blue, REG, rhodamine green, rhodamine red, renog Raffin, ROX, TAMRA, TET, tetramethylrhodamine, Texas Red, A F350, AF405, AF532, AF488, AF647, AF680, AF750 , Cy5, Cy5.5, Cy7, indocyanine green (ICG), green fluorescent protein (GFP), red fluorescent protein (RFP), and a fluorophore selected from the group consisting of dsRED is. In some embodiments, A comprises one, two, or three, independently selected fluorophores.

[0114] In some embodiments, B is an optional linking group comprising one or more amino acid residues . In some embodiments, B is from about 1 to about 100 amino acid residues, such as from about 1 to about 100 , from about 1 to about 80, from about 1 to about 60, from about 1 to about 40, from about 1 to about 20, from about 1 to about 10, from about 1 to about 5, from about 5 to about 100, from about 5 to about 80, from about 5 to about 60, from about 5 to about 40, from about 5 to about 20, from about 5 to about 10, from about 10 to about 100, from about 10 to about 80, from about 10 to about 60, from about 10 to about 40, from about 1 0 to about 20, from about 20 to about 100, from about 20 to about 80, from about 20 to about 60, from about 20 to about 40, from about 40 to about 100, from about 40 to about 80, from about 40 to about 60, from about 60 to about 100, from about 60 to about 80 , or an optional linking group comprising from about 80 to about 100 amino acid residues.

[0115] In some embodiments, B is one or more alkylene groups, one or more amine groups, one or more amide groups, one or more alkyleneoxy groups, one or more thiol groups, one or more carbohydrate groups, or any combination thereof, an optional linking group comprising. In some embodiments, B is one or more alkylene groups, one or more optionally selected linking group. or a plurality of amine groups, one or more amide groups, one or more alkyleneoxy groups , an optional linking group containing one or more thiol groups, or any combination thereof There is. In some embodiments, B is one or more C 1~50 alkylene groups, one or is a plurality of amine groups, one or more amide groups, one or more C 1~50 alkylene oxy groups, one or more C 1~50 thiol groups, or any combination thereof containing an optional linking group. In some embodiments, B is one or more C 1~30 al kylene groups, one or more amine groups, one or more amide groups, one or more C 1~30 alkyleneoxy groups, one or more C 1~30 thiol groups, or any combination thereof containing an optional linking group.

[0116] In some embodiments, B is an optional linking group containing one or more -(OCH 2 CH 2 )- groups. In some embodiments, B is a linking group of the formula -(OCH CH 2 CH 2 ) p - of optional linking group, and p is from about 1 to about 100, for example, from about 1 to about 100, from about 1 to about 80, from about 1 to about 60, from about 1 to about 40, from about 1 to about 20, from about 1 to about 10, from about 1 to about 5, from about 5 to about 100, from about 5 to about 80, from about 5 to about 60, from about 5 to about 40, from about 5 to about 20, from about 5 to about 10, from about 10 to about 1 00, from about 10 to about 80, from about 10 to about 60, from about 10 to about 40, from about 10 to about 20, from about 20 to about 100, from about 20 to about 80, from about 20 to about 60, from about 20 to about 40, from about 40 to about 100, from about 40 ~ about 80, about 40 to about 60, about 60 to about 100, about 60 to about 80, or about 80 to about 10 In some embodiments, p is an integer from about 10 to about 40. In some embodiments, p is an integer from about 20 to about 40. In some embodiments, p is an integer from about 25 to about The integer 35.

[0117] In some embodiments, C is an antibody that binds to granzyme B. C is an antibody that binds to granzyme B, including clone GB11 and clone GrB The antibody is selected from the group consisting of NCL-L-Gran-B, NCL-L-Gran-7, and NCL-L-Gran-B. In an embodiment, the antibody that binds granzyme B is clone GrB-7.

[0118] In some embodiments, C is an antibody fragment that binds to granzyme B. In the present study, C was clone GB11, clone GrB-7, or NCL-L-Gran- In some embodiments, C is an antibody fragment selected from the group consisting of the antibody fragment of clone GrB-7. It is a body fragment.

[0119] In some embodiments, C is an antibody fragment that binds to granzyme B, in which case anti The body fragments are GTEAAAASSCFVVAE (SEQ ID NO: 13); GTEAAAASACFVVAE (SEQ ID NO:14); GTEAAAASSAFVVAE (SEQ ID NO: 15); GTEAAAASSCAVVAE (SEQ ID NO: 16); GTEAAAASSCFAVAE (SEQ ID NO: 17); GTEAAAASSCFVAAE (SEQ ID NO:18); GTEAAAASSCFVVGE (SEQ ID NO: 19); and GTEAAAASSCFVVAD (SEQ ID NO: 20) comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 9 5%, at least 98%, at least 99%, at least 99.5% sequence identity) to a sequence selected from the group consisting of .

[0120] In some embodiments, C is an antibody fragment that binds to granzyme B, in which case the anti body fragment is GTEAAAASSCFVVAE (SEQ ID NO: 13); GTEAAAASACFVVAE (SEQ ID NO: 14); GTEAAAASSAFVVAE (SEQ ID NO: 15); GTEAAAASSCAVVAE (SEQ ID NO: 16); GTEAAAASSCFAVAE (SEQ ID NO: 17); GTEAAAASSCFVAAE (SEQ ID NO: 18); GTEAAAASSCFVVGE (SEQ ID NO: 19); and GTEAAAASSCFVVAD (SEQ ID NO: 20) selected from the group consisting of

[0121] In some embodiments, C is a polypeptide that binds to granzyme B (e.g., protease inhibitor 9 (PI-9)). In some embodiments, C is a polypeptide that binds to granzyme B , and one or more proteins (e.g., two, three, four, or five proteins). In some embodiments, C is a polypeptide that binds to granzyme B , and one protein. In some embodiments, C is a polypeptide that binds to granzyme B, and serpin P9

[0122] In some embodiments, C is a polypeptide that binds to granzyme B, in which case the polypeptide is X1 -X 2 -X 3 -X 4 -X 5 -X 6 -D (array number 1) comprises an amino acid sequence having at least 90% sequence identity, wherein X 1 , X 2 , and X 3 are each independently selected amino acids (e.g., natural amino acids or unnatural amino acids), X 4 is selected from the group consisting of I and V, X 5 is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D is selected.

[0123] In some embodiments, X 1 , X 2 , and X 3 are each independently, beta A (i.e., , beta-alanine), G, Q, N, S, T, Y, C, R, D, and E selected from the group consisting of. In some embodiments, X 1 , X 2 , and X 3 are each independently, beta A (i.e., beta-alanine) and G selected from the group consisting of. In some embodiments is, X 1 is beta A. In some embodiments, X 2 is G. In some embodiments in, X 3 is G. In some embodiments, X 2 and X 3 are each G. In some embodiments, X 1 is beta A, X 2 and X 3 are each G. In some embodiments In the state, X 1 , X 2 , and X 3 are each G.

[0124] In some embodiments, X 1 , X 2 , and X 3 are each independently selected non-charged hydrophilic amino acids. In some embodiments, X 1 , X 2 , and X 3 are each independently selected non-charged hydrophilic amino acids selected from the group consisting of G, Q, N, S, and T and are non-charged hydrophilic amino acids selected therefrom.

[0125] In some embodiments, X 4 is I. In some embodiments, X 4 is V.

[0126] In some embodiments, X 5 is E. In some embodiments, X 5 is G. In some embodiments, X 5 is D. In some embodiments, X 5 is S.

[0127] In some embodiments, X 6 is P. In some embodiments, X 6 is S. In some embodiments, X 6 is T. In some embodiments, X 6 is Q. In some embodiments, X 6 is N. In some embodiments, X 6 is A. In some embodiments, X 6 is H. In some embodiments, X 6 is V. In some embodiments X 6It is D.

[0128] In some embodiments, C is a polypeptide that binds to granzyme B, in which case the polypeptide has an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 1 and includes. In some embodiments, C is a polypeptide that binds to granzyme B, and in this case, the polypeptide has an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 1 and includes. In some embodiments, C is a polypeptide that binds to granzyme B, in which case the polypeptide has an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 1 and includes. In some embodiments, C is a polypeptide that binds to granzyme B, in which case the polypeptide has an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO: 1 and includes.

[0129] In some embodiments, the polypeptide beta A-G-G-I-E-F-D (SEQ ID NO: 2); G-G-G-I-E-F-D (SEQ ID NO: 3); and beta A-G-G-I-E-P-D (SEQ ID NO: 4) and has an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of and includes.

[0130] In some embodiments, the polypeptide beta A-G-G-I-E-F-D (SEQ ID NO: 2); G-G-G-I-E-F-D (SEQ ID NO: 3); beta A-G-G-I-E-P-D (SEQ ID NO: 4); beta A-G-G-G-I-E-P-D (SEQ ID NO: 22); beta A-G-G-G-T-E-A-A-A-A-S-S-C-F-I-E-F-D( SEQ ID NO: 23) An amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of is included.

[0131] In some embodiments, the polypeptide beta A-G-G-G-I-E-P-D (SEQ ID NO: 22); and beta A-G-G-G-T-E-A-A-A-A-S-S-C-F-I-E-F-D( SEQ ID NO: 23) An amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of is included.

[0132] In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO: 2. In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. is. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3. is.

[0133] In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3. Then, C is a polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 3. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 3. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO: 3. In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.

[0134] In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 4. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO: 4. In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 4.

[0135] In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 22. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 22. In some embodiments, C is an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 22. It is a polypeptide containing. In some embodiments, C is at least 99% a polypeptide comprising an amino acid sequence having sequence identity with SEQ ID NO: 22. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99.5% sequence identity with SEQ ID NO: 22 a polypeptide. In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 22 a polypeptide.

[0136] In some embodiments, C is a polypeptide having at least 90% sequence identity with SEQ ID NO: 23 an amino acid sequence. In some embodiments, C is at least a polypeptide comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 23. In some embodiments C is a polypeptide comprising an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 23 a polypeptide. In some embodiments, C is at least 99% a polypeptide comprising an amino acid sequence having sequence identity with SEQ ID NO: 23. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99.5% sequence identity with SEQ ID NO: 23 a polypeptide. In some embodiments, C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 23 a polypeptide.

[0137] In some embodiments, C is a polypeptide that binds to granzyme B, in which case , the polypeptide is X 4 -X 5 -X 6 -D (SEQ ID NO: 5) comprises an amino acid sequence having at least 90% sequence identity with, wherein X 4 is selected from the group consisting of I and V, X 5 is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D and is so selected.

[0138] In some embodiments, X 4 is I. In some embodiments, X 5 is E. In one In some embodiments, X 6 is P or F. In some embodiments, X 6 is P .

[0139] In some embodiments, C is a polypeptide that binds to granzyme B, in which case the polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 5 and is so included. In some embodiments, C is a polypeptide that binds to granzyme B, in this case the polypeptide comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 5 and is so included. In some embodiments, C is a polypeptide that binds to granzyme B and comprises a polypeptide having an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 5 and is so included. In some embodiments, C is a polypeptide that binds to granzyme B and comprises a polypeptide having an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO: 5 and is so included.

[0140] In some embodiments, the polypeptide is I-E-F-D (SEQ ID NO: 6); and I-E-P-D (SEQ ID NO: 7) and comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of and is so included.

[0141] In some embodiments, C has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6 It is a polypeptide containing an amino acid sequence. In some embodiments, C is at least a polypeptide containing an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments C is a polypeptide containing an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 6. In some embodiments, C is a polypeptide containing an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 6. In some embodiments, C is a polypeptide containing an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO: 6. In some embodiments, C is a polypeptide containing the amino acid sequence of SEQ ID NO: 6. .

[0142] In some embodiments, C is a polypeptide that binds to granzyme B, in which case the polypeptide is X -X -X identity to SEQ ID NO: 7. In some embodiments, C is a polypeptide containing an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO: 7. In some embodiments, C is a polypeptide containing the amino acid sequence of SEQ ID NO: 7. .

[0143] In some embodiments, C is a polypeptide that binds to granzyme B, in which case the polypeptide is X 1 -X 2 -X 3-X 4 -X 5 -X 6 -D-X 7 (Array number 8) comprises an amino acid sequence having at least 90% sequence identity, wherein X 1 , X 2 , and X 3 are each independently selected amino acids (e.g., natural amino acids or unnatural amino acids), X 4 is selected from the group consisting of I and V, X 5 is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D is selected, X 7 is an electrophilic group.

[0144] In some embodiments, X 1 , X 2 , and X 3 are each independently beta A (i.e., , beta-alanine), G, Q, N, S, T, Y, C, R, D, and E selected from the group. In some embodiments, X 1 , X 2 , and X 3 are each independently selected from the group consisting of beta A (i.e., beta-alanine) and G. In some embodiments is, X 1 is beta A. In some embodiments, X 2 is G. In some embodiments in, X 3 is G. In some embodiments, X 2 and X 3 are each G. In some embodiments, X 1 is beta A, X 2 and X 3is each G. In some embodiments state, X 1 X 2 and X 3 is each G.

[0145] In some embodiments, X 1 X 2 and X 3 are each independently selected non-charged hydrophilic amino acids. In some embodiments, X 1 X 2 and X 3 are each independently selected non-charged hydrophilic amino acids selected from the group consisting of G, Q, N, S, and T selected non-charged hydrophilic amino acids.

[0146] In some embodiments, X 4 is I. In some embodiments, X 4 is V.

[0147] In some embodiments, X 5 is E. In some embodiments, X 5 is G. One In some embodiments, X 5 is D. In some embodiments, X 5 is S.

[0148] In some embodiments, X 6 is P. In some embodiments, X 6 is S. One In some embodiments, X 6 is T. In some embodiments, X 6 is Q. In some embodiments, X 6 is N. In some embodiments, X 6 is A. In some embodiments form, X 6 is H. In some embodiments, X 6 is V. In some embodiments then X​6 is D.

[0149] In some embodiments, X 7 comprises the C-terminus of the amino acid sequence of SEQ ID NO: 8. In some embodiments, X 7 is -C(O)H, -C(O)C 1~6 alkyl, -C(O)C 1~6 halo lower alkyl, -C(O)C 1~6 alkoxy, -C(O)C 1~6 haloalkoxy, -C (O)-(C 1~6 alkyl)-(5- to 10-membered heteroaryl), -C(O)-(C 1 ~6 haloalkyl)-(5- to 10-membered heteroaryl), -C(O)-(C 1~6 alko xy)-(5- to 10-membered heteroaryl), and -C(O)-(C 1~6 haloalkoxy xy)-(5- to 10-membered heteroaryl) and is an electrophilic group selected from the group consisting of. In some embodiments, X 7 is -C(O)H. In some embodiments, X 7 is selected from the group of substituents presented in column A of Table 1.

[0150] In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 8. In some embodiments C is a polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 8. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99.5% sequence identity to SEQ ID NO: 8. In some embodiments, C is It is a polypeptide comprising the amino acid sequence of SEQ ID NO: 8.

[0151] In some embodiments, C is a polypeptide that binds to granzyme B, in which case , the polypeptide is X 4 -X 5 -X 6 -D-X 7 (SEQ ID NO: 21) comprises an amino acid sequence having at least 90% sequence identity to, wherein X 1 , X 2 , and X 3 are each independently selected amino acids (e.g., natural amino acids or unnatural amino acids), X 4 is selected from the group consisting of I and V, X 5 is selected from the group consisting of E, G, D, and S, X 6 is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D selected, X 7 is an electrophilic group.

[0152] In some embodiments, X 1 , X 2 , and X 3 are each independently beta A (i.e., , beta-alanine), G, Q, N, S, T, Y, C, R, D, and E selected from the group consisting of. In some embodiments, X 1 , X 2 , and X 3 are each independently beta A (i.e., beta-alanine) and G selected from the group consisting of. In some embodiments is, X 1 is beta A. In some embodiments, X 2 is G. In some embodiments in, X 3is G. In some embodiments, X 2 and X 3 are each G. In some embodiments, X 1 is beta A, and X 2 and X 3 are each G. In some embodiments X 1 X 2 and X 3 are each G.

[0153] In some embodiments, X 1 X 2 and X 3 are each independently selected non-charged hydrophilic amino acids. In some embodiments, X 1 X 2 and X 3 are each independently selected non-charged hydrophilic amino acids selected from the group consisting of G, Q, N, S, and T and are non-charged hydrophilic amino acids.

[0154] In some embodiments, X 4 is I. In some embodiments, X 4 is V.

[0155] In some embodiments, X 5 is E. In some embodiments, X 5 is G. In some embodiments, X 5 is D. In some embodiments, X 5 is S.

[0156] In some embodiments, X 6 is P. In some embodiments, X 6 is S. In some embodiments, X 6 is T. In some embodiments, X 6 is Q. In some embodiments, X 6is N. In some embodiments, X 6 is A. In some embodiments, X 6 is H. In some embodiments, X 6 is V. In some embodiments, X 6 is D.

[0157] In some embodiments, X 7 includes the C-terminus of the amino acid sequence of SEQ ID NO: 9. In some embodiments, X 7 is -C(O)H, -C(O)C 1~6 alkyl, -C(O)C 1~6 halo alkyl, -C(O)C 1~6 alkoxy, -C(O)C 1~6 haloalkoxy, -C (O)-(C 1~6 alkyl)-(5- to 10-membered heteroaryl), -C(O)-(C 1 ~6 haloalkyl)-(5- to 10-membered heteroaryl), -C(O)-(C 1~6 alko xy)-(5- to 10-membered heteroaryl), and -C(O)-(C 1~6 haloalkox y)-(5- to 10-membered heteroaryl) and is an electrophilic group selected from the group consisting of. In some embodiments, X 7 is -C(O)H. In some embodiments, X 7 is selected from the group of substituents presented in column A of Table 1.

[0158] In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 21. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 21. In some In one form, C is a polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 21. In some embodiments, C is a polypeptide comprising an amino acid sequence having at least 99. 5% sequence identity to SEQ ID NO: 21. In some embodiments , C is a polypeptide comprising the amino acid sequence of SEQ ID NO: 21.

[0159] In some embodiments, C is a small organic molecule that binds to granzyme B. As used herein the term "small organic molecule" refers to an organic compound having a low molecular weight (e.g., less than 900 daltons, 80 0 daltons or less, 700 daltons or less, 600 daltons or less, or 500 daltons or less ). In some embodiments, C, which is a small organic molecule, is a peptidomimetic. As used herein the term "peptidomimetic" refers to a compound that represents a natural or synthetic amino acid sequence and contains one or more functional groups (e.g., amine, amide, carboxy, a ldehyde, etc.).

[0160] In some embodiments, C has the following structure:

[0161]

Chemical formula

[0162] is a small organic molecule, or a pharmaceutically acceptable salt thereof.

[0163] In some embodiments, C has the following structure:

[0164]

Chemical formula

[0165] is a small organic molecule, or a pharmaceutically acceptable salt thereof.

[0166] In some embodiments, C is an organic small molecule that binds to granzyme B, each of whose disclosures is incorporated herein by reference in its entirety from the group of compounds presented in International Application Publication No. WO 2014 / 153667 and International Application Publication No. WO 2003 / 065987 which is selected

[0167] In some embodiments, C is an organic small molecule of formula III:

[0168]

Chemical formula

[0169] or a pharmaceutically acceptable salt thereof, wherein n is 0, 1, or 2; m is 0, 1, or 2; R 1 and R 2 are each independently selected from the group consisting of hydrogen, C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl, C 6~10 aryl, HET, and -N(R 10 ) 2 wherein each C alkyl, C 1~6 alkoxy, C 1~6 cycloalkyl is optionally substituted with one, two, or three 3~6 substituents independently selected from the group consisting of halo and hydroxy, and each C aryl and HET are independently oxo, halo, hydroxy, C 6~10 alkyl, and C haloalkyl 1~4 from the group 1~4 consisting of optionally substituted with one, two, or three substituents selected from; or R 1 and R 2 together with the carbon atom to which they are attached form, each independently, a 5- to 6-membered 10 cycloalkyl group or 5- to 6-membered heterocycloalkyl group, optionally substituted with one, two, or three R groups; ; each R 3 and R 7 is independently hydrogen, C 1~4 alkyl, and C 1~4 haloalkyl selected from the group consisting of; each R 4 , R 5 , R 6 , and R 8 is independently hydrogen, halo, hydroxy, C 1~4 alkyl, and C haloalkyl selected from the group consisting of; 1~4 ; R 9 is independently HET optionally substituted with one, two, or three substituents selected from the group consisting of oxo, halo, hydroxy, C 1~4 alkyl, and C 1~4 halo alkyl; ; R 10 is hydrogen, C 1~4 alkyl, or -C(O)C 1~4 alkyl selected from the group consisting of, where -C(O)C alkyl is optionally substituted with -N(R 1~4 ), H 11 ET, and C 2 aryl, where C aryl is optionally substituted with one, two, or three halo groups; 6~10 ; 6~10 optionally; ; Each HET is an independently selected monocyclic or bicyclic 5- to 10-membered heteroaryl group, or a monocyclic or bicyclic 5- to 10-membered heterocycloalkyl group, where in this case, each HET contains one, two, three, or four hetero atoms selected from O, S, and N, and is optionally substituted with one or two oxo groups; R 11 is selected from hydrogen, C 1~4 alkyl, and C 1~4 haloalkyl.

[0170] In some embodiments, when m is 0, n is 0. In some embodiments, when n is 0, m is 0. In some embodiments, when m is 0, n is 0, and when n is 0, m is 0.

[0171] In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, n is 0. In some embodiments n is 1. In some embodiments, n is 2. In some embodiments, m and n are each 0.

[0172] In some embodiments, R 1 and R 2 are each independently selected from the group consisting of hydrogen, C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl, phenyl, HET, and -N(R 10 ) 2 wherein each C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl is independently selected from the group consisting of one, two 、 or optionally substituted by three substituents, and each phenyl and HET is, independently, oxo, halo, hydroxy, C 1~4 alkyl, and C 1~4 haloalkyl, is optionally substituted by one, two, or three substituents selected from the group consisting of.

[0173] In some embodiments, R 1 and R 2 are each, independently, C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cycloalkyl, phenyl, HET, and -N(R 10 ) 2 selected from the group consisting of, wherein each C 1~6 alkyl, C 1~6 alkoxy, C 3~6 cyclo alkyl is independently optionally substituted by one, two, or three substituents selected from the group consisting of halo and hydroxy, and each phenyl and HET is, independently, oxo , halo, hydroxy, C 1~4 alkyl, and C 1~4 haloalkyl, and is optionally substituted by one, two, or three substituents selected from the group consisting of.

[0174] In some embodiments, R 1 and R 2 are each, independently, C 1~6 alkyl, C 3~6 cycloalkyl, phenyl, pyridyl, 2-oxopyrrolidine, and -N(R 10 ) 2 selected from the group consisting of, wherein each C 1~6 alkyl, C 3~6 cycloalkyl is independently by one, two, or three groups selected from the group consisting of halo and hydroxy optionally substituted, and phenyl, pyridyl, and 2-oxopyrrolidine are each independently optionally substituted by halo, hydroxy, and one, two, or three halo groups C 1~4 optionally substituted by one, two, or three groups selected from the group consisting of alkyl .

[0175] In some embodiments, R 1 and R 2 are each independently selected from the group consisting of C 1~6 alkyl, C 3~6 cycloalkyl, phenyl, pyridyl, 2-oxopyrrolidine, and -N(R 10 ) 2 , wherein each C 1~6 alkyl, C 3~6 cycloalkyl is independently optionally substituted by one, two, or three groups selected from the group consisting of halo and hydroxy optionally substituted, and phenyl, pyridyl, and 2-oxopyrrolidine are each independently optionally substituted by halo, hydroxy, and one, two, or three halo groups C 1~4 optionally substituted by one, two, or three groups selected from the group consisting of alkyl , R 10 is selected from the group consisting of hydrogen, C 1~4 alkyl, and -C(O)C 1~4 alkyl, and -C(O)C alkyl is optionally substituted by N(R 1~4 ) 11 , 2 pyrrolidinyl, piperidinyl, morpholinyl, benzothiophenyl, and phenyl, and phenyl is optionally substituted by one, two, or three halo groups .​ .

[0176] In some embodiments, R 3 is selected from the group consisting of H and C 1~4 alkyl. In some embodiments, R 3 is H.

[0177] In some embodiments, each R 4 is selected from the group consisting of H, halo, C 1~6 alkyl, and C 1~6 halo alkyl. In some embodiments, each R 4 is independently H or C 1~6 alkyl. In some embodiments, each R 4 is H.

[0178] In some embodiments, each R 5 is selected from the group consisting of H, halo, C 1~6 alkyl, and C 1~6 halo alkyl. In some embodiments, each R 5 is independently H or C 1~6 alkyl. In some embodiments, each R 5 is H.

[0179] In some embodiments, each R 6 is selected from the group consisting of H, halo, C 1~6 alkyl, and C 1~6 halo alkyl. In some embodiments, each R 6 is independently H or C 1~6 alkyl. In some embodiments, each R 6 is H.

[0180] In some embodiments, R 7 is selected from the group consisting of H and C 1~4 alkyl. In some embodiments, R 7 is H.

[0181] In some embodiments, each R 8 is selected from the group consisting of H, halo, C 1~6 alkyl, and C 1~6 halo alkyl. In some embodiments, each R 8 is independently H or C 1~6 alkyl. In some embodiments, each R 8 is H.

[0182] In some embodiments, each R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are H.

[0183] In some embodiments, each HET contains one, two, three, or four heteroatoms each independently selected from O, S, and N, and is optionally substituted with one or two oxo groups, and is independently selected from monocyclic, 5- to 6-membered heteroaryl, bicyclic, 8- to 10-membered heteroaryl, monocyclic, 4- to 6-membered heterocycloalkyl, or bicyclic, 8- to 10 membered heterocycloalkyl groups. In some embodiments, R is an unsubstituted HET group. In some embodiments, R

[0184] is independently an HET group substituted with one or two groups selected from oxo, halo, hydroxy, C 9 alkyl, and C 9 haloalkyl. In some embodiments, R 1~4 is pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl 1~4 alkyl, and C alkyl. In some embodiments, R 9 is selected from the group consisting of pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl , thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, 1,2,4-tri zolyl, 1,2,3-triazolyl, and tetrazolyl. In some embodiments, R 9 is, each independently, halo, hydroxy, C 1~4 alkyl, and C 1~4 haloalkyl, and is optionally substituted by one, two, or three substituents selected from the group consisting of pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, imidazo ryl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, 1 ,2,4-triazolyl, 1,2,3-triazolyl, and tetrazolyl. Selected from the group.

[0185] In some embodiments, each HET is, each independently, halo, oxo, hydroxy, C 1~ 4 alkyl, and C 1~4 haloalkyl, and is optionally substituted by one or two substituents selected from the group consisting of benzimidazolyl, benzofuranyl, benzopyr zolyl, benzotriazolyl, benzothiophenyl, benzoxazolyl, cinnolinyl, furanyl, imidazolyl, indolinyl, indolyl, indradinyl, indazolyl, isobenzofuranyl, isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl yl, naphthyridinyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyr dopyridinyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinoli yl, quinoxalinyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, tetraz ril, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl, Pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl, dihydro benzofuranyl, dihydrobenzothiophenyl, dihydrobenzoxazolyl, dihydro furanyl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydro pyrazinyl, dihydropyrazolyl, dihydropyridinyl, dihydropyrimidinyl, dihydro pyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydro thiazolyl, dihydrothienyl, dihydrotriazolyl, tetrahydrofuranyl, and tetrahydrothienyl, independently selected from the group consisting of.

[0186] In some embodiments, the compound of formula III, or a pharmaceutically acceptable salt thereof, is of formula II I-a:

[0187]

Chemical formula

[0188] [wherein, R 9 is as defined above for the compound of formula III] of the compound, or a pharmaceutically acceptable salt thereof.

[0189] In some embodiments, the compound of formula III is Table 1

[0190]

Table 3-1

[0191]

Table 3-2

[0192]

Table 3-3

[0193]

Table 3-4

[0194] In some embodiments, the compound of Formula III is the one presented in Table 2

[0195]

Table 4

[0196] In some embodiments, the compound of Formula III is the one presented in Table 3

[0197]

Table 5

[0198] [wherein,

[0199]

Chemical Structure

[0200] represents a bond connecting group C to an optional linker B or group A of Formula I] is selected from the group consisting of the compounds presented in, or a pharmaceutically acceptable salt thereof.

[0201] In some embodiments, A is an imaging agent comprising one or more of a paramagnetic ion, an x-ray imaging agent, a fluorophore, and a radioisotope, and B is one or more alkylene groups, one or more amine groups, one or more an amide group, one or more alkyleneoxy groups, one or more thiol groups, or an optional linking group containing any combination thereof, where C is selected from the group consisting of a polypeptide that binds to granzyme B, an antibody that binds to granzyme B, an antibody fragment that binds to granzyme B, and a small organic molecule that binds to granzyme B. In some embodiments, A contains a radioisotope,

[0202] and B is an optional linking group containing one or more C alkylene groups, one or more amine groups, one or 1~30 more amide groups, one or more C alkyleneoxy groups, one or more 1~30 C thiol groups, or any combination thereof, 1~30 where C is selected from the group consisting of a polypeptide that binds to granzyme B and a small organic molecule that binds to granzyme B. In some embodiments, A is

[0203] H, 3 C, 11 C, 14 C, 18 F, 32 P, 35 S, 36 C l, 51 Cr, 52 Fe, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 G a, 68 Ga, 75 Se, 76 Br, 77 Br, 89 Zr, 90 Y, 99m Tc, 111 In, 123 I,124 I, 125 I, 131 I, 152 Eu, 153 Sm, 166 Ho , 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 210 At, 211 At, 212 Bi, 213 Bi, and 225 radioisotopes selected from the group consisting of Ac, wherein B is an optional linking group comprising one or more C 1 ~ 30 alkylene groups, one or more amide groups, one or more C ~ 1 ~ 30 alkyleneoxy groups, or any combination thereof, and C is selected from the group consisting of a polypeptide that binds to granzyme B and an organic small molecule that binds to granzyme B.

[0204] In some embodiments, A is 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 36 C l, 51 Cr, 52 Fe, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 G a, 68 Ga, 75 Se, 76 Br, 77 Br, 89 Zr, 90 Y, 99m Tc, 111 ​ In, 123 I, 124 I, 125 I, 131 I, 152 Eu, 153 Sm, 166 Ho , 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 210 At, 211 At, 212 Bi, 213 Bi, and 225 radioisotopes selected from the group consisting of Ac, including, B is an optional linking group containing one or more -(-OCH 2 CH 2 )- groups, C is selected from the group consisting of a polypeptide that binds to granzyme B, and an organic small molecule that binds to granzyme B.

[0205] In some embodiments, A is an imaging agent comprising one or more of paramagnetic ions, x-ray imaging agents, fluorophores, and radioisotopes, wherein, B is an optional linking group containing from about 1 to about 100 amino acid residues, C is selected from the group consisting of a polypeptide that binds to granzyme B, an antibody that binds to granzyme B, an antibody fragment that binds to granzyme B, and an organic small molecule that binds to granzyme B.

[0206] In some embodiments, A contains a radioisotope, B is an optional linking group containing from about 1 to about 100 amino acid residues, C is selected from the group consisting of a polypeptide that binds to granzyme B, and an organic small molecule that binds to granzyme B.​It is selected from the group consisting of low molecules.

[0207] In some embodiments, A is 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 36 Cl, 51 Cr, 52 F e, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 S e, 76 Br, 77 Br, 89 Zr, 90 Y, 99m Tc, 111 In, 123 I, 12 4 I, 125 I, 131 I, 152 Eu, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 210 At, 211 At, 212 Bi, 2 13 Bi, and 225 selected from the group consisting of Ac, contains a radioisotope, B is an optional linking group containing about 1 to about 100 amino acid residues, C is a polypeptide that binds to granzyme B, and an organic that binds to granzyme B selected from the group consisting of low molecules.

[0208] In some embodiments, A is 3 H, 11 C, 14 C, 18 F, 32 P, 35 S, 36 Cl, 51 Cr, 52 F e, 57 Co, 58 Co, 59 Fe, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 S e, 76 Br, 77 Br, 89 Zr, 90 Y, 99m Tc, 111 In, 123 I, 12 4 I, 125 I, 131 I, 152 Eu, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 210 At, 211 At, 212 Bi, 2 13 Bi, and 225 selected from the group consisting of Ac, and contains a radioisotope B is an optional linking group containing from about 1 to about 100 amino acid residues, C is selected from the group consisting of a polypeptide that binds to granzyme B and an organic small molecule that binds to granzyme B.

[0209] In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is of formula I-b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I-l, a compound of I-m, I-n, or I-o:

[0210] [Chem.]

[0211] or a pharmaceutically acceptable salt thereof is.

[0212] In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is a compound of formula I-p or I-q: is.

[0213] [Chem.] is.

[0214] In some embodiments, the compound of formula I is 68 Ga-NOTA-betaA-G-G-I-E-F-D (Compound 1; SEQ ID NO: 9) 68 Ga-NOTA-(OCH 2 CH 2 ) 27 -G-G-G-I-E-F-D (Compound 2; SEQ ID NO: 10), and 68 Ga-NOTA-betaA-G-G-I-E-P-D (Compound 3; SEQ ID NO: 11) and is a compound selected from the group consisting of.

[0215] In some embodiments, the compound of formula I is 68 Ga-NOTA-betaA-G-G-I-E-F-D (Compound 1; SEQ ID NO: 9); 68 Ga-NOTA-(OCH 2 CH 2 ) 27 -G-G-G-I-E-F-D (Compound 2; Sequence number 10); 68 Ga-NOTA-betaA-G-G-I-E-P-D (Compound 3; Sequence number 11) ; and 68 Ga-NOTA-betaA-G-G-G-T-E-A-A-A-A-S-S-C- F-I-E-F-D (Compound 4, Sequence number 24) is a compound selected from the group consisting of.

[0216] In some embodiments, the compound of Formula I is 68 Ga-NOTA-betaA-G-G-I- E-P-D (Compound 3; Sequence number 11).

[0217] Synthesis As will be appreciated, the compounds presented herein, including their salts, can be prepared using known organic synthesis methods and can be synthesized according to any of a number of possible synthetic routes. For example, the compound of Formula III presented herein can be prepared according to the procedures described in U.S. Patent Application No. 10 / 503,155 the disclosure of which is incorporated herein by reference in its entirety. Once prepared, the compound of Formula III can be covalently bonded to Group A or, optionally, to Group B using standard techniques known in the art.

[0218] Peptide synthesis The compounds of Formula I presented herein, which include one or more polypeptides, can be prepared, for example , using standard methods for preparing peptide bonds (e.g., the solid-phase synthesis method described in Merrifield et al, Journal of the American Chemical Society 85.14 (1963): 2149-2154) ​​​​) can be prepared using. Those skilled in the art are well aware of peptide synthesis methods. For example, Bodanszky et al, Gastroenterology 71 (1976): 965-970; Houghten, Proceedings of the National Academy of Sciences 82.15 (1985): 5131-5135; Stewart et al, Soli d phase peptide synthesis. Pierce Chemical Company, 1984. Suitable protecting groups for use in such syntheses can be found in the above textbooks, as well as in McOmie, Protecti ve Groups in Organic Chemistry, (1973):98. These synthesis methods involve the sequential addition of one or more amino acid residues, or suitable, protected amino acid residues, to a growing peptide chain. Usually, the amino or carboxyl group of the first amino acid residue is protected with a suitable, selectively removable protecting group. For amino acids containing reactive side chain groups such as lysine, different, selectively removable protecting groups are utilized.

[0219] In some embodiments, the polypeptides of the present application can be prepared using solid-phase synthesis methods. For example, an amino acid is joined to an inert solid support via its unprotected carboxyl or amino group. Then, the protecting group of the amino or carboxyl group is selectively removed, and the next amino acid in the sequence, which has a complementary (e.g., amino or carboxyl) group appropriately protected, is mixed and reacted with the residue already joined to the solid support. Then, the protecting group of the amino or carboxyl group is newly added to the newly added residue, and the process is repeated until the desired peptide chain length is reached. Remove from the amino acid residues and then add additional amino acids (e.g., appropriately protected amino acids). Repeat this procedure until the desired polypeptide length is prepared. After ligating the desired amino acids in the proper sequence, any remaining terminal protecting groups and side chain protecting groups (and solid support) can be removed sequentially or simultaneously to yield the final peptide. In some embodiments, the polypeptide compounds of Formula I presented herein do not contain benzylated amino acid residues or methylbenzylated amino acid residues. As described elsewhere, reactions may also be necessary to form intramolecular linkages that restrict conformation.

[0220] Linking group Bifunctional crosslinking reagents are widely used for the preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. Homobifunctional reagents having two identical functional groups crosslink identical polypeptides as well as different polypeptides or residues of polypeptides and have been shown to be highly efficient when linking polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivity of the different functional groups, crosslinking can be controlled selectively and sequentially. For example, bifunctional crosslinking reagents can be classified according to the specificity of their functional groups, including but not limited to groups specific for amino, sulfhydryl, guanidino, indole, carboxyl. Many heterobifunctional crosslinking reagents contain a primary amine-reactive group and a thiol-reactive group.

[0221] For further examples of heterobifunctional cross-linking reagents and methods of using cross-linking reagents, see U.S. Patent No. 5,88 9,155, the disclosure of which is incorporated herein by reference in its entirety. The cross-linking reagent combines a nucleophilic hydrazide residue with an electrophilic maleimide residue and, in one example, enables the coupling of an aldehyde with a free thiol. The cross-linking reagent can be modified to cross-link a variety of functional groups and is thus useful for cross-linking polypeptides. Table 4 details certain heterobifunctional cross-linking agents that are believed to be useful for preparing compounds of formula I containing a linking group B.

[0222]

Table 6

[0223] For compounds in which a particular peptide contains residues in its native sequence that are not suitable for a particular cross-linking reagent, conservative genetic changes or synthetic amino acid changes within the primary sequence can be exploited.

[0224] Imaging agents In the art, many suitable imaging agents are known, similar to their methods of attachment to antibodies (see, for example, U.S. Patent No. 5,021,236; No. 4,938,948; and No. 4,472,509, the disclosures of each of which are incorporated herein by reference in their entirety). Radioactively labeled compounds of formula I presented herein can be prepared according to methods well known in the art. For example, then the monoclonal antibody can be iodinated by contacting it with a chemical oxidizing agent such as sodium and / or potassium iodide and sodium hypochlorite, or an enzymatic oxidizing agent such as lactoperoxidase. In a further example, the compounds of Formula I presented herein can be radiolabeled by radiometalation of a bifunctional chelating agent (e.g., NOTA, DOTA, or NODAGA) or similar derivatives thereof presented herein, with Ga. In the art, synthetic methods for incorporating radioisotopes into organic compounds are well known, and those skilled in the art will readily recognize other methods applicable to the compounds presented herein. Those skilled in the art will appreciate that the described procedures are not exclusive means for synthesizing the compounds presented herein, and that a wide repertoire of synthetic organic reactions are potentially available for use in the synthesis of the compounds presented herein. Those skilled in the art know how to select and implement an appropriate synthetic route. Appropriate synthetic methods for starting materials, intermediates, and products can be found in Advances in Heterocyclic Chemistry, Vols. 1- 107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.) Science of Synthe 68

[0225] 107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.) Science of Synthe tic Methods, Volumes 1-35 (Georg Thieme Verlag, 2000-2012); Comprehensive Organic Transformations, 2nd Edition (Wiley-VCH, 1995); Larock, Comprehensive Organic Transformations, 2nd Edition (Wiley-VCH, 1995); Carey and Sundberg, Advanced Organic Chemistry, 5th Edition (Springer, 2007); March, Advanced Organic Chemistry, 6th Edition (Wiley, 2007); and other standard references in the art.

[0226] Those skilled in the art will recognize that the described steps are not the exclusive means for synthesizing the compounds presented herein, and that a wide range of synthetic organic reactions are potentially available for use in the synthesis of the compounds presented herein. Those skilled in the art know how to select and implement an appropriate synthetic route. Appropriate synthetic methods for starting materials, intermediates, and products can be found in Advances in Heterocyclic Chemistry, Vols. 1- 107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.) Science of Synthe tic Methods, Volumes 1-35 (Georg Thieme Verlag, 2000-2012); Comprehensive Organic Transformations, 2nd Edition (Wiley-VCH, 1995); Larock, Comprehensive Organic Transformations, 2nd Edition (Wiley-VCH, 1995); Carey and Sundberg, Advanced Organic Chemistry, 5th Edition (Springer, 2007); March, Advanced Organic Chemistry, 6th Edition (Wiley, 2007); and other standard references in the art. The appropriate synthetic methods for starting materials, intermediates, and products can be found in Advances in Heterocyclic Chemistry, Vols. 1- 107 (Elsevier, 1963-2012); Journal of Heterocyclic Chemistry Vols. 1-49 (Journal of Heterocyclic Chemistry, 1964-2012); Carreira, et al. (Ed.) Science of Synthe tic Methods, Volumes 1-35 (Georg Thieme Verlag, 2000-2012); Comprehensive Organic Transformations, 2nd Edition (Wiley-VCH, 1995); Larock, Comprehensive Organic Transformations, 2nd Edition (Wiley-VCH, 1995); Carey and Sundberg, Advanced Organic Chemistry, 5th Edition (Springer, 2007); March, Advanced Organic Chemistry, 6th Edition (Wiley, 2007); and other standard references in the art. tic Methods, Volumes 1-35 (Georg Thieme Verlag, 2000-2012); Comprehensive Organic Transformations, 2nd Edition (Wiley-VCH, 1995); Larock, Comprehensive Organic Transformations, 2nd Edition (Wiley-VCH, 1995); Carey and Sundberg, Advanced Organic Chemistry, 5th Edition (Springer, 2007); March, Advanced Organic Chemistry, 6th Edition (Wiley, 2007); and other standard references in the art. ​​​sis, Vols. 1-48 (2001-2010) and Knowledge Updates KU2010 / 1-4; 2011 / 1-4; 2012 / 1-2 (Thieme, 2001-2012); Katritzky, et al. (Ed.) Comprehensive Organic Functional G roup Transformations, (Pergamon Press, 1996); Katritzky et al. (Ed.); Comprehens ive Organic Functional Group Transformations II (Elsevier, 2 nd Edition, 2004); Ka tritzky et al. (Ed.), Comprehensive Heterocyclic Chemistry (Pergamon Press, 1984 ); Katritzky et al., Comprehensive Heterocyclic Chemistry II, (Pergamon Press, 1 996); Smith et al., March's Advanced Organic Chemistry: Reactions, Mechanisms, a nd Structure, 6 th Ed. (Wiley, 2007); Trost et al. (Ed.), Comprehensive Organic S ynthesis (Pergamon Press, 1991), etc. can be identified by referring to the literature containing such references can be.

[0227] The reactions for preparing the compounds described in this specification can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis. Suitable solvents are the temperatures at which the reactions are carried out ( ), For example, at a temperature that can be in the range of the freezing point of the solvent to the boiling point of the solvent, the starting materials (reactants), intermediates, or products may be substantially non-reactive. A given reaction can be carried out in one solvent or in a mixture of more than one solvent. Depending on the particular reaction step, one of ordinary skill in the art can select a solvent suitable for the particular reaction step .

[0228] The preparation of the compounds described herein may involve the protection and deprotection of various chemical groups. One of ordinary skill in the art can readily determine the need for protection and deprotection, as well as the selection of appropriate protecting groups . For chemical reactions of protecting groups, see, for example, T. W. Greene and P. G. M. Wuts , Protective Groups in Organic Synthesis, 3 rd Ed., Wiley & Sons, Inc., New York (1999).

[0229] The reaction can be monitored according to any suitable method known in the art. For example, the formation of the product can be monitored by spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1 H or 13 C), infrared spectroscopy , spectrophotometry (e.g., UV-visible light), mass spectrometry, etc., and can also be monitored by chromatographic methods such as high performance liquid chromatography (HPLC), liquid chromatography- mass spectrometry (LCMS), or thin layer chromatography (TLC). One of ordinary skill in the art can purify the compound by various methods, including high performance liquid chromatography ( HPLC) and normal phase silica chromatography.

[0230] Throughout various places in this specification, divalent linking substituents are described. Each divalent linking substituent is specifically intended to include both the forward and reverse forms of the linking substituent. For example, -NR(CR’R’’) - includes both -NR(CR’R’’) n - and -(CR’R’ n - and -(CR’R’ ’) n NR-. When the structure clearly claims a linking group, the Markush variables listed for this group are also understood to be linking groups.

[0231] The term "n-membered", where n is an integer, typically describes the number of ring-forming atoms within a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydronaphthalene is an example of a 10-membered cycloalkyl group.

[0232] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. As used herein, the term "substituted" means that a hydrogen atom has been removed and replaced with a substituent. It should be understood that substitution at a given atom is limited by valence.

[0233] Throughout the definitions, the term "C n~m " refers to a range that includes the endpoints, where n and m are integers and indicate the number of carbons. Examples include C 1~4 , C 1~6 , and the like.

[0234] As used herein, "Cn~m The term "alkyl", alone or in combination with other terms, refers to a saturated hydrocarbon group which may be linear or branched and which has n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and higher homologues. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. The term "alkyl", alone or in combination with other terms, refers to a saturated hydrocarbon group which may be linear or branched and which has n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and higher homologues. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. The term "alkyl", alone or in combination with other terms, refers to a saturated hydrocarbon group which may be linear or branched and which has n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and higher homologues. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. The term "alkyl", alone or in combination with other terms, refers to a saturated hydrocarbon group which may be linear or branched and which has n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and higher homologues. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. The term "alkyl", alone or in combination with other terms, refers to a saturated hydrocarbon group which may be linear or branched and which has n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and higher homologues. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. The term "alkyl", alone or in combination with other terms, refers to a saturated hydrocarbon group which may be linear or branched and which has n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and higher homologues. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. The term "alkyl", alone or in combination with other terms, refers to a saturated hydrocarbon group which may be linear or branched and which has n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and higher homologues. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms. The term "alkyl", alone or in combination with other terms, refers to a saturated hydrocarbon group which may be linear or branched and which has n to m carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl and higher homologues. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0235] As used herein, the term "C n~m alkoxy", alone or in combination with other terms, refers to a group of the formula -O-alkyl [wherein the alkyl group has n to m carbon atoms]. Examples of alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, etc. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term "C alkoxy", alone or in combination with other terms, refers to a group of the formula -O-alkyl [wherein the alkyl group has n to m carbon atoms]. Examples of alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, etc. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. As used herein, the term "C alkoxy", alone or in combination with other terms, refers to a group of the formula -O-alkyl [wherein the alkyl group has n to m carbon atoms]. Examples of alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), tert-butoxy, etc. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0236] As used herein, the term "amino" refers to a group of the formula -NH 2

[0237] As used herein, the term "aryl", alone or in combination with other terms, refers to an aromatic hydrocarbon group which may be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). The term "C aryl", alone or in combination with other terms, refers to an aromatic hydrocarbon group which may be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). The term "C aryl", alone or in combination with other terms, refers to an aromatic hydrocarbon group which may be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). The term "C n~m aryl", alone or in combination with other terms, refers to an aromatic hydrocarbon group which may be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). The term "aryl" refers to an aryl group having n to m cyclic carbon atoms. The aryl group includes, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, etc. In some embodiments, the aryl group has 6 to 20 carbon atoms, 6 to 15 carbon atoms, or 6 to 10 carbon atoms. In some embodiments, the aryl group is a substituted phenyl or an unsubstituted phenyl.

[0238] As used herein, "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon including a cyclic alkyl group and / or a cyclic alkenyl group. The cycloalkyl group can include a monocyclic group or a polycyclic group (e.g., having 2, 3, or 4 fused rings) and a spiro ring. The cycloalkyl group can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbons (C ). The ring-forming carbon atoms of the cycloalkyl group can be optionally substituted by oxo or sulfide (e.g., C(O) or C(S)). The cycloalkyl group also includes cycloalkylidene. Examples of the cycloalkyl group include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, etc. In some embodiments, the cycloalkyl 3~ 10 is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl , or adamantyl. In some embodiments, the cycloalkyl has 6 to 10 ring -forming carbon atoms. In some embodiments, the cycloalkyl is adamantyl. The definition of cycloalkyl also includes one or more aromatic rings fused to the cycloalkyl ring (i.e., sharing a bond therewith), for example, benzo derivatives or thienyl derivatives of cyclopentane, cyclohexane, etc. A cycloalkyl group containing a fused aromatic ring can be joined through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. The term "halo" as used herein refers to F, Cl, Br, or I. In some embodiments, halo is F, Cl, or Br. The term "C haloalkyl" as used herein is used alone or in combination with other terms and refers to an alkyl group having from 1 to 2s + 1 halogen atoms, which may be the same or different, where "s" is the number of carbon atoms in the alkyl group and has n to m carbon atoms. In some embodiments, only the haloalkyl group is fluorinated. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. The term "heteroaryl" as used herein refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N within the heteroaryl moiety can be an N-oxide. In some embodiments, heteroaryl has 5 to 10 ring atoms and is independently selected from nitrogen, sulfur, and oxygen.

[0239]

[0240] n~m

[0241] ​ having 1, 2, 3, or 4 heteroatom ring members. In some embodiments, the hetero aryl has 5 to 6 ring atoms and 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5 -membered or 6-membered heteroaryl ring. A 5-membered heteroaryl ring is a heteroaryl having a ring with 5 ring atoms, wherein 1 or more (e.g., 1, 2, or 3 of) the ring atoms are independently selected from N, O, and S. Exemplary 5-membered heteroaryl rings include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1, 2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A 6-membered heteroaryl ring is a heteroaryl having a ring with 6 ring atoms, wherein 1 or more (e.g., 1, 2, or 3) of the ring atoms are independently selected from N, O, and S. Exemplary 6-membered heteroaryl rings include pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl. As used herein, "heterocycloalkyl" refers to a monocyclic or polycyclic non-aromatic heterocyclic ring having 1 or more ring-forming heteroatoms selected from O, N, or S. Heterocycloalkyl includes 4-, 5-, 6-, and 7-membered, monocyclic heterocyclo alkyls, as well as bicyclic and tricyclic heterocycloalkyls formed by fusing two or more of the foregoing monocyclic heterocycloalkyls. Exemplary monocyclic heterocycloalkyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and tetrahydrothiophenyl. Exemplary bicyclic heterocycloalkyls include indolinyl, isoindolinyl, chromanyl, isochromanyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and benzodioxolyl. Exemplary tricyclic heterocycloalkyls include carbazolyl, acridinyl, phenanthridinyl, and phenanthrolinyl. .

[0242] As used herein, "heterocycloalkyl" refers to a monocyclic or polycyclic non-aromatic heterocyclic ring having one or more ring-forming heteroatoms selected from O, N, or S. Heterocycloalkyl includes monocyclic heterocycloalkyls having 4, 5, 6, and 7 ring members, as well as bicyclic and tricyclic heterocycloalkyls formed by fusing two or more of the foregoing monocyclic heterocycloalkyls. Exemplary monocyclic heterocycloalkyls include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydropyranyl, and tetrahydrothiophenyl. Exemplary bicyclic heterocycloalkyls include indolinyl, isoindolinyl, chromanyl, isochromanyl, It contains an alkyl group. The heterocycloalkyl group may also contain a spiro ring. The heterocy Examples of the cloalkyl group include pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydrofuran, oxetanyl, azetidinyl, morpholino, thiomorpho lino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pi rolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolid nyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazapene ), etc. The ring-forming carbon atoms and ring-forming heteroatoms of the heterocycloalkyl group Optionally, they can be substituted by oxo or sulfide (e.g., C(O), S(O), C(S), or S(O) 2 ), etc. The heterocycloalkyl group can be joined via a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocy cloalkyl group contains 0 to 3 double bonds. In some embodiments, the hetero cycloalkyl group contains 0 to 2 double bonds. The definition of heterocycloalkyl also includes a part where one or more aromatic rings are fused to the cycloalkyl ring (i.e., share a bond), such as benzo derivatives of piperidine, morpholine, azepine or thienyl derivatives, etc. The heterocycloalkyl group containing a fused aromatic ring can be joined via any ring-forming atom including the ring-forming atoms of the fused aromatic ring . In some embodiments, the heterocycloalkyl is 4 to 10, 4 to 7 or 4 to 6 ring atoms with 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur has a child and has one or more oxidized ring members.

[0243] As used herein, the term "hydroxy" refers to a group of the formula -OH.

[0244] At certain places, the definition or embodiment refers to a specific ring (e.g., azetidine ring, pyr ridine ring, etc.). Unless otherwise indicated, these rings can be joined to any ring member, provided that the valence of the atoms is not exceeded. For example, an azetidine ring can be joined at any position of the ring, while a pyridine-3-yl ring is joined at the 3-position.

[0245] As used herein, the term "compound" is intended to include all stereoisomers , geometric isomers, tautomers, and isotopes of the depicted structure. As used herein, a compound identified as a particular tautomeric form by name or structure is intended to include other tautomeric forms unless otherwise specified.

[0246] The compounds presented herein also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond and the accompanying movement of a proton. Tautomeric forms are prototropic tautomers, which are isomeric protonation states having the same empirical formula and total charge. Examples of prototropic tautomers are keto-enol pairs, am ide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which a proton occupies two or more positions of a complex ring system, such as 1H-imidazole and 3H-imidazole, 1H-1,2,4-triazole, 2H-1,2,4-tri azole. Zole, and 4H-1,2,4-triazole, 1H-isoindole and 2H-isoindole, and 1H-pyrazole and 2H-pyrazole. Tautomers may be in an equilibrium state and may be stereochemically fixed into one form by appropriate substitution.

[0247] Unless specifically defined otherwise, the compounds presented herein may also include all isotopes of all atoms that occur in the intermediates or the final compounds. Isotopes include atoms having the same number of atoms but different mass numbers. Unless stated otherwise, an atom is understood to include an isotope or radioisotope (e.g., deuterium, C], 11 F]) 18 when referred to as an isotope or radioisotope, is understood to include the isotope or radioisotope in an amount that is at least 3000 times the natural abundance of the isotope or radioisotope. For example, when an atom is referred to as "D" or "deuterium", the position is understood to have deuterium at an abundance that is at least 3000 times the natural abundance of deuterium, which is 0.015% (i.e., at least 45% of the amount of deuterium incorporated).

[0248] All compounds and their pharmaceutically acceptable salts can also be found and isolated in combination with other substances such as water and solvents (e.g., hydrates and solvates).

[0249] In some embodiments, the preparation of the compounds may involve the addition of an acid or a base, for example, which affects the desired reaction or catalyzes the formation of a salt form such as an acid addition salt.

[0250] Examples of acids can be inorganic acids, can be organic acids, and include strong acids and weak acids, but these​​​​​​​​ is not limited to. Some examples of acids include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, 4-nitrobenzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid, and nitric acid. Some weak acids include acetic acid, propionic acid, butanoic acid, benzoic acid, tartaric acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and decanoic acid, but are not limited thereto.

[0251] Examples of bases include lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, and sodium bicarbonate. Some strong bases include hydroxides, alkoxides, metal amides, metal hydrides, metal dialkylamides, and aryl amines, but are not limited thereto. In this case, alkoxides include lithium salts, sodium salts, and potassium salts of methoxide, ethoxide, and t-butoxide; metal amides include sodium amide, potassium amide, and lithium amide ; metal hydrides include sodium hydride, potassium hydride, and lithium hydride ; metal dialkylamides include lithium salts, sodium salts, and potassium salts of amides substituted with methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, trimethylsilyl, and cyclohexyl.

[0252] In some embodiments, the compounds or salts presented herein are substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it is formed or detected. Partially Separation may involve, for example, compositions enriched in the compounds presented herein. Substantial separation may involve compositions containing, by weight, at least about 50 %, at least about 60%, at least about 70%, at least about 80%, at least about 90 %, at least about 95%, at least about 97%, or at least about 99% of the compounds or salts thereof presented herein. In the art, methods for isolating compounds and their salts are well established methods.

[0253] In the art, the expressions “ambient temperature” and “room temperature” or “rt” as used herein are understood and generally refer to a temperature, for example, approximately the temperature of the room in which the reaction is carried out, the reaction temperature, for example, a temperature of about 20 °C to about 30 °C.

[0254] As used herein, the term “pharmaceutically acceptable” is invoked to refer to compounds, materials, compositions, and / or dosage forms that are within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0255] This application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds in which the existing acidic or basic moiety is modified by converting it to its salt form. Examples of pharmaceutically acceptable salts include inorganic or organic acid salts of basic residues such as amines; and alkali or organic salts of acidic residues such as carboxylic acids, among others. is not limited thereto. The pharmaceutically acceptable salts of the present application are conventional non-toxic salts of the parent compound, and include, for example, salts formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form or basic form of these compounds with a stoichiometric amount of an appropriate salt group or acid in water, an organic solvent, or a mixture of the two. Preferred solvents for this purpose are generally non-aqueous media such as ethers, ethyl acetate, alcohols (e.g., methanol, ethanol, isopropanol, or butanol), or acetonitrile (MeCN). A list of suitable salts can be found in Remington’s Pharmaceutical Sciences, 17th ed. , Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977). Conventional methods for preparing salt forms are , for example, described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley -VCH, 2002.

[0256] Method of Use The present application further presents a method for imaging granzyme B. In some embodiments, the imaging method is performed on cells, tissues, cell samples, tissue samples, or subjects. As used herein, the term "subject" includes mammals and invertebrates, including, Refers to any animal. For example, mouse, rat, other rodents, rabbit, dog, cat, pig , cow, sheep, horse, primate, fish, and human. In some embodiments, the subject is human. In some embodiments, the subject is a mouse. In some embodiments, the sub ject is fish (e.g., zebrafish). In some embodiments, the method comprises adminis tering to the subject an effective amount of a compound presented herein (e.g., a com pound of Formula I), or a pharmaceutically acceptable salt thereof. In some embodi ments, the method is an in vitro

[0257] This application is a method for imaging granzyme B in cells or tissues, compris ing:

[0258]

Chemical formula

[0259] (i) contacting the cell or tissue with a compound of Formula I: (ii) imaging the cell or tissue with an appropriate imaging method, thereby imag ing granzyme B within the cell or tissue ; wherein A comprises one or more imaging agents, B is an optional linking group, C is a group that binds to granzyme B. The application further provides a method.

[0260] This application is a method for imaging granzyme B in a cell sample or a tissue sample, compris ing: (i) contacting a sample of the cell or tissue with a compound of Formula I: (ii)

[0261] [Chemical formula]

[0262] contacting with a compound of formula (I) or a pharmaceutically acceptable salt thereof; ii) imaging the cell sample or tissue sample with an appropriate imaging method, thereby imaging granzyme B in the cell sample or tissue sample; comprising, wherein A comprises one or more imaging agents; B is an optional linking group; C is a group that binds to granzyme B, further provided is a method.

[0263] This application is a method for imaging granzyme B in a subject, comprising: i) administering to the subject a compound of formula I:

[0264] [Chemical formula]

[0265] or a pharmaceutically acceptable salt thereof; ii) imaging the subject with an appropriate imaging method, thereby imaging granzyme B in the subject; comprising, wherein A comprises one or more imaging agents; B is an optional linking group; C is a group that binds to granzyme B, further provided is a method.

[0266] This application is a method for imaging an immune response in a cell or tissue sample, comprising: i) contacting the cell or tissue sample with a compound of formula I:

[0267] [Chemical formula]

[0268] contacting the compound of formula I, or a pharmaceutically acceptable salt thereof, with the cell or tissue sample; ii) imaging the cell or tissue sample with an appropriate imaging method, thereby imaging the immune response in the cell or tissue sample; and wherein, in formula I, A comprises one or more imaging agents; B is an optional linking group; C is a group that binds to granzyme B. Also provided is a method.

[0269] This application provides a method for imaging an immune response in a subject, comprising: i) administering to the subject a compound of formula I:

[0270]

Chemical formula

[0271] or a pharmaceutically acceptable salt thereof; ii) imaging the subject with an appropriate imaging method, thereby imaging the immune response in the subject; and wherein, in formula I, A comprises one or more imaging agents; B is an optional linking group; C is a group that binds to granzyme B. Also provided is a method. C is a group that binds to granzyme B. Also provided is a method.

[0272] This application provides a method for monitoring the treatment of a disease in a subject, comprising: i) administering to the subject a compound of formula I:

[0273]

Chemical formula

[0274] administering a compound of formula I or a pharmaceutically acceptable salt thereof; and ii) imaging the subject by an appropriate imaging method; comprising, wherein A comprises one or more imaging agents; B is an optional linking group; C is a group that binds to granzyme B, further provided is a method.

[0275] The present application is a method for monitoring an immune response in the treatment of a disease in a subject, comprising , i) administering to the subject a compound of formula I:

[0276]

Chemical formula

[0277] administering a compound of formula I or a pharmaceutically acceptable salt thereof; and ii) imaging the subject by an appropriate imaging method; comprising, wherein A comprises one or more imaging agents; B is an optional linking group; C is a group that binds to granzyme B, further provided is a method.

[0278] In some embodiments, the compound of formula I is of formula I-a:

[0279]

Chemical formula

[0280] [wherein A comprises one or more imaging agents; C is a group that binds to granzyme B] or a pharmaceutically acceptable salt thereof.

[0281] In some embodiments, the groups A, B, and C of formula I are defined for the compounds of formula I according to the provisions presented herein and in accordance with the provisions presented herein. In some embodiments, the compounds of formula I are of formula I-a, I -b, I-c, I-d, I-e, I-f, I-g, I-h, I-i, I-j, I-k, I -l, I-m, I-n, or compounds of I-o, or pharmaceutically acceptable salts thereof are. In some embodiments, the compounds of formula I are compounds of formula I-p or formula I-q, or pharmaceutically acceptable salts thereof.

[0282] In some embodiments, the methods presented herein further include a sufficient waiting time for the compound to accumulate in a cell or tissue site associated with the disease (e.g., a cell or tissue site in a subject) prior to imaging . In some embodiments, the methods presented herein further include a sufficient waiting time for the compound to bind to granzyme B in a cell or tissue site associated with the disease (e.g., a cell or tissue site in a subject) prior to imaging. In some embodiments, the sufficient time is from about 30 seconds to about 24 hours , e.g., from about 30 seconds to about 24 hours, from about 30 seconds to about 12 hours, from about 30 seconds to about 6 hours , from about 30 seconds to about 2 hours, from about 30 seconds to about 1 hour, from about 30 seconds to about 30 minutes, from about 30 seconds to about 10 minutes, from about 10 minutes to about 24 hours, from about 10 minutes to about 12 hours, from about 10 minutes to about 6 hours , from about 10 minutes to about 2 hours, from about 10 minutes to about 1 hour, from about 10 minutes to about 30 minutes, from about 30 minutes to about 24 hours, from about 30 minutes to about 12 hours, from about 30 minutes to about 6 hours, from about 30 minutes to about 2 hours , from about 30 minutes to about 1 hour, from about 1 hour to about 24 hours, from about 1 hour to about 12 hours, from about 1 hour to about 6 hours, from about 1 hour to about 2 hours, from about 2 hours to about 24 hours, from about 2 hours to about 12 hours, from about 2 hours to about 6 hours, from about 2 hours to about 4 hours, from about 4 hours to about 24 hours, from about 4 hours to about 12 hours, from about 4 hours to about 6 hours, etc. About 6 hours, about 1 hour to about 2 hours, about 2 hours to about 24 hours, about 2 hours to about 12 hours, about 2 hours to about 6 hours, about 6 hours to about 24 hours, about 6 hours to about 12 hours, or about 12 hours to about 24 hours.

[0283] In some embodiments, a suitable imaging method is a non-invasive imaging method. Some embodiments, a suitable imaging method is a minimally invasive imaging method. As used in this specification, the term "minimally invasive imaging method" refers to the use of internal probes or imaging methods that employ the injection of compounds or radioactive tracers via a syringe. Examples of imaging methods include fluoroscopic imaging, x-ray imaging, magnetic resonance imaging (MRI), ultrasonic imaging, photoacoustic imaging, thermographic imaging, tomographic imaging, echocardiography, positron emission tomography (PET) imaging, PET with computed tomography (CT) imaging, PET-MRI, single photon emission computed tomography (SPECT), and ultrasonic imaging including, but not limited to, these. In some embodiments, a suitable imaging method is selected from the group consisting of PET imaging, PET-CT, PET-MRI, and SPECT. In some embodiments, a suitable imaging method is selected from the group consisting of positron emission tomography (PET) imaging, positron emission tomography with computed tomography imaging, and positron emission tomography (PET) with magnetic resonance imaging (MRI). In some embodiments, a suitable imaging method is dedicated positron emission tomography (PET) imaging.

[0284] In some embodiments, the diseases described herein are selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancer, and cardiovascular disorders.

[0285] In some embodiments, the disease is cancer. In some embodiments, the cancer is a solid tumor including. In some embodiments, the cancer is a blood cancer (e.g., leukemia, lymphoma, etc.) is. In some embodiments, the cancer is a brain tumor, breast cancer, cervical cancer, colorectal cancer, lung cancer , lymphoma, melanoma, bladder cancer, renal cell carcinoma, multiple myeloma, pancreatic cancer, and prostate cancer selected from the group consisting of. In some embodiments, the cancer is hairy cell leukemia, Kaposi's sarcoma , follicular lymphoma, chronic myeloid leukemia, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, T-cell prolymphocytic leukemia, classical Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, myelodysplastic syndrome, primary myelofibrosis, essential thrombocythemia post myelofibrosis, polycythemia vera post myelofibrosis, melanoma, renal cell carcinoma, prostate cancer, non-small cell lung cancer, small cell lung cancer, glioblastoma, hepatocellular carcinoma, urothelial carcinoma, esophageal cancer, gastroesophageal cancer , gastric cancer, multiple myeloma, colon cancer, rectal cancer, head and neck squamous cell carcinoma, epithelial ovarian cancer (E OC), primary peritoneal cancer, fallopian tube cancer, HER2+ breast cancer, ER+ / PR+ / HER2-breast cancer cancer, triple negative breast cancer, gastric cancer, pancreatic cancer, bladder cancer, Merkel cell cancer, nasopharyngeal head cancer, adrenocortical carcinoma, meningioma, neuroblastoma, retinoblastoma, osteosarcoma, rhabdomyosarcoma, you ng sarcoma, liposarcoma, fibrosarcoma, leiomyosarcoma, peripheral primitive neuroectodermal tumor, cervical squamous cell carcinoma of the part, squamous cell carcinoma of the vagina, and squamous cell carcinoma of the vulva selected from the group consisting of . In some embodiments, the cancer is colon cancer.

[0286] In some embodiments, the disease is graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus , Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, posterior uveitis , allergic encephalomyelitis, glomerulonephritis, rheumatic fever, post-infectious glomerulonephritis, psoriasis, atopic dermatitis, contact dermatitis, eczematous dermatitis, lipophilic dermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, erythema sclerosis, acne, alopecia areata, keratoconjunctivitis, vernal catarrh, uveitis associated with Behçet's disease , keratitis, herpes keratitis, keratoconus, corneal epithelial dysplasia, corneal leukoma, ocular pemphigus, Mooren's ulcer, scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis , pollen allergy, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma, dust asthma, chronic (chronic or inveterate) asthma , late-onset asthma and airway hyperresponsiveness, bronchitis, gastric ulcer, vascular damage caused by ischemic disease and thrombosis , ischemic bowel disease, inflammatory bowel disease, necrotizing enteritis, intestinal lesions associated with burns, celiac disease, proctitis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis, migraine headache, rhinitis, eczema, interstitial nephritis, Goodpasture syndrome, hemolytic uremic syndrome, diabetic nephropathy, polymyositis, Guillain-Barré syndrome, Meniere's disease, polyneuritis (polyneuritis, multiple neuritis), mononeuritis, radiculopathy, hyperthyroidism, Graves' disease, erythroleukemia, aplastic anemia (aplastic anemia, hypoplastic anemia), idiopathic thrombocytopenic purpura, autoimmune ​​Acute hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia, erythropoiesis imperfecta, osteoporosis, leukocytosis, pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo vulgaris, ichthyosis vulgaris, photoallergic hypersensitivity, cutaneous T-cell lymphoma, arteriosclerosis, atherosclerotic arteriosclerosis, large arterial inflammatory syndrome, polyarteritis nodosa, myocarditis, scleroderma, Wegener's granulomatosis, Sjogren's syndrome, hyperlipidemia, eosinophilic fasciitis, gingiva, periodontal tissue, alveolar bone, cementum lesions of teeth, glomerulonephritis, male pattern alopecia, senile alopecia due to hair loss, lack of hair germination, and / or senile alopecia due to decreased hair generation and hair growth, muscular dystrophy, impetigo syndrome, Addison's disease, organ injury due to ischemia-reperfusion, transplantation diseases, ischemic diseases, endotoxin shock, pseudomembranous colitis, colitis caused by drugs or radiation, acute ischemic renal failure, chronic renal failure, oxygen lung or poisoning caused by drugs, lung cancer emphysema, cataract, hemosiderosis, retinitis pigmentosa, age-related macular degeneration, vitreous scarring, corneal arcus caliburn, polymorphic erythematous dermatitis, linear IgA bullous dermatitis and cement dermatitis, gingivitis periodontitis, sepsis, pancreatitis, aging, carcinogenesis, metastasis of cancer tumors and mountain sickness, histamine or leukotriene C4 release-related diseases, Behcet's disease, autoimmune hepatitis, primary biliary cirrhosis sclerosing cholangitis, partial hepatectomy, acute liver necrosis, necrosis caused by toxins, viral hepatitis, shock, anoxia, hepatitis B virus, non-A / non-B hepatitis, cirrhosis, alcoholic cirrhosis, liver failure, fulminant liver failure, late-onset liver failure, acute exacerbation of chronic hepatitis, cytomegalo virus infection, HCMV infection, AIDS, senile dementia, trauma, chronic bacterial infection, lymph system-derived malignant tumors, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphoblastic lymphoma It is selected from the group consisting of myeloma and chronic lymphocytic lymphoma.

[0287] In some embodiments, the disease is systemic lupus erythematosus, rheumatoid arthritis, type I diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis, Crohn's disease, ulcerative colitis pemphigoid, sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis, ichthyosis, Graves' ophthalmopathy, asthma, scleroderma, and Sjögren's syndrome. It is selected from the group consisting of.

[0288] In some embodiments, the disease is selected from the group consisting of bone marrow rejection, organ graft rejection, and graft-versus-host disease. It is selected from the group consisting of.

[0289] As used herein, the term "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical agent that elicits a biological or medical response that is sought by a researcher, veterinarian, physician, or other clinician in a tissue, system, animal, individual, or human. In some embodiments, the dosage of a compound, or a pharmaceutically acceptable salt thereof, administered to a subject or individual is about 1 μg to about 2 g, such as, for example, about 1 μg to about 2 g, about 1 μg to about 1000 mg, about 1 μg to about 500 mg, about 1 μg to about 100 mg, about 1 μg to about 50 mg, about 1 μg to about 1 mg, about 1 μg to about 500 μg, about 1 μg to about 100 μg, about 1 μg to about 10 μg, about 10 μg to about 2 g, such as, for example, about 10 μg to about 2 g, about 10 μg to about 1000 mg, about 10 μg to about 5 00 mg, about 10 μg to about 100 mg, about 10 μg to about 50 mg, about 10 μg to about 1 mg about 10 μg to about 500 μg, about 10 μg to about 100 μg, about 100 μg to about 2 g, such as for example ​then about 100 μg to about 2 g, about 100 μg to about 1000 mg, about 100 μg to about 500 m g, about 100 μg to about 100 mg, about 100 μg to about 50 mg, about 100 μg to about 1 mg , about 100 μg to about 500 μg, about 500 μg to about 2 g, for example, about 500 μg to about 2 g , about 500 μg to about 1000 mg, about 500 μg to about 500 mg, about 500 μg to about 10 0 mg, about 500 μg to about 50 mg, about 500 μg to about 1 mg, about 1 mg to about 2 g, about 1 mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg to about 100 mg, about 1 mg to 50 mg, or about 50 mg to about 500 mg.

[0290] As used herein, the term "treating" or "treatment" means (1) inhibiting a disease; e.g., inhibiting a disease, condition, or disorder in an individual who is undergoing or presenting the pathology or symptoms of the disease, condition, or disorder (i.e., stopping further development of the pathology and / or symptoms); and (2) alleviating a disease; e.g., reducing the severity of a disease, or reducing or alleviating one or more symptoms of a disease, such as in an individual who is undergoing or presenting the pathology or symptoms of a disease, condition, or disorder (i.e., antagonizing the pathology and / or symptoms), and refers to one or more of the following: alleviating a disease; e.g., reducing the severity of a disease, or reducing or alleviating one or more symptoms of a disease, such as in an individual who is undergoing or presenting the pathology or symptoms of a disease, condition, or disorder (i.e., antagonizing the pathology and / or symptoms).

[0291] Combination therapy When employed in a method of treating a disease, the compounds presented herein can be administered in combination with one or more of the additional agents presented herein . Examples of therapeutic agents include anti-inflammatory agents, steroids, immunotherapeutic agents, chemotherapeutic agents, and therapeutic antibodies . including but not limited to these.

[0292] In some embodiments, administration of the therapeutic agent induces an immune response in a sample of cells or tissue or in a subject. In some embodiments, the therapeutic agent is a compound of Formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutic agent is a compound of Formula I or a pharmaceutically acceptable salt thereof. wherein A is a radioisotope.

[0293] In some embodiments, the therapeutic agent is a compound of Formula I or a pharmaceutically acceptable salt thereof. wherein A comprises a radioisotope (e.g., a therapeutic radioisotope). In some embodiments, the therapeutic agent is a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein A comprises a toxic radioisotope. Examples of toxic radioisotopes are alpha emitters ( e.g., 211 At, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 227 Th) and beta emitters (e.g., 90 Y, 131 I, and 177 Lu) including but not limited to these. In some embodiments, the toxic radioisotope is a beta emitter. In some embodiments, the toxic radioisotope is 90 Y, 131 I, and 177 Lu selected from the group consisting of a beta emitter. In some embodiments, the toxic radioisotope is an alpha emitter. 211 At , 212 Pb, 212 Bi, 213 Bi,225 Ac, 227 selected from the group consisting of Th is an alpha emitter.

[0294] This application is a method for treating a disease in a subject, i) administering to the subject a first compound of formula I:

[0295]

Chemical formula

[0296] [wherein A comprises a non-toxic imaging agent (e.g., a non-toxic radioisotope), and B and C are defined according to the provisions presented herein for the compound of formula I] or a pharmaceutically acceptable salt thereof; ii) imaging the subject by an appropriate imaging method; iii) administering to the subject a second compound of formula I:

[0297]

Chemical formula

[0298] [wherein A comprises a toxic radioisotope, and B and C are defined according to the provisions presented herein for the compound of formula I] or a pharmaceutically acceptable salt thereof, thereby treating the disease in the subject and further provides a method comprising.

[0299] This application is a method for treating a disease in a subject, i) administering to the subject a first compound of formula I:

[0300]

Chemical formula

[0301] [wherein, A contains a non-therapeutic imaging agent (for example, a non-therapeutic radioisotope), and B and C are defined according to the regulations presented herein for the compound of formula I] administering a compound of formula I, or a pharmaceutically acceptable salt thereof; ii) imaging the subject by an appropriate imaging method; iii) administering to the subject a second compound of formula I:

[0302]

Chemical formula

[0303] [wherein, A contains a therapeutic radioisotope, and B and C are defined according to the regulations presented herein for the compound of formula I] administering a compound of formula I, or a pharmaceutically acceptable salt thereof, thereby treating a disease in the subject and presenting a method further comprising .

[0304] In some embodiments, the group A of the first compound of formula I, or a pharmaceutically acceptable salt thereof, is , 3 H, 11 C, 14 C, 18 F, 35 S, 52 Fe, 58 Co, 64 Cu, 68 Ga, 76 Br, 77 Br, 89 Zr, 111 In, 123 I, 124 I, 125 I, 131 I , 186 Re, 188 Re, 201 Tl, a non-toxic radioisotope selected from the group consisting of It contains an element.

[0305] In some embodiments, the group A of the compound of the second formula I, or a pharmaceutically acceptable salt thereof, is , 211 At, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 227 Th, 90 Y , 131 I, and 177 contains a toxic radioisotope selected from the group consisting of Lu.

[0306] In some embodiments, the method further comprises determining whether the compound of the first formula I, or a pharmaceutically acceptable salt thereof, binds to the cells or tissues of the subject to be treated before the administration of step iii). In some embodiments, the method is that of step iii) before the administration, further comprises determining whether the compound of the first formula I, or a pharmaceutically acceptable salt thereof, binds to granzyme B .

[0307] In some embodiments, the subject has, is identified and / or diagnosed with, the disease to be treated before the administration of step i). In some embodiments, after the imaging of step ii), the subject is identified and / or diagnosed as having the disease to be treated. For example , the disease to be treated is selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancer , and cardiovascular disorders as described herein.

[0308] In some embodiments, the subject has been treated with one or more immunotherapeutic agents before the administration of step i). In some embodiments, the disease is administered before the administration of step i) has been determined to be resistant to one or more immunotherapeutic agents.

[0309] In some embodiments, the method iv) administering one or more immunotherapeutic agents after administration of a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, steps i) - iv) are repeated multiple times.

[0310] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof and the second compound of formula I or a pharmaceutically acceptable salt thereof are the same. In some embodiments the compound of formula I or a pharmaceutically acceptable salt thereof is administered to the subject at a higher concentration compared to the amount of the compound of formula I or a pharmaceutically acceptable salt thereof that has been administered to the subject. In some embodiments the compound of formula I or a pharmaceutically acceptable salt thereof is administered to the subject at a lower concentration compared to the amount of the compound of formula I or a pharmaceutically acceptable salt thereof that has been administered to the subject. In some embodiments the compound of formula I or a pharmaceutically acceptable salt thereof is administered to the subject at approximately the same concentration compared to the amount of the compound of formula I or a pharmaceutically acceptable salt thereof that has been administered to the subject.

[0311] In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof and the second compound of formula I or a pharmaceutically acceptable salt thereof are different.

[0312] In some embodiments, group A of the compound of formula I or a pharmaceutically acceptable salt thereof is the same as group A of the compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments In an embodiment, group B of the compound of the first formula I, or a pharmaceutically acceptable salt thereof, is the same as group B of the compound of the second formula I, or a pharmaceutically acceptable salt thereof. In some embodiments , group C of the compound of the first formula I, or a pharmaceutically acceptable salt thereof, is the same as group C of the compound of the second formula I , or a pharmaceutically acceptable salt thereof. In some embodiments, groups B and C of the compound of the first formula I, or a pharmaceutically acceptable salt thereof, are the same as groups B and C of the compound of the second formula I , or a pharmaceutically acceptable salt thereof. In some embodiments, groups A and B of the compound of the first formula I, or a pharmaceutically acceptable salt thereof, are the same as groups A and B of the compound of the second formula I , or a pharmaceutically acceptable salt thereof. In some embodiments , groups A and C of the compound of the first formula I, or a pharmaceutically acceptable salt thereof, are the same as groups A and C of the second formula I, or a pharmaceutically acceptable salt thereof.

[0313] In some embodiments, group A of the compound of the first formula I, or a pharmaceutically acceptable salt thereof , is different from group A of the compound of the second formula I, or a pharmaceutically acceptable salt thereof. In some embodiments , group B of the compound of the first formula I, or a pharmaceutically acceptable salt thereof, is different from group B of the compound of the second formula I . In some embodiments, group C of the compound of the first formula I, or a pharmaceutically acceptable salt thereof, is different from group C of the compound of the second formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, groups B and C of the compound of the first formula I , or a pharmaceutically acceptable salt thereof, are different from groups B and C of the compound of the second formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of the first formula I or the groups A and B of the pharmaceutically acceptable salts thereof are different from the compound of formula I of the second or the pharmaceutically acceptable salts thereof. In some embodiments, the groups A and C of the compound of formula I of the first or the pharmaceutically acceptable salts thereof are different from the compound of formula I of the second or the pharmaceutically acceptable salts thereof. The groups A and B of the pharmaceutically acceptable salts are different. or the groups A and C of the pharmaceutically acceptable salts thereof are different from the compound of formula I of the second or the pharmaceutically acceptable salts thereof.

[0314] This application provides a method for determining the likelihood that a subject having a disease will exhibit a positive response to treatment with a therapeutic agent (e.g., a compound of formula I of the second or a pharmaceutically acceptable salt thereof), comprising: i) administering to the subject a compound of formula I of the first:

[0315]

Chemical formula

[0316] or a pharmaceutically acceptable salt thereof; ii) imaging the subject by an appropriate imaging method; iii) determining whether the compound of formula I of the first or a pharmaceutically acceptable salt thereof binds to the cells or tissues being treated wherein A comprises a non-therapeutic imaging agent (e.g., a non-therapeutic radioisotope), and B and C are defined according to the provisions presented herein for the compound of formula I, further presenting a method.

[0317] This application provides a method for predicting the efficacy of treatment with a therapeutic agent (e.g., a compound of formula I of the second or a pharmaceutically acceptable salt thereof), comprising: i) administering to the subject a compound of formula I of the first:

[0318]

Chemical formula

[0319] administering a compound of formula I or a pharmaceutically acceptable salt thereof; ii) imaging the subject with a suitable imaging method; iii) determining whether the compound of formula I or a pharmaceutically acceptable salt thereof binds to the cells or tissue being treated; wherein A comprises a non-therapeutic imaging agent (e.g., a non-therapeutic radioisotope), and B and C are defined according to the definitions provided herein for the compound of formula I. The method further comprises: In some embodiments, the method further comprises, when it is determined that the compound of formula I or a pharmaceutically acceptable salt thereof binds to the cells or tissue being treated: iv) administering a therapeutic agent to the subject to thereby treat a disease in the subject.

[0320] In some embodiments, the method further comprises, when it is determined that the compound of formula I or a pharmaceutically acceptable salt thereof binds to the cells or tissue being treated: iv) administering to the subject a compound of formula I:

[0321] In some embodiments, the method further comprises, when it is determined that the compound of formula I or a pharmaceutically acceptable salt thereof binds to the cells or tissue being treated: iv) administering to the subject a compound of formula I:

[0322]

Chemical formula

[0323] [wherein A comprises a therapeutic radioisotope, and B and C are defined according to the definitions provided herein for the compound of formula I] or a pharmaceutically acceptable salt thereof to thereby treat a disease in the subject. and further comprises.​​​​​​

[0324] In some embodiments, the disease to be treated is selected from the group consisting of autoimmune disorders, inflammatory disorders, skin disorders, cancer, and cardiovascular disorders as described herein. In some embodiments the disease is cancer as described herein.

[0325] In some embodiments, a compound of Formula I or a pharmaceutically acceptable salt thereof is administered to a subject in a therapeutically effective amount.

[0326] In some embodiments, the method presented herein further comprises administering a therapeutic agent prior to the administration of step i). In some embodiments, the method presented herein further comprises administering a therapeutic agent after the imaging of step ii). In some embodiments, the method presented herein iii) administering a therapeutically effective amount of a therapeutic agent after the imaging of step ii); and iv) repeating steps i) and ii) of the method presented herein. In some embodiments, the therapeutic agent is a compound other than the compound of Formula I or a pharmaceutically acceptable salt thereof as presented herein.

[0327] In some embodiments, steps i) - iv) are repeated multiple times.

[0328] In some embodiments, the compounds presented herein and one or more additional therapeutic agents are administered over a period of time according to an administration regimen. Exemplary administration regimens are the following combinations:

[0329]

Table 7

[0330] wherein, "A" represents administration of a compound presented herein (e.g., a compound of formula I, or a pharmaceutically acceptable salt thereof), and "B" represents administration of a further therapeutic agent (e.g., a compound of formula I, or a pharmaceutically acceptable salt thereof, where group A includes a toxic radioisotope element and / or a therapeutic radioisotope, or a therapeutic agent presented herein other than the compound of formula I, or a pharmaceutically acceptable salt thereof). In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a compound presented herein. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a further therapeutic agent. wherein, "A" represents administration of a compound presented herein (e.g., a compound of formula I, or a pharmaceutically acceptable salt thereof), and "B" represents administration of a further therapeutic agent (e.g., a compound of formula I, or a pharmaceutically acceptable salt thereof, where group A includes a toxic radioisotope element and / or a therapeutic radioisotope, or a therapeutic agent presented herein other than the compound of formula I, or a pharmaceutically acceptable salt thereof). In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a compound presented herein. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a further therapeutic agent. including but not limited to, a toxic radioisotope element and / or a therapeutic radioisotope, or a therapeutic agent presented herein other than the compound of formula I, or a pharmaceutically acceptable salt thereof). In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a compound presented herein. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a further therapeutic agent. a toxic radioisotope element and / or a therapeutic radioisotope, or a therapeutic agent presented herein other than the compound of formula I, or a pharmaceutically acceptable salt thereof). In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a compound presented herein. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a further therapeutic agent. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a compound presented herein. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a further therapeutic agent. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a compound presented herein. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a further therapeutic agent. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a further therapeutic agent. In some embodiments, cells, cell samples, tissues, tissue samples, or subjects are imaged by an appropriate imaging method after administration of a further therapeutic agent.

[0331] In some embodiments, the present application is a method for treating a disease in a subject, comprising: i) administering to the subject a compound of formula I:

[0332]

Chemical formula

[0333] [wherein, A includes a non-toxic imaging agent (e.g., a non-toxic radioisotope), and B and C are defined according to the regulations presented herein for the compound of formula I] of the compound, or a pharmaceutically acceptable salt thereof; ii) imaging the subject by an appropriate imaging method; iii) administering a therapeutic agent to the subject to thereby treat the disease in the subject and presenting a method comprising the steps above. and presenting a method comprising the steps above.

[0334] In some embodiments, the present application is a method for treating a disease in a subject, comprising: i) administering to the subject a compound of formula I:

[0335] [wherein A comprises a non-therapeutic imaging agent (e.g., a non-therapeutic radioisotope), and B

[0336] and C are defined according to the provisions presented herein for the compounds of formula I] or a pharmaceutically acceptable salt thereof; ii) imaging the subject by an appropriate imaging method; iii) administering a therapeutic agent to the subject to thereby treat the disease in the subject . In some embodiments, the method further comprises determining, prior to the administration of step iii), whether the first compound of formula I,

[0337] or a pharmaceutically acceptable salt thereof, binds to the cells or tissues of the subject to be treated. In some embodiments, the method further comprises determining, prior to the administration of step iii), whether the first compound of formula I, or a pharmaceutically acceptable salt thereof, binds to granzyme B or a pharmaceutically acceptable salt thereof, binds to granzyme B . . In some embodiments, the subject is identified and / or diagnosed as having the disease to be treated prior to the administration of step i). In some embodiments, after the imaging of step ii), the subject is identified and / or diagnosed as having the disease to be treated.

[0338] In some embodiments, the subject is identified and / or diagnosed as having the disease to be treated prior to the administration of step i). In some embodiments, after the imaging of step ii), the subject is identified and / or diagnosed as having the disease to be treated. . .

[0339] In some embodiments, the subject has been treated with one or more immunotherapeutic agents prior to administration in step i). In some embodiments, the disease has been determined to be resistant to one or more immunotherapeutic agents administered prior to administration in step i).

[0340] In some embodiments, the method further comprises iv) administering one or more immunotherapeutic agents after administration of the therapeutic agent in step iii). In some embodiments, steps i) - iv) are repeated multiple times.

[0341] In some embodiments, a further therapeutic agent is administered to the subject in a therapeutically effective amount.

[0342] In some embodiments, the therapeutic agent is an antibody. Examples of antibodies for use in combination therapy include trastuzumab (e.g., anti-HER2), ranibizumab (e.g., anti-VEGF-A ), bevacizumab (e.g., anti-VEGF), panitumumab (e.g., anti-EGFR), cetuximab (e.g., anti-EGFR), rituximab (anti-CD20), antibodies directed against c-MET , and antibody inhibitors of granzyme B (e.g., clone GB11, clone GrB- 7, and NCL-L-Gran-B), ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), atezolizumab (anti-PD-1), erlotinib (anti-PD-L1), elotuzumab (anti-SLAM7), and daratumumab (anti-CD38), including but not limited to these.

[0343] In some embodiments, the therapeutic agent is a steroid. Examples of steroids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone. ​​​It contains corticosteroids such as prednisone. In some embodiments, the additional agent is a corticosteroid.

[0344] In some embodiments, the therapeutic agent is an anti-inflammatory compound. Examples of anti-inflammatory compounds are aspirin, choline salicylate, celecoxib, diclofenac potassium, diclofenac sodium, diclofenac sodium with misoprostol, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, meclofenamic acid sodium, mefenamic acid, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, salsalate, sodium salicylate, sulindac, tolmetin sodium, and valdecoxib.

[0345] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents are cytostatic agents, cisplatin, doxorubicin, taxol, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epothilone, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, SCH 66336, R1 15777, L778,123, BMS 214662, gefitinib, erlotinib hydrochloride salt, antibody against EGFR, imatinib mesylate, interferon, ara-C, gemcitabine, uracil mustard, chloromethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramide, busulfan, carmustine, lomustine, streptozocin, dacarbazine, fluorouracil, Gem, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, o xaliplatin, folic acid, pentostatin, vinblastine, vincristine, vin desine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epir bicin, idarubicin, mitomycin, deoxycoformycin, mitomycin C, L-asparaginase, teniposide, 17α-ethinyl estradiol, diethylstil bestrol, testosterone, prednisone, fluoxymesterone, propionic acid dromostanolone, testolactone, megestrol acetate, methylprednisolone, methy lutetestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxy cyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, tamoxifen, goserelin, carboplatin , hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, le vamisole, vinorelbine, anastrozole, letrozole, capecitabine, lelox fin, hexamethylmelamine, bevacizumab, bexarotene, belotecan, zevalin, to lsenox, zeloda, vinorelbine, porfimer, erbitux, liposome agent , thiotepa, altretamine, melphalan, trastuzumab, fulvestrant, eki semestane, ifosfamide, rituximab, C225, alemtuzumab, clofarab in, cladribine, aphidicolin, sunitinib, dasatinib, tesacytabine, Sml1 , triapine, didox, trimidox, amidox, 3-AP, MDL-101 ,731, bendamustine, ofatumumab, and GS-1101 (also, CAL-1 including, but not limited to, those also known as 01).

[0346] In some embodiments, the chemotherapeutic agent is an alkylating agent (e.g., busulfan, chlorambucil, carmustine, cisplatin, cyclophosphamide (cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan), a nitrosourea (e.g., carmustine, lomustine, semustine, and streptozocin), a triazine (e.g., dacarbazine), an antimetabolite (e.g., 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate), a purine analogue (e.g., 6-mercaptopurine, 6-thioguanine, and pentostatin (2-deoxycoformycin)), a mitotic inhibitor (e.g., docetaxel, etoposide (VP16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine), an antitumor antibiotic (e.g., bleomycin, dactinomycin, daunorubicin, doxorubicin, mitomycin, plicamycin, and idarubicin), a platinum chemotherapeutic agent (e.g., cisplatin and carboplatin), an anthracycline (e.g., mitoxantrone), a toxin (e.g., ricin A chain (Burbage, Leukemia research, 21.7 (1997): 681-690), diphtheria toxin A (Massuda et al., Proceedings of the National Academy of Sciences, 94.26 (1997): 14701-14706; Lidor, Am J. Clin. Oncol., 16.11 (1998): 3404-3412), and others known in the art) including, but not limited to, those also known in the art). kemia research, 21.7 (1997): 681-690), diphtheria toxin A (Massuda et al., Proceedings of the National Academy of Sciences, 94.26 (1997): 14701-14706; Lidor, Am rican Journal of Obstetrics and Gynecology, 177.3 (1997): 579-585), pertussis toxin The A subunit, the Escherichia coli (E. coli) enterotoxin toxin A subunit, cholera toxin The A subunit, and the C terminus of a Pseudomonas toxin, and a gene A therapeutic vector (e.g., a signaling protein (e.g., Src, Abl, and Ra s), Jun, Fos, and Myc) selected from the group consisting of

[0347] In some embodiments, the therapeutic agent is an immunotherapeutic agent. Immunotherapeutic agents generally target and destroy cells (e.g., cancer cells) to induce immune effector cells and molecules to do so. Immune effectors can be, for example, antibodies specific for markers on the surface of cells (e.g., tumor cells). When an antibody is used alone as a therapeutic effector it may also mobilize other cells to effect cell killing. A variety of effector cells include, but are not limited to, cytotoxic T cells and NK cells.

[0348] Examples of immunotherapeutic agents are azathioprine, chlorambucil, cyclophosphamide, cyclos porin, daclizumab, infliximab, methotrexate, tacrolimus, immunostimulatory agents (e.g., IL-2, IL-4, IL-12, GM-CSF, tumor necrosis factor; inter feron alpha, interferon beta, and interferon gamma; F42K and other cytokine analogs; MIP-1, MIP-1β, MCP-1, RANT ES, chemokines such as IL-8; or growth factors such as FLT3 ligand), antigenic peptides A peptide, antigenic polypeptide, or antigenic protein, or an autologous or allogeneic tumor cell composition (see, e.g., Ravindranath & Morton, International reviews of immunology , 7.4 (1991): 303-329), hormone therapy, adrenocorticosteroids, progestins (e.g., hydroxyprogesterone caproate, medroxypro gesterone acetate, and megestrol acetate), estrogens (e.g., diethyl stilbestrol and ethinyl estradiol), antiestrogens (e.g., te stosterone propionate and fluoxymesterone), antiandrogens (e.g ., flutamide), and gonadotropin releasing hormone analogs (e.g., leuprolide ), but are not limited thereto. Further immunotherapeutic agents are known in the art, e.g., as found in Rosenberg et al, New England Journal of Medicine, 319.25 (1988): 1676-16 80; and Rosenberg et al, Annals of surgery, 210.4 (1989): 474.

[0349] The therapeutic agents presented herein may be effective over a wide range of dosages and are generally administered in effective amounts. However, the amount of the therapeutic agent actually administered will typically be determined by a physician in accordance with the circumstances involved, including the condition being imaged, the route of administration selected, the actual compound being administered, the age, weight, and individual

[0350] Pharmaceutical Compositions When employed as a medicine, the compounds and therapeutic agents presented herein can be administered in the form of a pharmaceutical composition. These compositions can be prepared as described herein or elsewhere, and depending on whether local treatment is desired, systemic treatment is desired, and depending on the area to be treated, can be administered via various routes. Administration can be local administration (including delivery to the mucosa including transdermal delivery, epidermal delivery, intravitreal delivery, as well as intranasal delivery, intravaginal delivery, and rectal delivery), pulmonary administration (e.g., administration by inhalation or insufflation of a powder or aerosol, including nebulizers; intratracheal administration or intranasal administration), oral administration, or parenteral administration. Parenteral administration can be intravenous administration, intraarterial administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, or injection or infusion; or intracranial administration (e.g., intrathecal administration or intraventricular administration). Parenteral administration can be in the form of a single bolus dose, for example, or continuous perfusion by a pump. In some embodiments, the compounds presented herein (e.g., compounds of formula I) are suitable for parenteral administration. In some embodiments, the compounds presented herein (e.g., compounds of formula I) are suitable for intravenous administration. Pharmaceutical compositions and formulations for local administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc. may be necessary or desirable. In some embodiments, pharmaceutical compositions suitable for parenteral administration are presented herein. In some embodiments, compositions suitable for intravenous administration are presented herein.

[0351] Also provided is a pharmaceutical composition containing, as an active ingredient, a compound presented in this specification or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers (excipients). When preparing the composition presented in this specification, the active ingredient is typically mixed with the excipient, diluted with the excipient, or enclosed in such a carrier in the form of, for example, capsules, sachets, paper, or other containers. The excipient may act as a medium, carrier, or vehicle for the active ingredient, and may be a solid material, a semi-solid material, or a liquid material when used as a diluent. Thus, the composition can be in the form of tablets, pills, powders, troches, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in a solid medium or a liquid medium), ointments, soft gelatin capsules and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile powder packs. Some examples of suitable excipients include, without limitation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. The formulations may additionally include, without limitation, lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl benzoate and propyl hydroxybenzoate; sweetening agents; flavoring agents, or combinations thereof. Kit

[0352]

[0353] This application further provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the kit further comprises one or more additional therapeutic agents presented herein.

[0354] In some embodiments, the kit comprises one or more components of the compounds presented herein (e.g., one or more imaging agents, one or more chelating agents, one or more linking groups, and one or more peptides or small organic molecules that bind to granzyme B). In some embodiments, each component of the kit is stored in a separate container (e.g., a separate vial) within the kit. In some embodiments, the components of the kit can be packaged in an aqueous medium, or can be packaged in a lyophilized form.

[0355] In some embodiments, the kit further comprises instructions, such as a package insert or label, indicating the quantity of the administered composition, guidelines for administration, and / or guidelines for mixing the components of the kit to prepare a compound of formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the instructions further comprise instructions for performing one or more of the methods presented herein.

[0356] The kits presented herein can, if desired, further comprise one or more conventional pharmaceutical kit components, such as, for example, a container with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art.

Examples

[0357] The present invention will now be described in more detail by way of specific examples. The following examples are provided for illustrative purposes. It is illustrative and is not intended to limit the invention in any manner. Those skilled in the art will recognize that there are various modifications and variations that can be made to the method that will produce essentially the same results. , one will easily recognize the less important parameters.

[0358] Cell lines and reagents CT-26 cells were obtained from ATCC (Manassas, VA) and were The cells were cultured in RPMI medium. BRAF V600E mutant melanoma cells were transfected with Tyr:CreE R;BrafCA;Ptenlox / lox mice (see, e.g., Dankort et al., Nature Genomics, 2014). netics, 41.5, (2009):544-552) and is derived from David The cells were kindly provided by Dr. Fisher and cultured in DMEM with 10% FBS. Anti-PD1 (clone RMP1-14) and anti-mouse CTLA4 (clone 9D9) were Dabrafenib was obtained from Bio X Cell (West Lebanon, NH). and purchased from LC Laboratories (Woburn, MA). For lot analysis, anti-CD8 (Ab108292, Abcam, Cambridge, MA), anti-CD3 (sc-20047, Santa Cruz Biotechnolo gy, Dallas, TX), anti-CD4 (sc-19643, Santa Cruz), Anti-FoxP3(12653s, Cell Signaling Technologie s, Danvers, MA), anti-pSTAT (700349, ThermoFisher , Waltham, MA), anti-granzymes B (4275s, Cell Signali ng Technologies), and beta-actin (Cell Signal ing Technologies, 4970S) were purchased at 1:1000.

[0359] Granzyme B was purchased from R&D systems (Minneapolis, MN) and activated using cathepsin B (R&D systems) according to the manufacturer's protocol. Enzyme activity was evaluated by cleavage of BOC-Ala-Ala-Asp-SBZL (SBZL = thiobenzyl ester) (Sigma) and measurement of absorbance at 405 nm (NanoDrop 2000 Spectrophotometer, Thermo Scientific) in a reaction with 5,5'-dithio-bis(2-nitrobenzoic acid) (Si gma). Serial dilutions of radiolabeled NOTA-GZP with non-radioactive gallium were used to inhibit granzyme B by incubation for 30 minutes at 37°C prior to substrate addition. Additionally, 68 Ga-NOTA-GZP (Example 1) was incubated with activated forms of granzyme B, granzyme A (R&D Systems), granzyme H (R&D Systems), granzyme K (Enzo Life Scie nces), and progranzyme B to evaluate peptide specificity. After incubation for 30 minutes at 37°C, the enzymes were purified by size exclusion chromatography and the bound radioactivity was evaluated by a gamma counter (Wiz ard 2480, Perkin Elmer).

[0360] Mouse treatment protocol Mice were cultured at Massachusetts General Hospital Institute of Medicine. Titional Animal Care and Use Committee The animals were cultured in accordance with an animal protocol approved by the Center for Compara tive Medicine at Massachusetts General H The cells were cultured and maintained by ospital. CT-26 cells 1 × 10 6 pcs, Matr The antibody was diluted 1:1 with igel and injected into the right shoulder of female BALB / C mice.

[0361] For imaging studies, mice were cultured at 3, 6, and 9 days after inoculation of CT-26 tumors. In each group, the mice were treated with vehicle, 200 μg of anti-PD-1 mouse therapeutic antibody (anti-PD1), or 200 μg of anti-PD-1 mouse therapeutic antibody (anti-PD1). μg of anti-PD1 and 100 μg of anti-CTLA-4 mouse therapeutic antibody (anti-CTLA-4) The mice were treated with 1 × 10 BP cells via intraperitoneal injection of either 6 1 piece, Matrigel, The antibody was diluted at 1:1 and injected into the right shoulder of female C57BL / 6 mice. Tumors grew to a diameter of 3–5 mm. Once reached, mice were placed in a 30-mL bolus of either vehicle or 0.5% hydroxypropyl methylcellulose (HPMC). of dabrafenib prepared in aqueous suspension with MC) and 0.2% Tween; Mice were treated by daily oral gavage. One day after initiating BRAF inhibition, mice were They were further randomized to receive 200 μg of anti-PD1 intraperitoneally. and immunohistochemistry alone or with subsequent imaging for correlative analysis. For this purpose, mice were sacrificed on the indicated days.

[0362] For imaging studies involving correlation growth curve analysis, as described above, CT-26 was injected into BALB / C mice. After inoculation, the mice were treated with 200 μg of anti-PD 1 and 100 μg of anti-CTLA-4 or anti-PD1 alone every three days until day 30. The mice were imaged on day 14 and the tumor size was measured three times a week.

[0363] For the imaging studies described in Examples 9-12, anti-mouse PD-1 (clone RPM1-14) therapy, anti-mouse CTLA-4 (clone 9H10) therapy, and anti-mouse TIM-3 (clone RPM3-23) therapy were obtained from Bio X Cell (West Lebanon , NH).

[0364] Biochemical analysis of tumors For the studies described in Examples 9-12, on day 12 after tumor inoculation, for ex vivo analysis the tumors were excised from sacrificed mice and dissolved in 1% SDS solution before analysis by Western blot . Anti-granzyme B antibody (4275S, Cell Signaling Technologies , Danvers, MA), anti-CTLA-4 antibody (ab134090, Abcam, Cambridge, MA), anti-PD- 1 antibody (12A7D7, ThermoFisher, Waltham, MA), anti-TIM -3 antibody (ab185703, Abcam Cambridge, MA), and anti-β- actin antibody (4970S, Cell Signaling Technologies , Danvers, MA) were used, and then, following the manufacturer's recommendations, horseradish peroxidase conjugated goat anti-rabbit polyclonal antibody (ab6721, Abcam, Cambridge, MA) was used. Detected by Cambridge, MA. SignalFire™ ECL R eagent (Cell Signaling Technologies, Danve rs, MA) was used to detect bands and Kodak I n-Vivo Multispectral Imaging System, Car estream MI software (Carestresam Health Inc. ., Woodbridge CT) was used for imaging. For each sample, the images were quantitatively analyzed by the net relative optical intensity ratio of the target β-actin to the optical intensity of the target target.

[0365] PET Imaging Mice were imaged on a GE Triumph PET / CT. After CT acquisition, PET images were obtained at two supine positions for 15 minutes. 3D-OSEM (4 iterations, 16 subsets) was used to construct the images and correct for scatter and random coincidences. For each tumor, the mean standardized uptake value (SUVmean) was calculated within the target 3D region and automatically contoured around the tumor using an isocontour at the 30% threshold. The target region was also calculated for the left ventricle, liver, lungs, muscle, and kidneys. VivoQuant (InviCRO, Boston, MA) was used for post-processing of the images. region. For the imaging studies described in Examples 9-12, on day 12 post-inoculation, mice were

[0366] injected intravenously with , 68 Ga-NOTA-GP and imaged 1 hour later. Imaging was performed on a Triumph PET / CT for rodents (GE Healthcare ). , was performed using (Wilmington, MA). PET images were collected over 15 minutes followed by CT collection. Reconstruction was achieved using 3D-MLEM (4 iterations, 20 sub- sets) and corrected for scattered and random coincidences. Image processing and analysis were performed using VivoQuant software (InviCRO, Boston, MA). Individual tumors were manually identified by delineating the target 3D region using CT-anatomical correlation. Background blood pool radioactivity was measured by identifying the left ventricle as the target region. 68 Specific uptake of GaNOTA-GP was quantified by dividing the total uptake by the tumor by the background blood pool to derive the tumor-to-blood ratio (TBR).

[0367] In vivo distribution study Approximately 1 mCi of 68 Ga-NOTA-GP was injected via the tail vein (see Example 1 if desired), and the mice were sacrificed at 1 or 2 hours. Mouse urine, blood, tumors, and vital organs were sampled and radioactivity was evaluated on a gamma counter .

[0368] Statistical analysis Statistical analysis was performed using Graphpad Prism Version 4 software. PA non-linear regression was used for competitive binding analysis. Pearson correlation tests were performed to correlate total tumor granzyme B expression, determined by Western blot, with the tumor-to-blood ratio (TBR). The TBR for treated tumors was compared to the TBR for untreated tumors by paired t-test.

[0369] GraphPad Prism Version 6 software (GraphPad Software, Inc., La Jolla, CA) was used to perform the statistical analysis and graphing described in Examples 9 - 1 2. For all comparisons between tumors treated with immunotherapy and tumors treated with vehicle, an unpaired t - test with Welch's correction was used. For each treatment arm, the response percentage was correlated with the tumor - to - blood ratio (TBR), which is the mean tumor - to - blood ratio, using weighted linear regression with group size. Based on Pearson's correlation coefficient, statistical estimation was used to define the 95% confidence interval of the best - fit straight line.

[0370] [Example 1] Synthesis of Granzyme B imaging agent ( 68 Ga - NOTA - GP)

[0371] [Chemical Structure]

[0372] Using standard Fmoc chemistry, NOTA - β - Ala - Gly - Gly - Il e - Glu - Phe - Asp - CHO (i.e., NOTA - GP; NOTA - GZP) was synthesized. The chemical purity was analyzed by HPLC and mass spectrometry. Calculated m / z: 1 176.949. Furthermore, HPLC analysis of the peptide showed a purity of greater than 90%, which was sufficient for all experiments.

[0373] 68 Ga was 68 obtained by eluting from a Ge / 68 Ga generator (iThemba Labs, South Africa) with 0.6 N HCl. The eluate was adjusted to approximately 100 μ It was added to a buffer system with 2 M HEPES at pH 3.5 - 4.0 with NOTA-GP of g. The labeling reaction was carried out at room temperature for 10 minutes. The reaction product was loaded onto a reverse-phase C18 Sep-Pak mini cartridge and eluted with 200 μL of 70% ethanol. The final formulation was adjusted to 10% ethanol in physiological saline. 68 Ga-NOTA-GP (i.e., 68 Ga-NOTA-GZP) in terms of chemical purity and radiochemical purity was measured via radio thin-layer chromatography (TLC). Radioactive labeling resulted in a yield of 67 ± 11%, a radiochemical purity of > 95%, and an average specific activity of 5190 ± 1100 MBq / mg.

[0374] [Example 1A] Synthesis of biotinylated human granzyme B peptide Biotin-β-Ala-Gly-Gly-Gly-Ile-Glu-Pro-Asp- CHO (hGZP; SEQ ID NO: 25) was synthesized according to the procedure described in Example 1 above. Calculated m / z: 907.010.

[0375] [Example 2] Differential expression of granzyme B in response to immunotherapy To quantify the difference in the expression of granzyme B over the course of treatment with immunotherapy CT26 colon cancer-bearing mice (BALB / C) were treated with 200 ng of anti-PD-1 antibody and 100 ng of anti-CTLA-4 antibody on days 3, 6, and 9 after tumor inoculation. On day 1 0, a subset of mice (4 treated mice and 4 untreated mice) were sacrificed and their tumors were excised and prepared for SDS-PAGE. Western Tumblot analysis was performed. The expression of granzyme B was quantified and standardized against the expression of beta-actin as a loading control. The results confirmed that granzyme B was expressed at low levels in both treated and untreated mice, with no statistical difference between the two groups. On day 14 post-inoculation, the mice were sacrificed and analyzed by Western blot in the same manner as the day 10 mice. The treated mice on day 14 showed a 100-fold higher expression of granzyme B, with a significant (4-fold) difference between the treated and untreated mice, as shown in Figure 1. Initial analysis of immune cell infiltration and activation into tumors on day 14 was performed by Western blotting in CT26 tumors treated with a dual-agent immunotherapy regimen (anti-PD1, anti-CTLA4), or vehicle alone as a control. As shown in Figure 2A, there was no significant difference in the expression levels of CD8 or CD3 between the treated and untreated mice. CD4 expression was present at low levels in the treated mice and undetectable in the untreated mice. Further exploration of mechanistic markers, including granzyme B, revealed a difference in the expression of granzyme B (i.e., GZB) between the treated and untreated mice. Quantification of Western blot data standardized against the expression of beta-actin revealed significantly higher levels of GZB (GZB:beta-actin = 20.4 ± 4.1) in treated tumors compared to untreated tumors (4.7 ± 1.6), as shown in Figure 2B. In comparison, CD8:beta-actin was 21.5 ± 5.0 in the treated mice and 14.3 ± 3.0 in the untreated mice. In both treated and untreated mice, granzyme B was expressed at low levels, and no statistical difference was found between the two groups. On day 14 after inoculation, the mice were sacrificed and analyzed by Western blot in the same manner as the day 10 mice. The treated mice on day 14 showed a 100-fold higher expression of granzyme B, with a significant (4-fold) difference between the treated and untreated mice, as shown in Figure 1. The treated mice on day 14 showed a 100-fold higher expression of granzyme B, with a significant (4-fold) difference between the treated and untreated mice, as shown in Figure 1. There was a significant (4-fold) difference between the treated and untreated mice.

[0376] Initial analysis of immune cell infiltration and activation into tumors on day 14 was performed by Western blotting in CT26 tumors treated with a dual-agent immunotherapy regimen (anti-PD1, anti-CTLA4), or vehicle alone as a control. Initial analysis of immune cell infiltration and activation into tumors on day 14 was performed by Western blotting in CT26 tumors treated with a dual-agent immunotherapy regimen (anti-PD1, anti-CTLA4), or vehicle alone as a control. Initial analysis of immune cell infiltration and activation into tumors on day 14 was performed by Western blotting in CT26 tumors treated with a dual-agent immunotherapy regimen (anti-PD1, anti-CTLA4), or vehicle alone as a control. As shown in Figure 2A, there was no significant difference in the expression levels of CD8 or CD3 between the treated and untreated mice. CD4 expression was present at low levels in the treated mice and undetectable in the untreated mice. CD4 expression was present at low levels in the treated mice and undetectable in the untreated mice. Further exploration of mechanistic markers, including granzyme B, revealed a difference in the expression of granzyme B (i.e., GZB) between the treated and untreated mice. Further exploration of mechanistic markers, including granzyme B, revealed a difference in the expression of granzyme B (i.e., GZB) between the treated and untreated mice. Quantification of Western blot data standardized against the expression of beta-actin revealed significantly higher levels of GZB (GZB:beta-actin = 20.4 ± 4.1) in treated tumors compared to untreated tumors (4.7 ± 1.6), as shown in Figure 2B. Quantification of Western blot data standardized against the expression of beta-actin revealed significantly higher levels of GZB (GZB:beta-actin = 20.4 ± 4.1) in treated tumors compared to untreated tumors (4.7 ± 1.6), as shown in Figure 2B. Quantification of Western blot data standardized against the expression of beta-actin revealed significantly higher levels of GZB (GZB:beta-actin = 20.4 ± 4.1) in treated tumors compared to untreated tumors (4.7 ± 1.6), as shown in Figure 2B. In comparison, CD8:beta-actin was 21.5 ± 5.0 in the treated mice and 14.3 ± 3.0 in the untreated mice. Both the expression level and the CD8 expression level were higher compared to those on the 10th and 12th days. Among the treated tumors, a dynamic range of GZB expression was observed, and 1 out of 4 treated tumors showed low GZB expression at a level similar to that of untreated mice. Without being bound by theory, the heterogeneity of GZB expression suggests that the level of secreted protease can be used as a biomarker for the immune response. To further investigate the transient expression of GZB, treated and untreated mice were sacrificed on the 10th and 12th days after inoculation and subjected to Western blotting in the manner described above, as shown in FIGS. 3A - 3B.

[0377] [Example 3] 68 Binding of Ga - NOTA - GP to purified granzyme B Granzyme B was adsorbed onto plates, and the plates were blocked with skim milk powder. 68 Ga - NOTA - GP was allowed to bind for 1 hour, the wells were washed, and the bound Ga - NOTA - GP was recovered by incubation with 2N NaOH and counted by a gamma counter. As shown in FIG. 4, the peptide showed significantly (P < 0.05) higher binding to granzyme B than to the control protein (BSA). The bars in FIG. 4 represent the mean ± SD of six replicates. As shown in FIG. 5, in vitro activity studies showed that NOTA - GP inhibits granzyme B activity with a Ki of 47 ± 54 nM. 6 8

[0378] [Example 4] 68 Binding of Ga - NOTA - GP to cells​​​​​​​​​​​​ CT26 cells were seeded in a 96-well plate and grown to 70% confluence. 68 Ga-NOTA-GP was diluted to 100 μCi / mL in 1% BSA + RPMI buffer and pre-incubated with granzyme B (60 nM) or medium before adding to the wells. The peptide was incubated with the cells for 30 minutes before washing and elution with 2M NaOH. The wells were counted using a gamma counter and the average of six wells was plotted with ±SD as shown in Figure 6. 68 It is confirmed that Ga-NOTA-GP binds to granzyme B and is specifically taken up by cancer cells.

[0379] [Example 5] Tumor specificity ex vivo 68 After confirming that Ga-NOTA-GP binds to purified granzyme B and specifically accumulates in target cells, the same model used to generate the Western blot data from Example 2 (see Figure 1) was used to explore the accumulation in tumors in vivo in CT26 tumor-bearing mice. On day 14 after inoculation, the mice were injected with 370 MBq of Ga-NOTA-GP and sacrificed 1 hour after injection. Tumors and blood were collected from 7 mice treated with combination therapy (anti-PD-1 and anti-CTLA-4). Tumors were grouped as responders (weight < 0.8 mg) or non-responders (weight ≥ 0.8 mg) based on size. A gamma counter was used to measure the radioactivity in both the tumors and the blood. 68 68 ​​​​​​​​​​​​​Quantification of the amount of Ga-NOTA-GP and adjusted based on weight, and the tumor blood ratio (TBR) of the injection dose per gram of tumor was plotted for tumors within each group. As shown in Figure 7, each mouse that was a responder showed a higher uptake (range: 6 .06 - 13.74) compared to non-responders (range: -0.95 - 2.48), which 68 indicated that Ga-NOTA-GP is specific for tumors that respond to immunotherapy .

[0380] After imaging, the mice were sacrificed and the GZB expression in tumors was quantified by Western blot . Comparison of TBR with GZB expression revealed a significant correlation (P < 0.001) between uptake and GZB expression, which indicated that the uptake of Ga-NOTA-G 68 P by tumors depends on the expression of GZB. Further ex vivo biodistribution studies at 1 hour and 2 hours after injection confirmed differential uptake by tumors and rapid renal clearance. As shown in Figure 7B, the uptake by treated tumors was 1.15% of the injection dose per gram of tumor at 1 hour and 1.02% of the injection dose per gram of tumor at 2 hours, compared to 0.70 and 0.55% of the injection dose per gram of tumor in untreated control tumors .

[0381] [Example 6] In vivo PET imaging for granzyme B expression In non-invasive imaging methods, to evaluate the ability of Ga-NOT 68 A-GP to detect the expression of granzyme B , Ga-NOTA-GP was used in the same manner as in Example 5 68 ​Injected. One hour after injection, the mice were subjected to PET imaging. PET imaging was then performed for 30 minutes, followed by CT acquisition for 2 minutes. 3D-O SEM (repeated 4 times, 16 subsets) was used to reconstruct the images, correct for accidental and scattered coincidences, and are shown in Figure 8. The images showed a marked difference in tumor uptake (T) between treated and untreated mice. Uptake in the kidneys of mice in both groups was also observed, which corresponded to the clearance of the peptide. This imaging analysis 68 validated the value of 68Ga-NOTA-GP for detecting expression in vivo using

[0382] [Example 7] In vivo PET imaging of granzyme B response to immunotherapy in tumor-bearing mice To further assess the predictive nature of granzyme B expression in tumor-bearing mice treated with combination checkpoint inhibitor therapy, imaging was performed in 4 treated mice and 3 vehicle-only mice in the same manner as previously described. Tumor uptake was clearly differentiated in both mouse groups. As shown in Figure 9, high uptake was observed in 3 out of 4 treated mice, which was considered responsive to checkpoint therapy, while one mouse showed low uptake, which would predict non-responsiveness to treatment. As shown in Figure 10, untreated mice, as expected given the absence of treatment, showed 2 mice with low tracer uptake. One untreated was supported, but this would represent a spontaneous immune response not related to treatment.

[0383] On day 14 after inoculation, mice were intravenously injected with approximately 37 MBq of purified 68 Ga-NOTA-GP and imaged 1 hour after injection. PET imaging revealed uptake within the treated tumor, kidney and bladder, with the latter two consistent with the renal clearance characteristic of small peptides. By subtracting the uptake by the left ventricle from the uptake by the tumor, the uptake of Ga-NOTA-GP by PET imaging was standardized to derive the tumor-to-blood ratio (TBR). As shown in Figure 11, the mean TBR of treated mice was calculated to be 1.69 ±0.26, while the mean TBR of untreated mice was 0.93 ± 0.11 68 and a significant increase (83%, P < 0.05) was seen from untreated to treated mice. (TBR). As shown in Figure 11, the mean TBR of treated mice was calculated to be 1.69 ±0.26, while the mean TBR of untreated mice was 0.93 ± 0.11 and a significant increase (83%, P < 0.05) was seen from untreated to treated mice. (TBR). As shown in Figure 11, the mean TBR of treated mice was calculated to be 1.69 68 Ga-NOTA-GP uptake was observed in both treated and untreated tumors. The correlation with GZB expression evaluated by Western blot ex vivo supported that this correlation was due to variation in GZB expression between tumors rather than variation in probe distribution. Ga-NOTA-GP uptake was observed in both treated and untreated tumors. The correlation with GZB expression evaluated by Western blot ex vivo supported that this correlation was due to variation in GZB expression between tumors rather than variation in probe distribution. Ga-NOTA-GP uptake was observed in both treated and untreated tumors. The correlation with GZB expression evaluated by Western blot ex vivo supported that this correlation was due to variation in GZB expression between tumors rather than variation in probe distribution. Ga-NOTA-GP uptake was observed in both treated and untreated tumors. The correlation with GZB expression evaluated by Western blot ex vivo supported that this correlation was due to variation in GZB expression between tumors rather than variation in probe distribution.

[0384] [Example 8] Immunofluorescence staining to evaluate granzyme B expression in tumors after immunotherapy Before CTL activation, GZB is contained within cytotoxic granules and is released at the immune synapse between CTL and its target cells after activation. However, in the context of cancer immunotherapy as well as the fate of GZB after release, the location (e.g., intracellular vs. extracellular) is On day 14 after inoculation, mice were intravenously injected with approximately 37 MBq of purified Not fully understood. To better understand the location of GZB within the tumor microenvironment tumors were analyzed by immunohistochemistry and immunofluorescence.

[0385] Slices of treated and untreated tumors were stained for GZB in addition to CD3 to evaluate whether GZB is sequestered within T cells or secreted. CD 3 staining showed increased T cell infiltration in treated tumors and low levels in untreated tumors. Granzyme B staining also showed further differences between untreated and treated tumors and two distinct patterns of GZB location. The first pattern observed was a pattern of low intensity and small surface areas, co-localized with CD3 staining and seen in both treated and untreated tumors, as shown in Figure 12, and was thought to represent GZB contained within CTL cell cytotoxic granules. The second pattern of GZB staining generally resulted in large, intense staining areas that did not co-localize with CD3. Fluorescence threshold analysis of GZB IF samples showed higher intensity staining in treated tumors compared to untreated (P<0.01). Without being bound by theory, the high intensity GZB staining areas correspond to prior exocytosis areas of GZB which are proposed to represent GZB that has already participated in tumor cell killing or GZB that has leaked from the immunological synapse and remains in the extracellular cavity. These results suggest that high GZB expression in treated tumors is due to active CTLs that secrete proteases in response to immunotherapy and are potential presented further evidence representing such biomarkers. As shown in Figure 13, IH C findings confirm the findings by immunofluorescence.

[0386] [Example 9] PD-1, PD-L1, CTLA-4, and T, which are targets of checkpoint inhibitors Western blot analysis of TIM-3 CT26 and MC38 were diluted 1:1 (v:v) in Matrigel (Corning, Tewksbury , MA) and injected into the right upper flank of balb / c mice or C57 BL / 6 mice, respectively. Mice were injected intraperitoneally with saline (i.e., vehicle), 200 μg of anti-PD1 (i.e., monotherapy), 200 μg of anti-PD1 and 100 μg of anti-CTLA4 (i.e., P +C combination therapy), or 200 μg of anti-PD1 and 250 μg of anti-TIM-3 (i.e., P+T combination therapy) on days 3, 6, and 9 after tumor inoculation. On day 12, mice were imaged or sacrificed for ex vivo Western blot analysis. In the imaging experiment of surviving mice, tumor size was measured with calipers starting from day 5 for MC38 and from day 10 for CT26, every 2-3 days. Mice were sacrificed when the tumor exceeded a volume of 50 0 mm or ulcerated. 0 mm 3 of volume or ulcerated.

[0387] Syngeneic tumors of MC38 and CT26 were excised and, as shown in Figures 14A-14C, on day 12, immunoblotting was performed to determine the baseline levels of the checkpoint inhibitor target molecules, PD-1, PD-L1, CTLA-4, and TIM-3. It was determined. The level of CTLA-4 was 2-fold within the MC38 tumor compared to that within the CT26 tumor ( CTLA-4:β-actin = 0.225 vs. 0.121, p = 0.043). For PD-1, the expression levels of PD-L1 and TIM-3 did not differ significantly between each tumor type . To evaluate the feasibility of imaging granzyme B within the MC38 tumor, the granzyme B levels were also assayed, and it was found that granzyme B expression was significantly higher within the MC38 tumor (0. 013, p = 0.014) than within the CT26 tumor (granzyme B:β- actin = 0.138).

[0388] [Example 10] Granzyme B PET imaging for CT26 tumor-bearing mice and MC38 tumor-bearing mice On day 12, 68 using Ga-NOTA-GP, granzyme B levels were non-invasively evaluated by imaging CT26 tumor-bearing mice and MC38 tumor-bearing mice treated with anti-PD1 monotherapy, P+C combination therapy, P+T combination therapy, or saline (see Example 9). PET imaging showed a pattern of accumulation in the kidneys and bladder, consistent with renal clearance characteristic of small peptides, with a common uptake pattern by non-tumor organs in all mice analyzed. However, as shown in FIGS. 15A - 17D, the degree of uptake by the tumor varied based on the tumor model and treatment regimen. For example, in the CT26 model, vehicle-treated mice did not show a difference in tracer accumulation within the tumor and left ventricle, as reflected by an average TBR of 0.97±0.07.

[0389] ​ In comparison, CT26 tumor-bearing mice receiving PD-1 monotherapy and P+C combination therapy The mice showed high tumor uptake, with mean TBRs of 1.32 ± 0.1, respectively. 5 (p<0.05) and 1.48±0.19 (p<0.005). Mice bearing CT26 tumors treated with P+T combination therapy showed tumor-induced tracing. The results did not support an increase in T accumulation compared to vehicle-treated mice (as shown in FIG. 17C). BR=0.99±0.14). Among the three treatment regimens, the TBR threshold was significantly lower for predicting response. To experimentally define the value, the highest TBR for vehicle-treated CT26 tumors was Tumors above 1.27 were classified as high uptake, and tumors below 1.27 were classified as low uptake. This cutoff for the CT26 model was defined as the value at which the patient was classified as having low uptake. 4 out of 12 mice treated with monotherapy and 6 out of 12 mice treated with P+C combination therapy , and 0 out of 6 P+T combination therapy treated mice were classified as high uptakers.

[0390] MC38 tumor-bearing mice also showed differential tumor uptake based on treatment regimen. Saline-treated mice bearing MC38 tumors had a mean HR of 1.37 ± 0.016. The mean TBR of the CT26 tumor-bearing mice treated with saline was significantly higher than that of the saline-treated mice. The results were highly correlated with the phenotype of MC38 tumors (p<0.005), as shown in FIG. 17B. Indicating high baseline granzyme B levels, consistent with Western blot results As in the CT26 model, treatment with either monotherapy or P+C combination therapy was performed. MC38 tumor-bearing mice treated with CT showed significantly increased gliomas compared with control mice, respectively. resulted in a high TBR of 1.99 ± 0.22 (p < 0.05) and 2.17 ± 0.21( p < 0.005). However, unlike the CT26 model, the MC38 tumors treated with the P+T combination therapy showed the lowest average TBR among the three treatments, as shown in Figure 17D. However, this group also resulted in higher uptake than the control tumors (TBR = 1.74 ± 0.12, p < 0.05). With the exception of a single vehicle-treated tumor that showed delayed growth compared to all other vehicle-treated tumors with high uptake, the same rationale described above for CT26 tumors was used to define the high and low uptake thresholds for MC38 tumors as 1.45. The number of high-uptake MC38 tumors included 4 out of 5

[0391] [Example 11] Analysis of growth curves Tumors either completely regressed by day 20 or continued to grow until the end of the study, reached a pre-defined maximum volume, or ulcerated, so the growth of the CT26 tumors described in Examples 9-10 was dichotomous. Based on these growth patterns, mice were classified 3 as responders if the tumor volume decreased to 0 mm or as non-responders if the tumor volume continued to increase. Figures 18A-18C illustrate the transient changes in tumor volume for each treatment regimen, with vehicle-treated tumors represented in black. None of the vehicle-treated CT26 mice showed a decrease in size, but None of the 6 mice treated with the +T combination therapy regimen responded to treatment.

[0392] In the MC38 tumor models described in Examples 9 - 10, based on the response patterns, three groups were identified as complete responders, non - responders, and partial responders. Responding tumors shrank in size by day 20, non - responding tumors reached a size of 500 mm by day 14 or earlier, and partial - response tumors reached 500 mm 3 at a later time point than day 14. Figures 18D - 18F highlight these transient patterns of tumor volume change for MC38 based on the treatment regimen, with the growth curve of 3 vehicle - treated tumors shown in black. One of the 7 vehicle - treated MC38 tumors showed a significant delay in tumor growth compared to other vehicle - treated tumors, classifying it as a partial responder. This tumor showed a TBR greater than 1.45 and is itself shown as a black dotted line in Figures 18D - 18F. Compared to CT26 tumors, a higher percentage of MC38 tumors responded to treatment, with 4 out of 5 tumors treated with monotherapy and 7 out of 8 tumors treated with the P + C combination therapy showing complete size regression. Unlike CT26, MC38 tumors responded to the P + T combination therapy, with 1 out of 7 tumors showing a complete response and 4 out of 7 tumors showing a delay in growth characteristic of a partial response in support of treatment response.

[0393] [Example 12] Prediction of response using granzyme B PET imaging Over multiple tumor models and treatments described in Examples 9 - 11, granzyme B Metrics for the accuracy of PET (i.e., GZP PET)-imaging-based response prediction For CT26 tumors, a TBR threshold of 1.27 was used, and for MC38 tumors, a TBR threshold of 1.45 was used to utilize the classification of uptake. Using this classification system, 25 out of 27 responder mice were classified as high-uptake cases by GZP PET imaging, while 34 out of 38 mice that did not respond to treatment were classified as low-uptake cases. This reflects 68 the overall sensitivity and specificity of Ga-GZP PET for predicting response to immunotherapy at 93% and 89%, respectively. The sensitivity and specificity of the probe for each treatment type are shown in Table 5.

[0394]

Table 8

[0395] To compare whether the accuracy of granzyme B PET imaging for individual-based image response enables group-based prediction of efficacy, the average TBR for each treatment was correlated with overall survival. The treatment with the best survival outcome also showed the highest average TBR based on the Kaplan-Meier plot for survival of each treatment, as shown in Figure 19A. To determine whether the relationship between the response percent and the average tumor uptake is correlated, the response percent was 2 plotted against the TBR, as shown in Figure 19B. The resulting curve showed a linear relationship with R = 0.84 and a significantly non-zero slope (p < 0.0005), suggesting that the average TBR of the treatment group is linearly correlated with the survival percent.

[0396] [Example 13] Synthesis of additional granzyme B peptides NOTA-betaA-PEG 27 -G-G-G-I-E-F-D (GZPPEG; sequence number 26; PEG = -(OCH 2 CH 2 )-), and NOTA-betaA-G-G-G -T-E-A-A-A-A-S-S-C-F-I-E-F-D-CHO (GZPSERP ; sequence number 27) were each prepared according to the standard FMOC chemical reaction described in Example 1 The calculated value of GZPSERP m / z [M+2H] 2+ = 1973.31

[0397] Effect of additional groups on pharmacokinetics and tumor-to-background ratio in mice Both peptides were designed to examine the effect on pharmacokinetics and tumor-to-background ratio in mice. Dynamic PET analysis was performed using NOTA-GP (Example 1), GZP PEG, and GZPSERP. Surprisingly GZPPEG showed an uptake reaction rate by tumors that was almost the same as that of NOTA-GP GZPSERP, which is a hybrid of the serpin P9-granzyme B binding sequence and the granzyme B peptide, resulted in a much lower tumor-to-background ratio Without being bound by any theory, these effects are thought to be due to the strong hydrophobic nature of the peptides, which resulted in significant aggregation and high uptake by the liver The time course of the PET signal in tumors / heart for NOTA-GP, GZPPEG, and GZPSERP is shown in Figure 20

[0398] [Example 14] Immunohistochemical analysis of human melanoma specimens​ After standard antigen retrieval in citrate buffer, immunohistochemistry was performed on formalin-fixed paraffin-embedded sections. Granzyme B expression was detected with an anti-Granzyme B antibody (ab5049, Abcam), or with biotinylated and humanized forms of GZP (hGZP, see Example 1A). Bound antibodies were detected with an HRP-conjugated goat anti-rabbit antibody and reacted with DAB substrate for IHC staining, or with AlexaFluor 488 goat anti-rabbit antibody or AlexaFluor 594 goat anti-rabbit antibody (Life Technologies) for visualization by immunofluorescence. Bound peptides were detected with HRP-conjugated streptavidin (Abcam) and then reacted with DAB substrate or Oregon Green-conjugated Neutravidin (Life Technologies). Patient samples were grouped as immunotherapy-treated or untreated, and treated specimens were further identified as responders or non-responders using modified RECIST criteria. Fluorescence quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD). A cohort of 9 human melanoma biopsy samples obtained from patients treated with anti-PD-1 checkpoint inhibitors (n = 6 nivolumab and n = 3 pembrolizumab) was retrospectively correlated with response based on modified RECIST criteria. Using an anti-human Granzyme B antibody, by IHC, as shown in Figure 21A, biopsy samples with treatment had a higher level of Granzyme B expression than untreated specimens, as shown in Figure 21B. The level of Granzyme B expression was positively correlated with the response of the tumor to treatment. In addition, Granzyme B expression was detected in tumor-infiltrating lymphocytes (TILs) and was also positively correlated with the response of the tumor to treatment. These results suggest that Granzyme B may be a useful biomarker for predicting the response of melanoma to anti-PD-1 checkpoint inhibitor treatment. higher level of Granzyme B expression than untreated specimens, as shown in Figure 21B. The level of Granzyme B expression

[0399] was positively correlated with the response of the tumor to treatment. In addition, Granzyme B expression was detected in tumor-infiltrating lymphocytes (TILs) and was also positively correlated with the response Significant differences in the amount and intensity of granzyme B staining between responders and non-responders were verified. To quantify the degree and magnitude of the differences between treated responders and non-responders, quantitative fluorescence microscopy was performed. Fluorescence microscopy analysis showed that granzyme B expression by treated responder specimens was up to about 1,000-fold that of treated non-responders, suggesting a continuous increase in granzyme B expression by treated specimens over time, as shown in Figure 21B. Finally, treated responder samples were analyzed by both immunohistochemistry and immunofluorescence to compare the ability of hGZP (Example 1A) to specifically detect granzyme B in human tissues and compared with anti-granzyme B antibodies. As shown in Figures 21C-21D, a strong correlation was observed by both techniques, indicating a highly specific binding of hGZP to human granzyme B. These results show that there are significant differences in granzyme B expression by tumors between human responders and non-responders, and that these differences are recognized by human granzyme B-specific probes. The difference between the treated responders and non-responders was verified. To quantify the degree and magnitude of the differences between treated responders and non-responders, quantitative fluorescence microscopy was performed. Fluorescence microscopy analysis showed that granzyme B expression by treated responder specimens was up to about 1,000-fold that of treated non-responders, suggesting a continuous increase in granzyme B expression by treated specimens over time, as shown in Figure 21B. Finally, treated responder samples were analyzed by both immunohistochemistry and immunofluorescence to compare the ability of hGZP (Example 1A) to specifically detect granzyme B in human tissues and compared with anti-granzyme B antibodies. As shown in Figures 21C-21D, a strong correlation was observed by both techniques, indicating a highly specific binding of hGZP to human granzyme B. These results show that there are significant differences in granzyme B expression by tumors between human responders and non-responders, and that these differences are recognized by human granzyme B-specific probes. Fluorescence microscopy analysis showed that granzyme B expression by treated responder specimens was up to about 1,000-fold that of treated non-responders, suggesting a continuous increase in granzyme B expression by treated specimens over time, as shown in Figure 21B. Finally, treated responder samples were analyzed by both immunohistochemistry and immunofluorescence to compare the ability of hGZP (Example 1A) to specifically detect granzyme B in human tissues and compared with anti-granzyme B antibodies. As shown in Figures 21C-21D, a strong correlation was observed by both techniques, indicating a highly specific binding of hGZP to human granzyme B. These results show that there are significant differences in granzyme B expression by tumors between human responders and non-responders, and that these differences are recognized by human granzyme B-specific probes. Fluorescence microscopy analysis showed that granzyme B expression by treated responder specimens was up to about 1,000-fold that of treated non-responders, suggesting a continuous increase in granzyme B expression by treated specimens over time, as shown in Figure 21B. Finally, treated responder samples were analyzed by both immunohistochemistry and immunofluorescence to compare the ability of hGZP (Example 1A) to specifically detect granzyme B in human tissues and compared with anti-granzyme B antibodies. As shown in Figures 21C-21D, a strong correlation was observed by both techniques, indicating a highly specific binding of hGZP to human granzyme B. These results show that there are significant differences in granzyme B expression by tumors between human responders and non-responders, and that these differences are recognized by human granzyme B-specific probes. As shown in Figures 21C-21D, a strong correlation was observed by both techniques, indicating a highly specific binding of hGZP to human granzyme B. These results show that there are significant differences in granzyme B expression by tumors between human responders and non-responders, and that these differences are recognized by human granzyme B-specific probes. Fluorescence microscopy analysis showed that granzyme B expression by treated responder specimens was up to about 1,000-fold that of treated non-responders, suggesting a continuous increase in granzyme B expression by treated specimens over time, as shown in Figure 21B. Finally, treated responder samples were analyzed by both immunohistochemistry and immunofluorescence to compare the ability of hGZP (Example 1A) to specifically detect granzyme B in human tissues and compared with anti-granzyme B antibodies. As shown in Figures 21C-21D, a strong correlation was observed by both techniques, indicating a highly specific binding of hGZP to human granzyme B. These results show that there are significant differences in granzyme B expression by tumors between human responders and non-responders, and that these differences are recognized by human granzyme B-specific probes.

[0400] Other embodiments Although the present invention has been described in detail, it should be understood that the foregoing description is intended to illustrate, and not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Although the present invention has been described in detail, it should be understood that the foregoing description is intended to illustrate, and not limit, the scope of the invention as defined by the appended claims. It is to be understood that the foregoing description is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for imaging granzyme B in a cell or tissue, comprising: i) subjecting the cell or tissue sample to a detection vector containing a compound of formula I: 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof; ii) imaging the cells or tissue with a suitable imaging technique, thereby imaging granzyme B in said cells or tissues; wherein A comprises one or more imaging agents; B is an optional linking group; A method wherein C is a group that binds to granzyme B.

2. 1. A method for imaging granzyme B in a subject, comprising: i) administering to the subject a compound of formula I: 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof; ii) imaging the subject with a suitable imaging modality, thereby imaging granzyme B in the wherein A comprises one or more imaging agents; B is an optional linking group; A method wherein C is a group that binds to granzyme B.

3. 1. A method for imaging an immune response in a cell or tissue sample, comprising: i) subjecting the cell or tissue sample to a detection vector containing a compound of formula I: 【Chemistry 3】 or a pharma- ceutically acceptable salt thereof; ii) imaging the cell or tissue sample with a suitable imaging technique, imaging said immune response in said cell or tissue sample by wherein A comprises one or more imaging agents; B is an optional linking group; A method wherein C is a group that binds to granzyme B.

4. 1. A method for imaging an immune response in a subject, comprising: i) administering to the subject a compound of formula I: 【Chemistry 4】 or a pharma- ceutically acceptable salt thereof; ii) imaging the subject with a suitable imaging modality, thereby imaging the immune response in the wherein A comprises one or more imaging agents; B is an optional linking group; A method wherein C is a group that binds to granzyme B.

5. 1. A method for monitoring treatment of a disease in a subject, comprising: i) administering to the subject a compound of formula I: 【Chemistry 5】 or a pharma- ceutically acceptable salt thereof; ii) imaging the subject with a suitable imaging modality; wherein A comprises one or more imaging agents; B is an optional linking group; A method wherein C is a group that binds to granzyme B.

6. 1. A method for monitoring an immune response in the treatment of a disease in a subject, comprising: i) administering to the subject a compound of formula I: 【Chemistry 6】 or a pharma- ceutically acceptable salt thereof; ii) imaging the subject with a suitable imaging modality; wherein A comprises one or more imaging agents; B is an optional linking group; A method wherein C is a group that binds to granzyme B.

7. A is a paramagnetic ion, an x-ray imaging agent, a fluorophore, or a radioisotope.

7. The method of claim 1 , further comprising administering to said patient one or more imaging agents selected from the group consisting of:

3. The method according to claim 1 .

8. The paramagnetic ions are chromium (III), manganese (II), iron (III), iron (II) ), cobalt(II), nickel(II), copper(II), neodymium(III), samary Mu(III), Ytterbium(III), Gadolinium(III), Vanadium(II ), terbium(III), dysprosium(III), holmium(III), and 8. The method of claim 7, wherein the metal is selected from the group consisting of erbium (III).

9. The x-ray imaging agent is selected from the group consisting of lanthanum (III), gold (III), lead (II), bismuth (II), and the like. and iodinated x-ray imaging agents. The method described above.

10. The radioisotope is ３ H. １１ C. １４ C. １８ F. ３２ P. ３５ S. ３６ Cl, ５１ Cr, ５２ Fe, ５７ Yes, ５８ Yes, ５９ Fe, ６４ Cu, ６７ Cu, ６７ Ga, ６８ Yes, ７５ Yes, ７６ Br, ７７ Br, ８９ Zr, ９０ Y、 ９９ｍ Tc、 １１１ I 、 １２３ I、 １２４ I、 １２５ I、 １３１ I、 １５２ Eu、 １５３ Sm、 １６６ Ho、 １ ７７ Lu、 １８６ Re、 １８８ Re、 ２０１ Tl、 ２０３ Pb、 ２１０ At、 ２１１ At , ２１２ Bi, ２１３ Bi, and ２２５ The compound according to claim 7, wherein the compound is selected from the group consisting of Method of posting.

11. A is a PET imaging agent, a SPECT imaging agent, and a computed tomography imaging agent.

7. Any of claims 1 to 6, wherein the imaging agent is selected from the group consisting of opioid imaging agents. The method according to any one of claims 1 to 5.

12. 7. The method of claim 1, wherein A is a PET imaging agent or a SPECT imaging agent.

2. The method according to claim 1 .

13. Aが、 ３ 8、 １１ 3、 １４ 3、 １８ 6、 ３５ 、 ５２ e、 ５８ Co、 ６４ Cu、 ６８ Ga、 ７６ Br、 ７７ Br、 ８９ Zr、 １１１ In、 １２３ I、 １２４ I、 １２５ I、 １ ３１ I, １８６ Re, １８８ Re, ２０１ A radioisotope selected from the group consisting of Tl 7. The method of claim 1, wherein the compound is a PET or SPECT imaging agent comprising:

2. The method according to claim 1 .

14. A, ６８ The PET imaging agent according to any one of claims 1 to 6, which is a Ga-containing PET imaging agent. The method described.

15. 15. The method of claim 1, wherein A further comprises a chelating agent.

16. The chelating agent is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4, 7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4 ,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), Diethylenetriaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexane xyl-1,2-diaminetetraacetic acid (CDTA), ethylene glycol-O,O'-bis( 2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydro (xybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetramine 1 triamine hexaacetic acid (TTHA), hydroxyethyldiamine triacetic acid (HEDTA), and ,4,8,11-Tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-cal Bamoylmethyl)-cyclododecane (TCMC), and desferrioxamine B (D 16. The method of claim 15, wherein the compound is selected from the group consisting of:

17. The chelating agent is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4, 7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-Triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) 16. The method of claim 15, selected from the group consisting of:

18. The chelating agent is 1,4,7-triazacyclononanetriacetic acid (NOTA). The method according to claim 15.

19. 7. The method of claim 1, wherein A is a fluorophore.

20. A is Alexa 350, Alexa 430, AMCA, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODPY-R6G, 1 3BODLPY-TMR, BODLPY-TRX, Cascade Blue, Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Quantum Dot, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renografin, ROX, T AMRA, TET, Tetramethylrhodamine, Texas Red, AF350, AF405 , AF532, AF488, AF647, AF680, AF750, Cy5, Cy5.5 , Cy7, indocyanine green (ICG), green fluorescent protein (GFP), red fluorescent protein A fluorophore selected from the group consisting of a photoprotein (RFP), and a dsRED. The method according to any one of claims 1 to 6.

21. 1. A method of treating a disease in a subject, comprising: i) administering to the subject a compound having a first formula I: 【Chemistry 7】 [In the formula, A comprises a non-toxic radioisotope; B is an optional linking group; C is a group that binds to granzyme B. or a pharma- ceutically acceptable salt thereof; ii) imaging the subject with a suitable imaging modality; iii) administering to the subject a compound represented by a second formula I: 【Chemistry 8】 [In the formula, A contains a toxic radioisotope; B is an optional linking group; C is a group that binds to granzyme B. or a pharma- ceutical acceptable salt thereof, thereby eliciting a pre-existing condition in said subject. Treating the disease. The method includes:

22. Prior to said administration of step iii), said first compound of formula I, or a pharma- ceutical acceptable salt thereof, A step of determining whether an acceptable salt binds to the cells or tissue of the subject being treated.

22. The method of claim 21 further comprising:

23. Prior to said administration of step iii), said first compound of formula I, or a pharma- ceutical acceptable salt thereof, The method further comprises the step of determining whether the acceptable salt binds to granzyme B. The method according to claim 21.

24. The group A of the first compound of formula I, or a pharma- ceutically acceptable salt thereof, is ３ H. １１ C. １４ C、 １８ F ３５ S、 ５２ ｦe ５８ Co ６４ C ６８ G ７６ Br、 ７７ B r、 ８９ Zr、 １１１ In、 １２３ I、 １２４ I、 １２５ I、 １３１ I、 １８６ Re、 １ ８８ Re, ２０１ 10. The method of claim 1, further comprising administering to said patient a radioisotope selected from the group consisting of 10, 11 and 12.

24. The method according to any one of items 21 to 23.

25. The group A of the first compound of formula I, or a pharma- ceutically acceptable salt thereof, further comprises a chelating agent.

25. The method of any one of claims 21 to 24, further comprising:

26. The chelating agent is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4, 7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4 ,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), Diethylenetriaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexane xyl-1,2-diaminetetraacetic acid (CDTA), ethylene glycol-O,O'-bis( 2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydro (xybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetramine 1 triamine hexaacetic acid (TTHA), hydroxyethyldiamine triacetic acid (HEDTA), and ,4,8,11-Tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-cal Bamoylmethyl)-cyclododecane (TCMC), and desferrioxamine B (D FO).

27. The group A of the second compound of formula I, or a pharma- ceutically acceptable salt thereof, is an alpha emitter. and beta emitters.

27. The method according to any one of claims 1 to 26.

28. The group A of the second compound of formula I, or a pharma- ceutically acceptable salt thereof, is ２１１ At, ２ １２ Pb、 ２１２ Bi、 ２１３ Bi、 ２２５ Ac、 ２２７ Th、 ９０ 9、 １７７ Lu、お Call １３１ 21 to 2, comprising a toxic radioisotope selected from the group consisting of I 7. The method according to any one of claims 6 to 6.

29. B is one or more amino acid residues, one or more carbohydrates, one or more an alkylene group, one or more amine groups, one or more amide groups, one or more alkyleneoxy groups, one or more thiol groups, or any combination thereof.

29. The method of claim 1 , wherein the optional linking group comprises

30. B is one or more C １～３０ an alkylene group, one or more amine groups, or more amide groups, one or more C １～３０ Alkyleneoxy group, one or more C １～３０ is an optional linking group comprising a thiol group, a thiol group, or any combination thereof; 29. The method of any one of claims 1 to 28.

31. B is one or more -(OCH ２ CH ２ )-group is an optional linking group.

29. The method according to any one of items 1 to 28.

32. B is a compound of the formula -(OCH ２ CH ２ ) ｐ - is an optional linking group, and p is an integer from 1 to 40.

29. The method of any one of claims 1 to 28, wherein

33. 33. The method of claim 32, wherein p is an integer from 10 to 40.

34. 33. The method of claim 32, wherein p is an integer from 20 to 40.

35. 33. The method of claim 32, wherein p is an integer from 25 to 35.

36. C is a polypeptide that binds to granzyme B, an antibody that binds to granzyme B, or a granzyme B-binding polypeptide. A group consisting of antibody fragments that bind to granzyme B, and small organic molecules that bind to granzyme B.

36. The method of any one of claims 1 to 35, wherein the compound is selected from the group consisting of

37. 36. The method of claim 1, wherein C is an antibody that binds to granzyme B. How to.

38. C is an antibody that binds to granzyme B, clone GB11, clone GrB-7 36. Any of claims 1 to 35, wherein the IL-11 is selected from the group consisting of NCL-L-Gran-B, and NCL-L-Gran-B. The method according to any one of claims 1 to 5.

39. 36. The method of claim 1, wherein C is an antibody fragment that binds to granzyme B. The method described.

40. 36. Any one of claims 1 to 35, wherein C is a polypeptide that binds to granzyme B. The method according to claim 5.

41. The polypeptide that binds to Granzyme B is about 4 to about 100 amino acid residues in length. The method of claim 36 or 40.

42. The polypeptide that binds to Granzyme B is about 4 to about 50 amino acid residues in length. The method of claim 36 or 40,

43. The polypeptide that binds to Granzyme B is about 4 to about 25 amino acid residues in length. The method of claim 36 or 40,

44. The polypeptide that binds to Granzyme B is about 4 to about 15 amino acid residues in length. The method of claim 36 or 40,

45. C is a polypeptide that binds to granzyme B, said polypeptide comprising: X １ -X ２ -X ３ -X ４ -X ５ -X ６ -D (SEQ ID NO: 1) comprising an amino acid sequence having at least 90% sequence identity to X １ , X ２ , and X ３ are each independently beta A, G, Q, N, S, T, Y, C, selected from the group consisting of R, D, and E; X ４ is selected from the group consisting of I and V; X ５ is selected from the group consisting of E, G, D, and S; X ６ is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D.

41. The method of claim 36 or 40,

46. X ４ The method of claim 45, wherein is I.

47. X ５ The method of claim 45, wherein is E.

48. X ６ The method of claim 45, wherein is F or P.

49. The polypeptide, beta A-G-G-I-E-F-D (SEQ ID NO:2); G-G-G-I-E-F-D (SEQ ID NO:3); and Beta A-G-G-IE-P-D (SEQ ID NO: 4) An amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of:

49. The method of any one of claims 45 to 48, comprising:

50. 46. ​​The method of claim 45, wherein C is a polypeptide comprising the amino acid sequence of SEQ ID NO:

2. 。

51. 46. ​​The method of claim 45, wherein C is a polypeptide comprising the amino acid sequence of SEQ ID NO:

3. 。

52. 46. ​​The method of claim 45, wherein C is a polypeptide comprising the amino acid sequence of SEQ ID NO:

4. 。

53. C is a polypeptide that binds to granzyme B, said polypeptide comprising: X １ -X ２ -X ３ -X ４ -X ５ -X ６ -D-X ７ (SEQ ID NO:8) comprising an amino acid sequence having at least 90% sequence identity to X １ , X ２ , and X ３ are each independently beta A, G, Q, N, S, T, Y, C, selected from the group consisting of R, D, and E; X ４ is selected from the group consisting of I and V; X ５ is selected from the group consisting of E, G, D, and S; X ６ is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D. And, X ７ is an electrophilic group comprising the C-terminus of the amino acid sequence of SEQ ID NO:8; or 40. The method according to claim 40.

54. X ４ The method of claim 53, wherein is I.

55. X ５ The method of claim 53 or 54, wherein is E.

56. X ６ The method of any one of claims 53 to 55, wherein is F or P.

57. X ７ is -C(O)H, -C(O)C １～６ Alkyl, -C(O)C １～６ Hello Aruki Ru, -C(O)C １～６ Alkoxy, -C(O)C １～６ Haloalkoxy, —C(O)— (C １～６ alkyl)-(5-10 membered heteroaryl), -C(O)-(C １～６ Halo alkyl)-(5-10 membered heteroaryl), -C(O)-(C １～６ Alkoxy)- (5-10 membered heteroaryl), and —C(O)—(C １～６ haloalkoxy)-( 53 to 55, wherein the electrophilic group is selected from the group consisting of 5- to 10-membered heteroaryl.

56. The method according to any one of claims 1 to 56.

58. X ７ The method of any one of claims 53 to 56, wherein is -C(O)H.

59. 36. Any one of claims 1 to 35, wherein C is a small organic molecule that binds to granzyme B. The method described above.

60. 60. The method of claim 36 or 59, wherein the small organic molecule is a small molecule peptidomimetic. 。

61. C is of formula III: 【Chemistry 9】 or a pharma- ceutically acceptable salt thereof, wherein n is 0, 1, or 2; m is 0, 1, or 2; R １ and R ２ Each independently represents hydrogen, C １～６ Alkyl, C １～６ Alkoxy, C ３～６ Cycloalkyl, C ６～１０ Aryl, HET, and -N(R １０ ) ２ Consists of wherein each C １～６ Alkyl, C １～６ Alkoxy, C ３～６ Cycloal Kyl is independently one, two, or three selected from the group consisting of halo and hydroxy. Optionally substituted by one or more substituents, each C ６～１０ Aryl and HET are independently Oxo, halo, hydroxy, C １～４ Alkyl, and C １～４ The group consisting of haloalkyl optionally substituted with one, two, or three substituents selected from Or R １ and R ２ are each of them together with the carbon atom to which they are attached. Each may optionally have one, two, or three R １０ 5 to 6 optionally substituted by a group It is possible to form a 5- or 6-membered cycloalkyl group or a 5- or 6-membered heterocycloalkyl group. can be; Each R ３ and R ７ are independently hydrogen, C １～４ Alkyl, and C １～４ Haloalkyl selected from the group consisting of: Each R ４ , R ５ , R ６ , and R ８ are independently hydrogen, halo, hydroxy, C １～４ Al Kill, and C １～４ haloalkyl; R ９ are independently oxo, halo, hydroxy, C １～４ Alkyl, and C １～４ Halo Optionally substituted with one, two, or three substituents selected from the group consisting of alkyl, HET which can be exchanged; R １０ is hydrogen, C １～４ Alkyl, and -C(O)C １～４ From the group consisting of alkyl wherein: １～４ Alkyl is optionally represented by -N(R １１ ) ２ , H E.T., and C. ６～１０ substituted by aryl, ６～１０ The aryl is optionally optionally substituted with 1, 2, or 3 halo groups; Each HET is an independently selected monocyclic or bicyclic 5-10 membered heteroaryl. or a monocyclic or bicyclic 5- to 10-membered heterocycloalkyl group, in which In this case, each HET is one, two, three, or four heteroatoms selected from O, S, and N. containing an oxo atom and optionally substituted by one or two oxo groups; R １１ is hydrogen, C １～４ Alkyl, and C １～４ haloalkyl; However, if m is 0, then n is 0, and if n is 0, then m is 0.

36. The method according to any one of claims 1 to 35.

62. C, 【Table 1】 or a pharma- ceutically acceptable salt thereof. 9 to 61. The method of any one of claims 9 to 61.

63. A is a paramagnetic ion, an x-ray imaging agent, a fluorophore, and a radioisotope. and wherein the imaging agent comprises one or more of B is one or more alkylene groups, one or more amine groups, one or more an amide group, one or more alkyleneoxy groups, one or more thiol groups, or are optional linking groups including any combination thereof; C is a polypeptide that binds to granzyme B, an antibody that binds to granzyme B, or a granzyme B-binding polypeptide. A group consisting of antibody fragments that bind to granzyme B, and small organic molecules that bind to granzyme B. The method according to any one of claims 1 to 6, wherein the

64. A comprises a radioisotope; B is one or more C １～３０ an alkylene group, one or more amine groups, or more amide groups, one or more C １～３０ Alkyleneoxy group, one or more C １～３０ optional linking groups including a thiol group, a thiol group, or any combination thereof; C is a polypeptide that binds to granzyme B, and an organic molecule that binds to granzyme B. The method of claim 1 , wherein the antibody is selected from the group consisting of small molecules.

65. Aが、 ３ H、 １１ C、 １４ C、 １８ F、 ３２ P、 ３５ S、 ３６ Cl、 ５１ Cr、 ５２ F e、 ５７ Yes, ５８ Yes, ５９ Fe, ６４ Cu, ６７ Cu, ６７ Ga, ６８ Ga, ７５ S e、 ７６ Br、 ７７ Br、 ８９ Zr、 ９０ Y、 ９９ｍ Tc、 １１１ In、 １２３ I、 １２ ４ I、 １２５ I、 １３１ I、 １５２ Uh １５３ S １６６ H １７７ Lu １８６ Re、 １８８ Re、 ２０１ Tl、 ２０３ Pb、 ２１０ At、 ２１１ At、 ２１２ Bi、 ２ １３ Bi, and ２２５ Ac, B is one or more C １ ~ ３０ an alkylene group, one or more amide groups, C of Numbers １ ~ ３０ Optional linkages including alkyleneoxy groups, or any combination thereof It is based on C is a polypeptide that binds to granzyme B, and an organic molecule that binds to granzyme B. The method of claim 1 , wherein the antibody is selected from the group consisting of small molecules.

66. Aが、 ３ H、 １１ C、 １４ C、 １８ F、 ３２ P、 ３５ S、 ３６ Cl、 ５１ Cr、 ５２ F e、 ５７ Yes, ５８ Yes, ５９ Fe, ６４ Cu, ６７ Cu, ６７ Ga, ６８ Ga, ７５ S e、 ７６ Br、 ７７ Br、 ８９ Zr、 ９０ Y、 ９９ｍ Tc、 １１１ In、 １２３ I、 １２ ４ I、 １２５ I、 １３１ I、 １５２ Uh １５３ S １６６ H １７７ Lu １８６ Re、 １８８ Re、 ２０１ Tl、 ２０３ Pb、 ２１０ At、 ２１１ At、 ２１２ Bi、 ２ １３ Bi, and ２２５ Ac, B is one or more -(-OCH ２ CH ２ is an optional linking group that includes a - group; C is a polypeptide that binds to granzyme B, and an organic molecule that binds to granzyme B. The method of claim 1 , wherein the antibody is selected from the group consisting of small molecules.

67. 67. The method of any one of claims 63 to 66, wherein A further comprises a chelating agent.

68. The chelating agent is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4, 7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4 ,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA), Diethylenetriaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexane xyl-1,2-diaminetetraacetic acid (CDTA), ethylene glycol-O,O'-bis( 2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydro (xybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), triethylenetetramine 1 triamine hexaacetic acid (TTHA), hydroxyethyldiamine triacetic acid (HEDTA), and ,4,8,11-Tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid (TETA), 1,4,7,10-tetraaza-1,4,7,10-tetra-(2-cal Bamoylmethyl)-cyclododecane (TCMC), and desferrioxamine B (D FO).

69. The chelating agent is 1,4,7-triazacyclononanetriacetic acid (NOTA), 1,4, 7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-Triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) 68. The method of claim 67, selected from the group consisting of:

70. The chelating agent is 1,4,7-triazacyclononanetriacetic acid (NOTA). The method of claim 67.

71. 71. Any of claims 63 to 70, wherein C is a polypeptide that binds to granzyme B.

13. The method according to claim 1.

72. The polypeptide that binds to Granzyme B is about 4 to about 100 amino acid residues in length.

71. The method of any one of claims 63 to 70.

73. The polypeptide that binds to Granzyme B is about 4 to about 50 amino acid residues in length.

71. The method of any one of claims 63 to 70,

74. The polypeptide that binds to Granzyme B is about 4 to about 25 amino acid residues in length.

71. The method of any one of claims 63 to 70,

75. The polypeptide that binds to Granzyme B is about 4 to about 15 amino acid residues in length.

71. The method of any one of claims 63 to 70,

76. C is a polypeptide that binds to granzyme B, said polypeptide comprising: X １ -X ２ -X ３ -X ４ -X ５ -X ６ -D (SEQ ID NO: 1) comprising an amino acid sequence having at least 90% sequence identity to X １ , X ２ , and X ３ are each independently beta A, G, Q, N, S, T, Y, C, selected from the group consisting of R, D, and E; X ４ is selected from the group consisting of I and V; X ５ is selected from the group consisting of E, G, D, and S; X ６ is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D.

71. The method of any one of claims 63 to 70, wherein

77. X ４ The method of claim 76, wherein is I.

78. X ５ The method of claim 76 or 77, wherein is E.

79. X ６ The method of any one of claims 76 to 78, wherein is F or P.

80. The polypeptide, beta A-G-G-I-E-F-D (SEQ ID NO:2); G-G-G-I-E-F-D (SEQ ID NO:3); and Beta A-G-G-IE-P-D (SEQ ID NO: 4) An amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of:

77. The method of claim 76, comprising:

81. 77. The method of claim 76, wherein C is a polypeptide comprising the amino acid sequence of SEQ ID NO:

2. 。

82. 77. The method of claim 76, wherein C is a polypeptide comprising the amino acid sequence of SEQ ID NO:

3. 。

83. 77. The method of claim 76, wherein C is a polypeptide comprising the amino acid sequence of SEQ ID NO:

4. 。

84. C is a polypeptide that binds to granzyme B, said polypeptide comprising: X １ -X ２ -X ３ -X ４ -X ５ -X ６ -D-X ７ (SEQ ID NO:8) comprising an amino acid sequence having at least 90% sequence identity to X １ , X ２ , and X ３ are each independently beta A, G, Q, N, S, T, Y, C, selected from the group consisting of R, D, and E; X ４ is selected from the group consisting of I and V; X ５ is selected from the group consisting of E, G, D, and S; X ６ is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D. And, X ７ is an electrophilic group comprising the C-terminus of the amino acid sequence of SEQ ID NO:

8.

10. The method according to any one of claims 0 to 9.

85. X ４ The method of claim 84, wherein is I.

86. X ５ The method of claim 84 or 85, wherein is E.

87. X ６ The method of any one of claims 84 to 86, wherein is F or P.

88. X ７ is -C(O)H, -C(O)C １～６ Alkyl, -C(O)C １～６ Hello Aruki Ru, -C(O)C １～６ Alkoxy, -C(O)C １～６ Haloalkoxy, —C(O)— (C １～６ alkyl)-(5-10 membered heteroaryl), -C(O)-(C １～６ Halo alkyl)-(5-10 membered heteroaryl), -C(O)-(C １～６ Alkoxy)- (5-10 membered heteroaryl), and —C(O)—(C １～６ haloalkoxy)-( 84 to 86, wherein the electrophilic group is selected from the group consisting of 5-10 membered heteroaryl.

87. The method according to any one of claims 87 to 87.

89. X ７ The method of any one of claims 84 to 87, wherein is -C(O)H.

90. 71. Any one of claims 63 to 70, wherein C is a small organic molecule that binds to granzyme B. The method according to claim 5.

91. 91. Any of claims 63 to 70 and 90, wherein the small organic molecule is a small molecule peptidomimetic. The method according to any one of claims 1 to 5.

92. C is of formula III: 【Chemistry 10】 or a pharma- ceutically acceptable salt thereof, wherein n is 0, 1, or 2; m is 0, 1, or 2; R １ and R ２ Each independently represents hydrogen, C １～６ Alkyl, C １～６ Alkoxy, C ３～６ Cycloalkyl, C ６～１０ Aryl, HET, and -N(R １０ ) ２ Consists of wherein each C １～６ Alkyl, C １～６ Alkoxy, C ３～６ Cycloal Kyl is independently one, two, or three selected from the group consisting of halo and hydroxy. Optionally substituted by one or more substituents, each C ６～１０ Aryl and HET are independently Oxo, halo, hydroxy, C １～４ Alkyl, and C １～４ The group consisting of haloalkyl is optionally substituted with one, two, or three substituents selected from Or R １ and R ２ are each of them together with the carbon atom to which they are attached. Each may optionally have one, two, or three R １０ 5 to 6 optionally substituted by a group It is possible to form a 5- or 6-membered cycloalkyl group or a 5- or 6-membered heterocycloalkyl group. can be; Each R ３ and R ７ are independently hydrogen, C １～４ Alkyl, and C １～４ Haloalkyl selected from the group consisting of: Each R ４ , R ５ , R ６ , and R ８ are independently hydrogen, halo, hydroxy, C １～４ Al Kill, and C １～４ haloalkyl; R ９ are independently oxo, halo, hydroxy, C １～４ Alkyl, and C １～４ Halo Optionally substituted with one, two, or three substituents selected from the group consisting of alkyl, HET which can be exchanged; R １０ is hydrogen, C １～４ Alkyl, and -C(O)C １～４ From the group consisting of alkyl wherein: １～４ Alkyl is optionally represented by -N(R １１ ) ２ , H E.T., and C. ６～１０ substituted by aryl, ６～１０ The aryl is optionally optionally substituted with 1, 2, or 3 halo groups; Each HET is an independently selected monocyclic or bicyclic 5-10 ring heteroaryl. or a monocyclic or bicyclic heterocycloalkyl group having 5 to 10 rings, In this case, each HET is one, two, three, or four heteroatoms selected from O, S, and N. containing an oxo atom and optionally substituted by one or two oxo groups; R １１ is hydrogen, C １～４ Alkyl, and C １～４ haloalkyl; However, if m is 0, then n is 0, and if n is 0, then m is 0.

92. The method of any one of claims 3 to 70, 90 and 91.

93. C, 【Table 2】 or a pharma- ceutically acceptable salt thereof.

91. The method according to any one of claims 91 to 91.

94. 94. The method of any one of claims 1 to 93, wherein C is an irreversible binder of granzyme B. Method of posting.

95. 95. The method of any one of claims 1 to 94, wherein C is an inhibitor of granzyme B. 。

96. 2. The method of claim 1 further comprising administering a therapeutic agent prior to said administering of step i).

95. The method according to any one of claims 1 to 95.

97. Administration of the therapeutic agent induces an immune response in a cell or tissue sample or in a subject.

97. The method of claim 96,

98. The therapeutic agent may be an anti-inflammatory agent, a steroid, an immunotherapeutic agent, a chemotherapeutic agent, or a therapeutic antibody.

98. The method of claim 96 or 97, selected from the group consisting of:

99. 98. The method of claim 96 or 97, wherein the therapeutic agent is a chemotherapeutic agent.

100. The disease comprises an autoimmune disorder, an inflammatory disorder, a skin disorder, a cancer, and a cardiovascular disorder.

100. The method of any one of claims 5 to 99, selected from the group:

101. 100. The method of any one of claims 5 to 99, wherein the disease is cancer.

102. The cancer is brain cancer, breast cancer, cervical cancer, colorectal cancer, lung cancer, lymphoma, melanoma , bladder cancer, renal cell carcinoma, multiple myeloma, pancreatic cancer, and prostate cancer. The method of claim 100 or 101,

103. 102. The method of claim 100 or 101, wherein the cancer is colon cancer.

104. The disease is graft-versus-host disease, rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis. , multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, posterior uveitis, allergies encephalomyelitis, glomerulonephritis, rheumatic fever, post-infectious glomerulonephritis, psoriasis, atopic dermatitis, infectious Tactile dermatitis, eczematous dermatitis, lipophilic dermatitis, lichen planus, pemphigus, bullous pemphigoid, epidermis Blisters, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, lupus erythematosus, acne , alopecia areata, keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's disease, keratitis, Herpetic keratitis, keratoconus, corneal epithelial dysplasia, corneal leukoplakia, ocular pemphigus, Mooren's ulcer, sclerosing keratitis, Ureitis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, pollen allergy Ghee, reversible obstructive airway disease, bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma , dust asthma, chronic or inveterate asthma, late onset asthma and Airway hyperresponsiveness, bronchitis, gastric ulcers, vascular damage caused by ischemic disease and thrombosis , ischemic bowel disease, inflammatory bowel disease, necrotizing enterocolitis, intestinal lesions associated with burns, celiac disease, enteritis, eosinophilic gastroenteritis, mastocytosis, Crohn's disease, ulcerative colitis, migraine, rhinitis, eczema, Interstitial nephritis, Goodpasture's syndrome, hemolytic uremic syndrome, diabetic nephropathy, polymyositis , Guillain-Barre syndrome, Meniere's disease, polyneuritis, m ultimate neuritis), mononeuritis, radiculopathy, hyperthyroidism, Graves' disease, pure red cell aplasia, aplastic anemia (hypop lastic anemia), idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, no Granulocytosis, pernicious anemia, megaloblastic anemia, red blood cell hypoplasia, osteoporosis, sarcoidosis, Pulmonary fibrosis, idiopathic interstitial pneumonia, dermatomyositis, vitiligo, ichthyosis vulgaris, photoallergic hypersensitivity. hypersensitivity, cutaneous T-cell lymphoma, arteriosclerosis, atherosclerosis, aortitis syndrome, Polyarteritis nodosa, cardiomyopathy, scleroderma, Wegener's granulomatosis, Sjogren's syndrome, adiposity , eosinophilic fasciitis, lesions of the gingiva, periodontium, alveolar bone, and dental cementum, glomerulonephritis, male Alopecia areata, senile alopecia due to hair loss, lack of hair germination and / or hair growth and development and decreased hair growth in senile alopecia, muscular dystrophy, pyoderma, and Sézary syndrome. , Addison's disease, ischemia-reperfusion organ damage, transplant disease, ischemic disease, endotoxin shock colitis, pseudomembranous colitis, drug- or radiation-induced colitis, acute ischemic renal failure , chronic renal failure, oxygen lung or drug-induced intoxication, lung cancer, emphysema, cataracts , Siderosis, Retinitis Pigmentosa, Age-Related Macular Degeneration, Vitreous Scarring, Corneal Alkali Burn, Polymorphism Erythematous dermatitis, linear IgA bullous dermatitis and cement dermatitis, gingivitis, periodontitis, sepsis , pancreatitis, aging, carcinogenesis, metastasis of cancer and altitude sickness, histamine or leukotriene C4 Release-related disease, Behcet's disease, autoimmune hepatitis, primary biliary cirrhosis, sclerosing cholangitis, Partial hepatectomy, acute hepatic necrosis, toxin-induced necrosis, viral hepatitis, shock, Anoxia, B viral hepatitis, non-A / non-B hepatitis, liver cirrhosis, alcoholic liver cirrhosis, liver failure, fulminant hepatic failure, delayed hepatic failure, acute exacerbation of chronic hepatitis, cytomegalovirus infection, HCMV infection, AIDS, senile dementia, trauma, chronic bacterial infection, malignant tumors originating from the lymphatic system , acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphocytic lymphoma, and chronic 104. The method according to any one of claims 5 to 103, wherein the tumor is selected from the group consisting of lymphocytic lymphomas. Method of posting.

105. The disease is systemic lupus erythematosus, chronic rheumatoid arthritis, type I diabetes, inflammatory bowel disease , biliary cirrhosis, uveitis, multiple sclerosis, Crohn's disease, ulcerative colitis, bullous pemphigoid Acne, sarcoidosis, psoriasis, autoimmune myositis, Wegener's granulomatosis, ichthyosis, Grave 5. The method according to claim 4, wherein the ophthalmopathy is selected from the group consisting of glaucoma, asthma, scleroderma, and Sjogren's syndrome.

104. The method according to any one of claims 1 to 103.

106. The disease is selected from the group consisting of bone marrow rejection, organ transplant rejection, and graft-versus-host disease.

104. The method of any one of claims 5 to 103,

107. The compound of formula I is ６８ Ga-NOTA-beta A-G-G-I-E-F-D (Compound 1; SEQ ID NO: 9); ６８ A-NOTA-(OC� ２ CH ２ ) ２７ -G-G-G-I-E-F-D(compound 2; SEQ ID NO:10); and ６８ Ga-NOTA-beta A-G-G-IE-P-D (Compound 3; SEQ ID NO: 11) The compound according to any one of claims 1 to 6 and 96 to 106, selected from the group consisting of How to.

108. Formula I: 【Chemistry 11】 or a pharma- ceutically acceptable salt thereof, wherein A comprises one or more imaging agents; B is an optional linking group; C is a group that binds to granzyme B, a) X １ -X ２ -X ３ -X ４ -X ５ -X ６ -D (SEQ ID NO: 1) [In the formula, X １ , X ２ , and X ３ are each independently beta A, G, Q, N, S, T, Y, C, selected from the group consisting of R, D, and E; X ４ is selected from the group consisting of I and V; X ５ is selected from the group consisting of E, G, D, and S; X ６ is selected from the group consisting of E, X, P, S, T, Q, N, A, H, V, F, and D. will be an amino acid sequence having at least 90% sequence identity to b) Formula III: 【Chemistry 12】 or a pharma- ceutically acceptable salt thereof, 、 n is 0, 1, or 2; m is 0, 1, or 2; R １ and R ２ Each independently represents hydrogen, C １～６ Alkyl, C １～６ Alkoxy, C ３～６ Cycloalkyl, C ６～１０ Aryl, HET, and -N(R １０ ) ２ Consists of wherein each C １～６ Alkyl, C １～６ Alkoxy, C ３～６ Cycloal Kyl is independently one, two, or three selected from the group consisting of halo and hydroxy. Optionally substituted by one or more substituents, each C ６～１０ Aryl and HET are independently Oxo, halo, hydroxy, C １～４ Alkyl, and C １～４ The group consisting of haloalkyl optionally substituted with one, two, or three substituents selected from Or R １ and R ２ are each of them together with the carbon atom to which they are attached. Each may optionally have one, two, or three R １０ 5 to 6 optionally substituted by a group It is possible to form a 5- or 6-membered cycloalkyl group or a 5- or 6-membered heterocycloalkyl group. can be; Each R ３ and R ７ are independently hydrogen, C １～４ Alkyl, and C １～４ Haloalkyl selected from the group consisting of: Each R ４ , R ５ , R ６ , and R ８ are independently hydrogen, halo, hydroxy, C １～４ Al Kill, and C １～４ haloalkyl; R ９ are independently oxo, halo, hydroxy, C １～４ Alkyl, and C １～４ Halo Optionally substituted with one, two, or three substituents selected from the group consisting of alkyl, HET which can be exchanged; R １０ is hydrogen, C １～４ Alkyl, and -C(O)C １～４ From the group consisting of alkyl wherein: １～４ Alkyl is optionally represented by -N(R １１ ) ２ , H E.T., and C. ６～１０ substituted by aryl, ６～１０ The aryl is optionally optionally substituted with 1, 2, or 3 halo groups; Each HET is an independently selected monocyclic or bicyclic 5-10 membered heteroaryl. or a monocyclic or bicyclic 5- to 10-membered heterocycloalkyl group, in which In this case, each HET is one, two, three, or four heteroatoms selected from O, S, and N. containing an oxo atom and optionally substituted by one or two oxo groups; R １１ is hydrogen, C １～４ Alkyl, and C １～４ haloalkyl; with the proviso that if m is 0 then n is 0, and if n is 0 then m is 0, or a pharma- ceutically acceptable salt thereof.

109. The compound of formula I is ６８ Ga-NOTA-beta A-G-G-I-E-F-D (Compound 1; SEQ ID NO: 9); ６８ A-NOTA-(OC� ２ CH ２ ) ２７ -G-G-G-I-E-F-D(compound 2; SEQ ID NO:10); and ６８ Ga-NOTA-beta A-G-G-IE-P-D (Compound 3; SEQ ID NO: 11) 109. The compound of claim 108, selected from the group consisting of:

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