Folate receptor-targeted conjugates with brush border membrane enzyme-cleavable linkers and methods of use in cancer imaging and therapy - Patents.com

Abstract

Conjugates of formula (I) FRTL-BBMecL-AA or (II) FRTL-Alb-BBMecL-AA, where FRTL is a folate receptor targeting ligand, BBMecL is a brush border membrane (BBM) enzyme cleavable linker, Alb is an albumin binding moiety, and AA is an active agent; compositions comprising same; and methods for imaging and / or treating tumors.

Description

[Technical field]

[0001] Priority This patent application claims priority benefit of U.S. Provisional Patent Application No. 63 / 188,910, filed May 14, 2021, and U.S. Provisional Patent Application No. 63 / 318,463, filed March 10, 2022. The contents of both of the foregoing applications are incorporated by reference in their entireties into this disclosure.

[0002] The present disclosure relates to conjugates comprising a receptor-targeting ligand, an enzyme-cleavable linker, and an agent for imaging or therapeutically treating a tumor or tumor-associated macrophages. [Background technology]

[0003] The folate receptor (FR) is a proven target for a variety of tumors and activated macrophages in humans. FRs bind folate conjugates with high affinity and are rapidly internalized, which are ideal attributes for receptor-mediated drug delivery.

[0004] Folate ligand targeting conjugates are used in imaging and therapy (e.g., cancer imaging and therapy). The disadvantage of their use is the high and long-lasting renal retention, which makes folate-based radiotherapy unusable due to the risk of damaging the radiosensitive kidney. Fluorescence or radioactive imaging of renal cancer is also ineffective with folate-based conjugates.

[0005] This problem is currently being addressed in a variety of ways: antifolates are administered to block renal uptake, folate ligand structures are modified, or plasma expanders or diuretics are administered to increase urination. Several strategies have already been used to improve the tumor-to-kidney ratio critical for folate-targeted theranostics in preclinical studies. These include pretreatment antifolates (e.g., pemetrexed) and the incorporation of albumin binders, but further optimization is required, especially for imaging and treatment of renal cancer, to enable the application of FR-targeted radiotherapy in human patients. Summary of the Invention [Problem to be solved by the invention]

[0006] In view of the above, the present disclosure seeks to provide a folate ligand targeting conjugate that can be used without further structural modification or administration of other agents to address renal retention. The conjugate can be used to image and treat tumors and tumor associated macrophages while providing improved pharmacokinetics. This and other objects and advantages, as well as additional inventive features, will become apparent from the detailed description provided herein. [Means for solving the problem]

[0007] Conjugates of Formula I or Formula II are provided. FRTL-BBMecL-AA (Formula I) or FRTL-Alb-BBMecL-AA (Formula II) [In the formula, FRTL is a folate receptor targeting ligand, BBMecL is a renal brush border membrane (BBM) enzyme-cleavable linker; Alb is the albumin binding moiety, AA is the activator.]

[0008] FRTL can have a molecular weight of less than 10,000. Alb can be non-covalently associated with serum albumin.

[0009] The FRTL may have the following structure: [ka] [In the formula, T is S, O, NR 4b and -HC=CH-; U, V and W are -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a )- (In the formula, R 6a and R 7a is hydrogen, halo and C1-C 12 alkoxy, or R 6a and R 7a represent a divalent moiety independently selected from the group consisting of: X and Y are halo, R 2 , OR 2 , S.R. 3 and N.R. 4 R 5 are each independently selected from the group consisting of: Q is C or CH; A 1 and A 2 are oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4b )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C12 Alkylene and C1-C 12 Alkyneoxy wherein Z is oxygen or sulfur; R 2 , R 3 , R 4 , R 4a , R 4b , R 5 , R 5b , R 6b and R 7b are hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 Alkoxy)carbonyl and (C1-C 12 each independently selected from the group consisting of: alkylamino)carbonyl; A 3 is an amino acid, R 1 are hydrogen, halo, C1-C 12 Heteroalkyl and C1-C 12 alkoxy; R 6 and R 7 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy, or R 6 and R 7 together form a carbonyl group, q is an integer from 1 to 3; p, r, s and t are each independently 0 or 1; * indicates the binding point with BBMecL or Alb.]

[0010] Q can be CH. X can be OH and Y can be NH2. W and U can be -N(R 4a )-, Q can be CH, V can be CH2, and A 1-N(R 4b )-, s can be 1, p can be 1, and t can be 0. R 4a and R 4b R may be independently alkyl or heteroalkyl. 4a and R 4b may be methyl.

[0011] The FRTL may have the following structure: [ka] [In the formula, [ka] indicates the binding site of FRTL with Alb or BBMecL.]

[0012] Alb may have the following structure: [ka] [In the formula, R 12-19 are independently -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -CN, -CHO, -B(OH)2, -C(O) alkyl, -C(O) aryl-, -C=CC(O) aryl, -C=CS(O) aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, or -SO2F; R 20 and R 21 are independently -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -OC 1-6 Alkyl, -CN, -CHO, -B(OH)2, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F or CF3,

[0013] [ka] JPEG2024518097000007.jpg126122

[0014] , ABD035, ABDCon, designed ankyrin repeat proteins (DARPins), dsFV CA645, nanobodies (single-domain antibodies (sdAbs)), or variable domain of new antigen receptor (VNAR) fused to anti-human serum albumin domain clone E06]

[0015] BBMecL is (i) Met-Val, (ii) X-Lys or X-Arg, where X=Gly or Arg, or a combination thereof; (iii) Gly-Tyr, (iv) Gly-Phe-(Lys), (v) Gly-Pro, (vi) Ala-X or Leu-X, where X is any amino acid, or a combination thereof; (vii) Asp-X or Glu-X, where X is any amino acid, or a combination thereof; (viii) γ-Glu-X, where X is any amino acid, and an acceptor peptide; (ix) sucrose, maltose, trehalose, lactose, palatinose, or a combination of two or more of the foregoing; (x) iodoinsulin B chain, (xi) phlorizin, (xii) p-nitrophenyl phosphate, or (xiii) A combination of two or more of the above may include:

[0016] AA may be an optical imaging agent, a radioactive imaging agent, or a radioactive therapeutic agent. The optical imaging agent may be a fluorescent dye. The fluorescent dye may be selected from the group consisting of S0456, fluorescein isothiocyanate (FITC), rhodamine, LS288, heptamethine cyanine dye (HMCD), SS180, acridine orange (AO), IRDye800CW, IR783, IR825, ZW800-1, or indocyanine green (ICG). When AA is a radioactive imaging agent or a radioactive therapeutic agent, AA may be: 18 F, 44 Sc, 47 Sc, 52 Mn, 55 Co, 64 Cu, 6 7Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, 90 Y, 99m Tc, 111 In, 114m In, 117m Sn, 124 I, 125 I, 131 I, 149 Tb, 153 Sm, 152 Tb, 155 Tb, 161 Tb, 177 Lu, 186 Re, 188 Re, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 Ab, 225 Ac and 227 The radioisotope may be selected from the group consisting of Th.

[0017] A.A. 68 Ga, 18 F, 90 Y,99m Tc, 111 In, 177 Lu, 225 Ac, 18 P, 124 I, 125 I, 131 I and 211 The radiolabeled prosthetic group may comprise a radioisotope selected from the group consisting of At. [ka] wherein R and R' are independently hydrogen or methyl and n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. The radiolabeled prosthetic group may comprise a structure selected from: [ka] wherein n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0018] The AA of the conjugates are DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine), and 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine.6]-eicosane-1,8-diamine), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (2,2'-(7-(4-((2-aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid), HYNIC (6-Hydrazinonicotinic acid) acid), NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl) acetic acid), TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid), HBED (N,N-bis(2-hydroxybenzyl)-ethylenediamine-N,N-diacetic acid), acid), 2,3-HOPO (3-hydroxypyridin-2-one), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid, 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid), DFO (desferrioxamine), DTPA (diethylenetriaminepentaacetic acid), OCTAPA (N,N-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N-diacetic acid), acid), H2macropa(N,N'-bis[(6-carboxy-2-pyridipmethyl]-4,13-diaza-18-crown-6), H2dedpa(1,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane), β-l-diaminopropionic acid, an EC20 head comprising aspartic acid and cysteine, and derivatives of any of the foregoing. AA may be. [ka] JPEG2024518097000011.jpg14395 The composition may include a chelating agent selected from the group consisting of:

[0019] The conjugate may have the following structure: [ka] (BBM1 (MVKC))

[0020] The conjugate may have the following structure: [ka] (BBM2 (MVKGYGKC))

[0021] The conjugate may have the following structure: [ka] (BBM3 (MVK))

[0022] The conjugate may have the following structure: [ka] BBM4(DOTA-MVK(EB-folic acid)-OH)

[0023] The conjugate may have the following structure: [ka] BBM5(DOTA-MVK(IP-folic acid)-OH

[0024] The conjugate may have the following structure: [ka]

[0025] The conjugate may have the following structure: [ka]

[0026] The conjugate may have the following structure: [ka]

[0027] The conjugate may have the following structure: [ka]

[0028] The conjugate may have the following structure: [ka]

[0029] The conjugate may have the following structure: [ka]

[0030] Further provided is a composition comprising the conjugate and a pharma- ceutically acceptable carrier.

[0031] Still further provided is a method of imaging, treating, or imaging and treating a tumor in a subject by targeted radioactivity to cells of the tumor, macrophages associated with the tumor (e.g., tumor-associated macrophages (TAMs)), or both, alone or further in combination with an optical imaging agent, comprising administering to the subject an effective amount of (i) a conjugate or (ii) a composition comprising a conjugate and a pharma- ceutically acceptable carrier. The method may further comprise imaging the tumor. The imaging may be by optical imaging, positron emission tomography (PET), or single photon emission computed tomography (SPECT).

[0032] Also provided is a kit for imaging, treating, or imaging and treating a tumor in a subject. In certain embodiments, the kit comprises at least one dosage unit of the conjugate or composition in a first container and at least one dosage unit of a second active agent or a composition comprising a second active agent. In certain embodiments, at least one dosage unit of the second active agent or a composition comprising a second active agent is contained in a first container. Alternatively, at least one dosage unit of the second active agent or a composition comprising a second active agent is contained in a second container. The kit can also further comprise a means for administering the conjugate or composition (e.g., a syringe, a stent, a cannula, a trocar, etc.). [Brief description of the drawings]

[0033] [Figure 1A] FIG. 13 shows flow cytometry results for validation of folate targeting for OTL38. [Figure 1B] FIG. 1 shows flow cytometry results for validation of folate targeting for BBM1. [Figure 1C] FIG. 1 shows flow cytometry results for validation of folate targeting for BBM2. [Figure 2A] FIG. 13 shows the folate conjugate binding curve for OTL38 (Kd=6.7 nM). [Figure 2B] FIG. 1 shows the folate conjugate binding curve for BBM1 (Kd=12.3 nM). [Figure 2C] FIG. 1 shows the folate conjugate binding curve for BBM2 (Kd=29.6 nM). [Figure 2D] FIG. 1 shows the folate conjugate binding curve for BBM3 (Kd=16.0 nM). [Figure 3A] ~ [Figure 3B]FIG. 3 shows the results of fluorescent scanning of organs from mice that were fed a folate deficient diet for over 3 weeks, injected with 1 nmol of OTL38, BBM1 or BBM2, and sacrificed 24 hours later; FIG. 3A shows the results of short-term exposure, and FIG. 3B shows the results of high-level exposure (the high fluorescence shown in FIG. 3A is further distinguished by the white circle). [Figure 4A] ~ [Figure 4B] FIG. 1 shows the results of fluorescent scanning of organs from mice maintained on a folate deficient diet for over 3 weeks, injected with 1 nmol of OTL38, BBM1 or BBM2 and sacrificed 1 hour later (high fluorescence is further distinguished by white rectangles). [Figure 5A] ~ [Figure 5B] FIG. 1 shows the results of fluorescent scanning of organs from mice maintained on a folate deficient diet for over 3 weeks, injected with 1 nmol of OTL38, BBM1 or BBM2, and sacrificed 3 hours later (high fluorescence is further distinguished by white rectangles). [Figure 6A] ~ [Figure 6B] FIG. 1 shows the results of fluorescent scanning of organs from mice maintained on a folate deficient diet for over 3 weeks, injected with 1 nmol of OTL38, BBM1 or BBM2 and sacrificed 6 hours later (high fluorescence is further distinguished by white rectangles). [Figure 7] FIG. 1 shows the results of fluorescence scanning of mice maintained on a folate deficient diet for over 3 weeks, injected with 1 nmol of OTL38 or BBM3, and scanned for fluorescence 1 hour post-injection (1 h pi) or 3 hours post-injection (3 h pi) (fluorescence is further distinguished by white circles). [Figure 8A] FIG. 1 shows the results of fluorescent scanning of organs from mice maintained on a folate deficient diet for over 3 weeks, injected with 1 nmol of OTL38 or BBM3, and sacrificed 3 hours later (highest fluorescence is further delineated by a black rectangle). [Figure 8B-1] ~ [Figure 8B-2]FIG. 1 shows the results of fluorescent scanning of organs from mice maintained on a folate deficient diet for over 3 weeks, injected with 1 nmol of OTL38, BBM1 or BBM2 and sacrificed 3 hours later (highest fluorescence is further demarcated by a white rectangle). [Figure 9A] FIG. 1 shows the results of scanning mice that were maintained on a folate deficient diet for over 3 weeks, injected with 5 nmol of OTL38 or BBM3, and scanned for fluorescence 1 hour (1 h pi) or 3 hours (3 h pi) after injection (highest fluorescence is further indicated by a black circle). [Figure 9B] FIG. 1 shows the results of scanning mice that were maintained on a folate deficient diet for over 3 weeks, injected with 5 nmol of OTL38 or BBM3, and scanned for fluorescence 6 hours post-injection (6 h pi) or 24 hours post-injection (24 h pi) (highest fluorescence is further indicated by a black circle). [Figure 10] FIG. 1 shows the results of scanning of organs from mice maintained on a folate deficient diet for over 3 weeks, injected with 5 nmol of OTL38 or BBM3, and sacrificed 24 hours later (highest fluorescence is further demarcated by a black rectangle). [Figure 11] FIG. 1 shows the results of organ scanning of athymic nude mice that were maintained on a folate deficient diet for over 3 weeks, injected with 5 nmol of OTL38, BBM1 or BBM2 and sacrificed 24 hours later. [Figure 12] FIG. 1 shows the results of scanning athymic nude mice that were maintained on a folate deficient diet for over 3 weeks, injected with 5 nmol of OTL38 or BBM3 (1-2 mice / conjugate) and scanned for fluorescence in the tumor (MDA-MD-231) versus kidney at 1 hour (1 hour pi), 3 hours (3 hours pi), 6 hours (6 hours pi) and 24 hours (24 hours pi) post-injection (fluorescence is further distinguished by black circles). [Figure 13]FIG. 1 shows the results of scanning athymic nude mice that were maintained on a folate deficient diet for over 3 weeks, injected via the tail vein with 5 nmol of OTL38 or BBM3 (1-2 mice / conjugate), and scanned for fluorescence in the tumor (MDA-MB-231) versus kidney at 1 hour (1 hour pi), 3 hours (3 hours pi), 6 hours (6 hours pi), and 24 hours (24 hours pi) post-injection (fluorescence is further distinguished by black circles). [Figure 14] FIG. 13 shows the results of organ scanning of athymic nude mice that were maintained on a folate deficient diet for over 3 weeks, injected via the tail vein with 5 nmol of OTL38 or BBM3 (1-2 mice / conjugate), and scanned for fluorescence in tumor, heart, lungs, liver, spleen, and kidneys 24 hours after injection. [Figure 15] This is a radiochromatogram of 111In-BBM4. [Figure 16] This is a radio-chromatogram of 111In-BBM5. [Figure 17A] FIG. 1 is a graph of concentration (nM) versus CPM showing binding of Indium-111 radiolabeled BBM4 conjugate in KB cells. [Figure 17B] FIG. 1 is a graph of concentration (nM) versus CPM showing binding of Indium-111 radiolabeled BBM5 conjugate in KB cells. [Figure 18] SPECT / CT images of Indium-111 radiolabeled BBM4 conjugate in a healthy mouse. 1 nmol was injected with approximately 250 uCi of In-111 radiolabeled. Yellow arrow indicates kidney. [Figure 19] SPECT / CT images of Indium-111 radiolabeled BBM5 conjugate in a healthy mouse. 1 nmol was injected with approximately 250 uCi of In-111 radiolabeled. Yellow arrow indicates kidney. [Figure 20]SPECT / CT images of Indium-111 radiolabeled BBM4 conjugate in a healthy mouse. 5 nmol of approximately 250 uCi of In-111 radiolabeled were injected. Yellow arrows indicate kidneys. [Figure 21] This is a radiochromatogram of 177Lu-BBM4. [Figure 22] This is a radiochromatogram of 177Lu-BBM5. [Figure 23A] 1 is a graph of days versus tumor volume (mm3) showing the total volume of KB tumors in mice treated with 177Lu radiolabeled BBM conjugate. [Figure 23B] 1 is a graph of relative tumor size versus days showing the relative size of KB tumors in mice treated with 177Lu radiolabeled BBM conjugate. [Figure 23C] 1 is a graph of days versus relative body weight (%) showing relative body weight of mice bearing KB tumors and treated with 177Lu radiolabeled BBM conjugate. [Figure 24] 1 shows SPECT / CT images of a mouse treated with 177Lu radiolabeled BBM4 conjugate. 5 nmol of approximately 500 uCi of Lu-177 radiolabeled was injected. White arrow indicates tumor, yellow arrow indicates kidney. [Diagram 25] 1 shows SPECT / CT images of a mouse treated with 177Lu radiolabeled BBM5 conjugate. 5 nmol of approximately 500 uCi of Lu-177 radiolabeled was injected. White arrow indicates tumor, yellow arrow indicates kidney. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of scope is intended by the description of these embodiments. On the contrary, the disclosure is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the disclosure.

[0035] The present disclosure is based, at least in part, on the discovery that the use of renal brush border membrane (BBM) enzyme substrates to link folate receptor targeting ligands with imaging or radiotherapy agents allows for rapid clearance of the conjugates from the kidney. Rapid clearance allows for renal cancer imaging and minimizes nephrotoxicity in radiotherapy of multiple tumors. In addition to rapid clearance from the kidney, the conjugates allow for specific targeting, high tumor penetration, and rapid clearance from receptor-negative tissues. Furthermore, since folate is expressed in cancer-associated macrophages of many solid tumors, the conjugates can be used to treat the stroma of many types of cancer.

[0036] Conjugates of Formula I or Formula II are provided. FRTL-BBMecL-AA (Formula I) or FRTL-Alb-BBMecL-AA (Formula II) [In the formula, FRTL is a folate receptor targeting ligand, BBMecL is a renal brush border membrane (BBM) enzyme cleavable linker; Alb is the albumin binding moiety, AA is the activator.] In certain embodiments, the FRTL has a molecular weight of less than 10,000. Alb can non-covalently associate with serum albumin.

[0037] The FRTL may have the following structure: [ka] During the ceremony, T is S, O, NR 4b and -HC=CH-; U, V and W are -(R 6a )C=, -N=, -(R 6a )C(R 7a )- and -N(R 4a )-, In the formula, R 6a and R 7a is hydrogen, halo and C1-C 12 alkoxy, or R 6a and R 7a together form a carbonyl group, X and Y are halo, R 2 , OR 2 , S.R. 3 and N.R. 4 R 5 are each independently selected from the group consisting of: Q is C or CH; A 1 and A 2 are oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R 4b )-, -C(Z)N(R 4b )-, -N(R 4b )C(Z)-, -OC(Z)N(R 4b )-, -N(R 4b )C(Z)O-, -N(R 4b )C(Z)N(R 5b )-, -S(O)-, -S(O)2-, -N(R 4b )S(O)2-, -C(R 6b )(R 7b )-, -N(C≡CH)-, -N(CH2C≡CH)-, C1-C 12 Alkylene and C1-C 12 alkyleneoxy, wherein Z is oxygen or sulfur; R 2 , R 3 , R 4 , R4a , R 4b , R 5 , R 5b , R 6b and R 7b are hydrogen, halo, C1-C 12 Alkyl, C1-C 12 Alkoxy, C1-C 12 Alkanoyl, C1-C 12 Alkenyl, C1-C 12 Alkynyl, (C1-C 12 Alkoxy)carbonyl and (C1-C 12 each independently selected from the group consisting of: alkylamino)carbonyl; A 3 is an amino acid, R 1 are hydrogen, halo, C1-C 12 Heteroalkyl and C1-C 12 alkoxy; R 6 and R 7 are hydrogen, halo, C1-C 12 Alkyl and C1-C 12 alkoxy, or R 6 and R 7 together form a carbonyl group, q is an integer from 1 to 3; p, r, s and t are each independently 0 or 1; * indicates the binding point with BBMecL or Alb.

[0038] "C1-C 12 "Alkyl" refers to a straight, branched or cyclic hydrocarbon chain containing 1 to 12 carbon atoms. 12Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, iso-propyl, cyclopropyl, butyl, iso-butyl, tert-butyl, sec-butyl, cyclobutyl, pentyl, cyclopentyl, iso-pentyl, neopentyl, hexyl, cyclohexyl, iso-hexyl, neohexyl, heptyl, cycloheptyl, iso-heptyl, neoheptyl, octyl, cyclooctyl, iso-octyl, neooctyl, nonyl, cyclononyl, iso-nonyl, neononyl, decyl, cyclodecyl, iso-decyl, neodecyl, undecyl, cycloundecyl, iso-undecyl, neoundecyl, dodecyl, cyclododecyl, iso-dodecyl, and neododecyl.

[0039] "C1-C 12 "Alkoxy" is a C1-C alkyl group with a single bond to oxygen. 12 It is an alkyl.

[0040] "C1-C 12 "Alkanoyl" is a C1-C alkyl group that is single-bonded to a carbonyl. 12 It is an alkyl.

[0041] "C1-C 12 Alkenyl is a C1-C alkyl group, including C=C. 12 It is an alkyl.

[0042] "C1-C 12 Alkynyl is a C1-C alkyl group, including C≡C. 12 It is an alkyl.

[0043] "(C1-C 12 "Alkoxy)carbonyl" refers to a C1-C bonded to a carbonyl. 12 It is an alkoxy.

[0044] "(C1-C 12 "Alkylamino)carbonyl" refers to a C1-C alkylamino group bonded to a carbonyl. 12 Alkylamino (i.e., C1-C bonded to amino 12 alkyl).

[0045] "C1-C 12 "Heteroalkyl" refers to a C-C alkyl group that contains at least one heteroatom (i.e., an atom other than carbon or hydrogen). 12 It is an alkyl.

[0046] "Halogen" and "halo" refer to fluorine, chlorine, iodine or bromine.

[0047] In certain embodiments, Q is CH. In certain embodiments, X is OH and Y is NH. In certain embodiments, W and U are -N(R 4a )-, Q is CH, V is CH2, and A 1 -N(R 4b )-, s is 1, p is 1, and t is 0 (e.g., A 1 is directly attached to the heterocycle. 4a and R 4b are each independently alkyl or heteroalkyl. In certain embodiments, R 4a and R 4b are each methyl.

[0048] The FRTL may have the following structure: [ka]

[0049] [In the formula, [ka] indicates the binding site of FRTL with Alb or BBMecL.]

[0050] Alb may have the following structure: [ka] [In the formula, [ka] indicates the binding site of Alb with FRTL, R 12-19 are independently -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -CN, -CHO, -B(OH)2, -C(O) alkyl, -C(O) aryl-, -C=CC(O) aryl, -C=CS(O) aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, or -SO2F; R 20 and R 21 are independently -H, -C1-C6 alkyl, -F, -Cl, -Br, -I, -OC 1-6 Alkyl, -CN, -CHO, -B(OH)2, -C=CC(O)aryl, -C=CS(O)2aryl, -CO2H, -SO3H, -SO2NH2, -PO3H2, -SO2F or CF3, [ka] JPEG2024518097000029.jpg126122 , ABD035 (Jonsson et al., Protein Eng. Des. Sel. 21, 515-527. doi: 10.1093 / protein / gzn028 (2008)), ABDCon (Jacobs et al., Protein Eng Des Sel 28(10): 385-393 (Oct 2015), designed ankyrin repeat proteins (DARPins), dsFV CA645 (Zorzi et al., Med Chem Comm 10: 1068-1081 (2019)), nanobodies (single domain antibodies (sdAbs)), or the variable domain of a novel antigen receptor (VNAR) fused to the anti-human serum albumin domain clone E06 (Barelle et al., Antibodies 4(3): 240-258 (2015)).

[0051] The BBMecL of the conjugate may be any suitable BBM enzyme-cleavable substrate (e.g., a linker). Examples include those shown in Table 1, although it will be appreciated that the BBMecL may include any suitable BBM enzyme-cleavable substrate now known or later developed.

[0052] [Table 1]

[0053] In certain embodiments, the BBMecL of the conjugate is (i) methionine-valine (Met-Val), (ii) X-Lys or X-Arg, or a combination thereof, where X is glycine (Gly) or arginine (Arg); (iii) glycine-tyrosine (Gly-Tyr), (iv) Glycine-phenylalanine-(lysine) (Gly-Phe-(Lys)); (v) glycine-proline (Gly-Pro), (vi) alanine-X (Ala-X) or leucine-X (Leu-X), or a combination thereof, where X is any amino acid; (vii) aspartic acid-X (Asp-X) or glutamic acid-X (Glu-X), or a combination thereof, where X is any amino acid; (viii) γ-glutamic acid-X (γ-Glu-X), where X is any amino acid, and an acceptor peptide; (ix) sucrose, maltose, trehalose, lactose, palatinose, or a combination of two or more of the foregoing; (x) iodoinsulin B chain, (xi) phlorizin, (xii) p-nitrophenyl phosphate, or (xiii) A combination of two or more of the above Includes.

[0054] In certain embodiments, the BBMecL of the conjugate comprises a kidney BBM cleavable substrate / linker. In certain embodiments, the BBM cleavable linker of the BBMecL is linked to at least AA of the conjugate and FRTL (e.g., the conjugate of formula I). ​​In certain embodiments, the BBM cleavable linker of the BBMecL is linked to at least AA of the conjugate and Alb (e.g., the conjugate of formula II). When such an embodiment of the conjugate is administered to a subject (e.g., systemically), the BBM linker can be cleaved, releasing AA from the remainder of the conjugate (e.g., FRTL and / or Alb). Thus, even if a portion of the conjugate can bind to a folate receptor (e.g., FRα) in the brush border membrane - e.g., in the kidney of a subject - the remainder of the conjugate is released and therefore is not taken up or retained in an organ (e.g., kidney).

[0055] The BBM linker, in certain embodiments, may include one or more unnatural amino acids (UAA), including, but not limited to, one or more of the D-amino acids, citrulline, hydroxyproline, norleucine, 3-nitrotyrosine, nitroarginine, naphthylalanine, aminobutyric acid (Abu), 2,4-diaminobutyric acid (DAB), methionine sulfoxide, methionine sulfone, and the like.

[0056] It should be appreciated that such physiological conditions that result in BBM cleavable linker rupture include standard chemical hydrolysis reactions that occur, for example, at physiological pH or as a result of compartmentalization into cellular organelles such as endosomes that have a pH lower than the cytoplasmic pH. By way of illustration, the BBM cleavable linkers described herein can undergo cleavage under other physiological or metabolic conditions.

[0057] The AA may be an optical imaging agent, a radioimaging agent, or a radiotherapeutic agent.

[0058] In certain embodiments, the AA is an optical imaging agent. The optical imaging agent can be any compound (or radical thereof) that emits a detectable signal (e.g., an electromagnetic signal (e.g., radio signal, fluorescent signal, gamma ray) or mass). Examples of optical imaging agents include, but are not limited to, a radio-imaging agent (e.g., a positron emission tomography (PET) imaging agent or a single photon emission computed tomography (SPECT) imaging agent), a fluorescent imaging agent (e.g., a fluorescent dye), and the like. The imaging agent can be a magnetic resonance (MR) agent. In some embodiments, the AA includes a radiolabel functional group (e.g., a radical thereof) suitable for PET imaging, SPECT imaging, other radioimaging techniques, magnetic resonance imaging, or radiotherapy. The AA can include a radical of a radioimaging, radiotherapeutic, or magnetic resonance isotope.

[0059] In certain embodiments, the optical imaging agent is an optical imaging agent comprising a fluorescent dye, which may be selected from the group consisting of S0456, fluorescein isothiocyanate (FITC), rhodamine, LS288, heptamethine cyanine dye (HMCD), SS180, acridine orange (AO), IRDye dye 800CW, IR783, IR825, ZW800-1, or indocyanine green (ICG).

[0060] In certain embodiments, AA is 18 F, 44 Sc, 47 Sc, 52 Mn, 55 Co, 64 Cu, 6 7Cu, 67 Ga, 68 Ga, 86 Y, 89 Zr, 90 Y,99m Tc, 111 In, 114m In, 117m Sn, 124 I, 125 I, 131 I, 149 Tb, 153 Sm, 152 Tb, 155 Tb, 161 Tb, 177 Lu, 186 Re, 188 Re, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 224 Ra, 225 Ab, 225 Ac and 227 A radioactive imaging or radiotherapeutic agent is a radioisotope selected from the group consisting of Th.

[0061] In certain embodiments, AA is 68 Ga, 18 F, 90 Y, 99m Tc, 111 In, 177 Lu, 225 Ac, 18 P, 124 I, 125 I, 131 I and 211 The radiolabeled prosthetic group includes a radioisotope selected from the group consisting of At. [ka] wherein R and R' are independently hydrogen or methyl and n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. The radiolabeled prosthetic group may comprise a structure selected from: [ka] wherein n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0062] AA may be a chelating group (e.g., a chelator (or radical thereof)). A "chelating group" refers to a multidentate chemical group that can bind to a central metal atom with multiple binding interactions by using two or more binding sites in the chelating group. The combination of a chelating group and a metal atom is a chelate. The binding of the chelating group to the metal atom can be through non-covalent interactions or bonds, and in some embodiments, the binding of the chelating group to the metal atom is through multiple coordinate bonds.

[0063] AA is a group consisting of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine, NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (2,2'-(7-(4-((2-aminoethyl) (N,N-bis(2-hydroxybenzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid), HYNIC (6-hydrazinonicotinic acid), NETA (4-[2-(bis-carboxymethylamino)-ethyl]-7-carboxymethyl-[1,4,7]triazonane-1-yl)acetic acid, TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid), HBED (N,N-bis(2-hydroxybenzyl)-1,4,7-triazonane-1,4-diyl)diacetic acid), amine-N,N-diacetic acid), 2,3-HOPO (3-hydroxypyridin-2-one), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid), DFO (desferrioxamine), DTPA (diethylenetriaminepentaacetic acid), OCTAPA (N,N-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N-diacetic acid), H2macropa (N,N'-bis[(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N-diacetic acid), AA may comprise a chelating group or agent selected from the group consisting of EC20 heads including 1,2-[[carboxy]-pyridylmethyl-4,13-diaza-18-crown-6), H2dedpa (1,2-[[carboxy]-pyridin-2-yl]-methylamino]ethane, β-1-diaminopropionic acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA) and cysteine, and derivatives of any of the foregoing. In certain embodiments, AA may comprise a radical of any of the foregoing chelators.

[0064] In certain embodiments, AA may be or include the radical of a group covalently bonded to an isotope (or metal) suitable for radioimaging, radiotherapy, or magnetic resonance imaging.

[0065] Representative chelating groups include: [ka] JPEG2024518097000034.jpg14395 (including the free base thereof, such as when one or more protons (H+) of COH(COOH) are removed to form COO-). In certain embodiments, each chelator may be coupled to an isotope (or metal) suitable for radioimaging, radiotherapy, or MR imaging.

[0066] Conjugates / compounds In some embodiments, the conjugate (eg, compound) has the structure: [ka] (BBM1 (MVKC))

[0067] In some embodiments, the conjugate has the structure: [ka] (BBM2 (MVKGYGKC))

[0068] In some embodiments, the conjugate may have the following structure: [ka] (BBM3 (MVK))

[0069] The conjugate may have the following structure: [ka] BBM4(DOTA-MVK(EB-folic acid)-OH)

[0070] The conjugate may have the following structure: [ka] BBM5(DOTA-MVK(IP-folic acid)-OH

[0071] The conjugate may have the following structure: [ka]

[0072] The conjugate may have the following structure: [ka]

[0073] The conjugate may have the following structure: [ka]

[0074] The conjugate may have the following structure: [ka]

[0075] The conjugate may have the following structure: [ka]

[0076] The conjugate may have the following structure: [ka]

[0077] The conjugate may contain one or more chiral centers or may otherwise exist as multiple stereoisomers, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. Unless otherwise specified, all stereoisomeric forms of the compound are intended to be contemplated. If the conjugate contains an alkene double bond, it is intended to include both E and Z geometric isomers (e.g., cis or trans), unless otherwise specified. Similarly, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are intended to be included.

[0078] The conjugates can exist as geometric isomers. The term "geometric isomer" refers to the E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. One of ordinary skill in the art will further appreciate that a compound can be "deuterated," meaning that one or more hydrogen atoms can be replaced with deuterium.

[0079] The conjugates can exist in unsolvated and solvated forms, including hydrated forms. In general, the solvated forms are equivalent to the unsolvated forms. The conjugates can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the intended use. The formula includes pharma-ceutically acceptable salts (e.g., acid addition and base salts), hydrates and / or solvates.

[0080] Compositions, routes of administration and dosages In certain embodiments, the pharmaceutical composition comprises a plurality of conjugates and a pharma- ceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" means a natural or synthetic organic or inorganic component with which the active ingredient is combined to facilitate application. A carrier may be an excipient. The selection of a carrier may depend on factors such as the particular mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form. Pharmaceutical compositions suitable for delivery of the conjugates as described herein and methods for their preparation may be found, for example, in Remington: The Science & Practice of Pharmacy, 21st edition (Lippincott Williams & Wilkins, 2005).

[0081] The components of the compositions are capable of being commingled with the compounds, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

[0082] The compositions can be prepared by combining one or more conjugates with a pharma- ceutically acceptable carrier, and optionally with one or more additional ingredients. The formulations can be administered in pharma- ceutically acceptable solutions, which may routinely contain pharma- ceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and may contain other therapeutic ingredients.

[0083] The conjugates can be administered as is (undiluted) or in the form of a pharma- ceutically acceptable salt, and may contain one or more other therapeutic agents. When used in medicine, the salt should be pharma- ceutically acceptable, but pharma-ceutically unacceptable salts may be conveniently used to prepare pharma-ceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts may also be prepared as alkali metal or alkaline earth salts, such as sodium, potassium, or calcium salts of the carboxylic acid group.

[0084] Suitable acid addition salts are formed from acids which form non-toxic salts. Illustrative examples include, but are not limited to, acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, sugarate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0085] Suitable base salts of the conjugates described herein are formed from bases that form non-toxic salts. Illustrative examples include, but are not limited to, arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, olamine salts, potassium salts, sodium tromethamine salts and zinc salts. Hemi-salts of acids and bases, such as hemisulfate salts and hemicalcium salts, can also be formed.

[0086] Compositions and / or dosage forms for administration may be prepared from the conjugate of at least about 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% purity. Compositions and / or dosage forms for administration may be prepared from the conjugate of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% purity.

[0087] Pharmaceutically acceptable carriers may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., and combinations thereof that are physiologically compatible. The carrier may be suitable for parenteral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Examples of such carriers (or excipients) include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. One or more other active agents may also be incorporated into the composition.

[0088] The composition can be formulated as a liquid, for example as a suspension or solution. Liquid formulations can contain water, ethanol, polyethylene glycol, propylene glycol, methylcellulose or suitable oil, and one or more emulsifying agents and / or suspending agents. Liquid formulations can be prepared by the reconstitution of a solid.

[0089] Pharmaceutical formulations (e.g., for parenteral administration) include aqueous solutions of the active compound in water-soluble form. In addition, suspensions of the active conjugate and / or compound may be prepared as appropriate oily injection suspensions. Aqueous suspensions may contain the conjugate, alone or further combined with one or more other active agents, in admixture with a suitable excipient. Excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as natural phospholipids, e.g., lecithin; condensation products of alkylene oxides with fatty acids, e.g., polyoxyethylene stearate; condensation products of ethylene oxide with long-chain aliphatic alcohols, e.g., heptadecaethyleneoxcycetanol; condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, e.g., polyoxyethylene sorbitol monooleate; or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, e.g., polyoxyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, e.g., ascorbic acid or ethyl, n-propyl or p-hydroxybenzoate, and one or more coloring agents. In certain embodiments, aqueous suspensions may further comprise a suitable lipophilic solvent or vehicle, including fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. The suspension may also contain suitable stabilizers or agents to increase the solubility of the conjugate and allow for the preparation of highly concentrated solutions.

[0090] Alternatively, the composition may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use. Dispersible powders and granules suitable for preparation of an aqueous suspension by adding water can provide the active ingredient in admixture with a suspending agent, dispersing or wetting agent, and one or more preservatives. Additional excipients, such as coloring agents, can also be present.

[0091] Suitable emulsifiers include natural gums such as gum acacia or gum tragacanth; natural phospholipids such as soybean lecithin; and partial esters derived from fatty acids and hexitol anhydrides, including sorbitan monooleate, and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, may be included in the composition. Prolonged absorption of an injectable composition may be achieved by including one or more agents that delay absorption, such as monostearate salts and gelatin, in the composition.

[0092] For use in therapy or treatment, an effective amount of the conjugate or composition can be administered to a subject by any mode that delivers the compound to the desired surface. Administering the composition can be accomplished by any means known to those skilled in the art. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., into a tumor or abscess), mucosal (e.g., topical to the eye), inhalation, and topical.

[0093] For oral administration, the conjugates can be easily formulated by combining the conjugates with pharma- ceutically acceptable carriers well known in the art. Such carriers can allow the conjugates to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipients, and the resulting mixture can be milled and, after adding suitable auxiliaries if desired, the mixture of granules can be processed to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and / or polyvinyl pyrrolidone (PVP, polyvinyl pyrrolidone). If desired, disintegrating agents can be added, such as the cross-linked PVP, agar, or alginic acid or a salt thereof, such as sodium alginate. Oral preparations may be formulated in saline or buffers, such as EDTA for neutralizing internal acid conditions, or may be administered without any carrier.

[0094] Oral dosage forms of the compounds are also contemplated. The conjugates may be chemically modified so that oral delivery of the derivatives is effective. In general, the contemplated chemical modifications are the attachment of at least one moiety to the compound itself, which (a) inhibits acid hydrolysis, and (b) allows uptake from the stomach or intestines into the bloodstream. Additionally or alternatively, the conjugates may be modified to increase their overall stability and circulation time in the body. Examples of moieties that may be used to increase stability and / or circulation time include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, PVP, and polyproline. See, for example, Abuchowski and Davis, "Soluble Polymer-Enzyme Adducts," In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383 (1981); Newmark et al., J Appl Biochem 4:185-189 (1982). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. For pharmaceutical uses, as indicated above, polyethylene glycol moieties are appropriate.

[0095] Colorants and / or flavoring agents may be included. For example, the compounds may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage, containing colorants and flavoring agents.

[0096] Illustrative forms for oral administration include, but are not limited to, tablets, capsules, elixirs, syrups, and the like.

[0097] In certain embodiments, the conjugates may be administered directly into the bloodstream, intramuscularly, or into internal organs. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, intranasal, and subcutaneous. Suitable means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and injection techniques. In embodiments where it is desired to deliver the conjugates and / or compositions systemically, the conjugates and / or compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form, e.g., in ampoules or multi-dose containers with added preservatives. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing, and / or dispersing agents.

[0098] Parenteral formulations are typically aqueous solutions which may contain carriers or excipients such as salts, carbohydrates and buffering agents (preferably at a pH of 3 to 9), although for some applications they may be more suitably formulated as sterile non-aqueous solutions or as a dry form for use in combination with a suitable vehicle, such as sterile pyrogen-free water.

[0099] The liquid formulation may be adapted for parenteral administration of the conjugate. Preparation of parenteral formulations under sterile conditions, for example by lyophilization under sterile conditions, may be easily accomplished using standard pharmaceutical techniques well known to those skilled in the art. The solubility of the conjugate may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility enhancers.

[0100] Formulations for parenteral administration may be formulated for immediate and / or modified release. The conjugate may be administered in a sustained release formulation, for example in a composition including a slow release polymer. The conjugate may be prepared with a carrier that will protect it against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic, polyglycolic copolymer (PGLA). Methods for the preparation of such formulations are generally known to those skilled in the art.

[0101] Sterile injectable solutions can be prepared by incorporating the conjugate alone or in combination with one or more other active agents in the required amount in a suitable solvent, with one or combination of the above-mentioned components as required, followed by sterilization by filtration.Typically, dispersions are prepared by incorporating the conjugate into a sterile medium, which contains a dispersion medium and any additional components described above.In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which obtain a powder of the active ingredient plus any additional desired ingredient from a solution that has been previously sterilized and filtered, or the ingredients are sterilized and filtered together.

[0102] The composition can be formulated as a liquid, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium, for example, containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants.

[0103] The conjugate, or a composition containing the conjugate, may be administered continuously, where appropriate.

[0104] kit Kits are also provided. When more than one conjugate is administered or when the conjugate is administered simultaneously or sequentially (in any order) with one or more other active agents, the conjugate and the active agent (or a composition comprising them) can be combined in a kit. In at least one embodiment, the kit comprises at least one dosage unit of the conjugate or a composition comprising the conjugate. In at least one embodiment, the kit comprises at least one dosage unit of a second active agent or a composition comprising a second active agent. "Dosage unit" refers to a composition or conjugate administered in one administration by one delivery operation. For example, in an embodiment where the composition is formulated for transmucosal administration by nasal delivery, the dosage unit is the volume of the composition administered or the amount of active agent administered by one delivery operation.

[0105] The conjugate and active agent may be in solid or liquid form and in the same or separate containers, e.g., vials, divided bottles, divided foil packets, etc. The kit may also contain instructions for use. The instructions may be printed on paper, supplied in electronically readable media, or accessed over the internet via email, text message, social media, website, etc. In certain embodiments, the kit further comprises a means for administration of the conjugate and / or active agent during treatment. Such administration means may include, for example, a syringe, a tourniquet, a stent, a cannula, and / or a trocar.

[0106] Imaging and / or Therapeutic Methods Also provided are methods of imaging, treating, or imaging and treating a tumor (e.g., cancer) in a subject by targeted radioactivity to cells of the tumor, tumor-associated macrophages (tumor-associated macrophages (TAMs)), or both, alone or in further combination with an optical imaging agent. In certain embodiments, the method comprises administering to the subject an effective amount of the conjugate, or a composition comprising the conjugate and a pharma- ceutically acceptable carrier. The method further comprises imaging the tumor. The imaging may be by optical imaging, PET, or SPECT.

[0107] In certain embodiments, a method of imaging, treating, or imaging and treating a tumor (e.g., cancer) in a subject includes contacting tumor cells (e.g., of a cancer patient) with a compound (e.g., a conjugate) of any of the formulas provided herein.

[0108] Thus, a method is provided for imaging a cancer (e.g., a tumor) in a patient with cancer. In certain embodiments, the method comprises administering to the subject (e.g., as part of a pharmaceutical composition or otherwise) an effective amount of a conjugate. In certain embodiments, the method further comprises imaging the subject. In certain embodiments, the method further comprises generating an image of the cancer (e.g., a tumor) in the subject (e.g., after or concurrently with administration of the conjugate).

[0109] Also provided is a method for optical imaging. In certain embodiments, the method for optical imaging of a subject comprises administering to the subject an effective amount of any of the conjugates (e.g., as part of a pharmaceutical composition or otherwise). The method may be, for example, for fluorescence-guided surgery. The method may be for radioimaging. The method may be for MRI.

[0110] The methods may be used in combination with one or more additional therapies and / or active agents, including, but not limited to, immunotherapy, administration of DNA damage response pathway inhibitors, chemotherapy, and / or surgery.

[0111] An "effective amount" refers to the amount of a conjugate or a composition comprising the same that induces the desired biological or pharmaceutical response in a subject (i.e., a tissue, organ, or organism, e.g., a vertebrate, e.g., a mammal, such as a human) desired by a researcher, veterinarian, physician, or other clinician, including, but not limited to, imaging and / or alleviation of signs and / or symptoms of the disease or disorder being treated. In one aspect, an effective amount is an amount of active agent that can treat or alleviate signs and / or symptoms of a disease at a reasonable benefit / risk ratio applicable to any medical treatment. An effective amount of a prodrug is an amount of an inactive prodrug that, when converted via normal metabolic processes, produces an amount of active drug that induces the desired biological or pharmaceutical response being sought in a subject. With respect to therapeutic use, an "effective amount" refers to the amount of compound in a preparation that, when administered (e.g., to a mammal such as a human) as part of a desired dosage regimen, alleviates the symptoms, ameliorates the condition, or slows the onset of the disease, according to clinically accepted criteria for the disorder or condition being treated, or for cosmetic purposes, e.g., with a reasonable benefit / risk ratio applicable to any medical treatment.

[0112] By selecting among the various active agents and weighting factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and mode of administration, in combination with the teachings provided herein, an effective protective or therapeutic treatment regimen can be designed that does not cause substantial undesirable toxicity, yet is effective to treat a particular subject.

[0113] A wide range of acceptable dosages is contemplated herein, including doses falling within the range of about 1 μg / kg to about 1 g / kg, depending on the type of cancer, the route of administration, and / or whether the conjugate is administered locally or systemically. The unit daily dosage may vary significantly depending on the condition of the patient, the cancer being treated, the route of administration, tissue distribution, and the possible co-administration of other therapeutic treatments, such as radiation therapy or additional drugs in combination therapy. The effective amount administered to the patient is based on body surface area, mass, and an assessment of the patient's condition by a physician. Therapeutically effective doses (also referred to herein as "therapeutically effective amounts" or "effective amounts") range from, for example, approximately 0.5 to 20.0 mg / m 2 can range from

[0114] For any conjugate, the effective amount may be determined first from an animal model. The effective dose may also be determined from human data for conjugates / compounds known to exhibit similar pharmacological activity, such as the compound and other related active agents tested in humans. Higher doses may be required for parenteral administration. The effective amount for any particular application may vary depending on factors such as the disease or condition being treated, the particular conjugate being administered, the size of the subject, or the severity of the disease or condition. Those skilled in the art may empirically determine the effective amount of a particular compound and / or other therapeutic agent without undue experimentation. A maximum dose, i.e., the highest safe dose subject to some medical judgment, may be used. Multiple doses per day may be used to achieve an appropriate systemic level of the compound. An appropriate systemic level may be determined, for example, by measuring the patient's peak or sustained plasma levels of the drug. "Dose" and "administration" are used interchangeably herein.

[0115] It is well within the ability of a person skilled in the art to adjust the dose to achieve maximum efficacy based on the methods described and other methods known in the art. The dose can be appropriately adjusted to achieve the desired local or systemic drug levels depending on the mode of administration. For example, the dose for intravenous administration can vary from one to several orders of magnitude lower per day. If the response in the subject is insufficient at such doses, higher doses (or effective higher doses by different more localized delivery routes) can be used to the extent that patient tolerance allows. Multiple doses per day are contemplated to achieve adequate systemic levels of the compound.

[0116] Any effective regimen for administering the conjugate may be used. Doses may be single or divided and may be administered according to a wide variety of protocols, including qd, bid, tid, or even every other day, biweekly (biw), weekly, monthly, quarterly, etc. In each of these cases, it is understood that the effective amounts described herein correspond to the dosage example, or alternatively, to the total daily, weekly, monthly, or quarterly dose as determined by the dosing protocol.

[0117] For example, the conjugate can be administered as a single dose, or the dose can be divided and administered as a multiple daily dose regimen. Additionally, a staggered regimen, e.g., 1-5 days per week, can be used as an alternative to daily treatment. Such intermittent or staggered daily regimens are considered equivalent to daily treatment. A patient can be treated with multiple injections of the conjugate to treat cancer. A patient can be injected multiple times (e.g., approximately 2-50 times) with the conjugate, e.g., at 12-72 hour intervals or 48-72 hour intervals. Additional injections of the conjugate can be administered to the patient at intervals of days or months after the first injection, and the additional injections can prevent recurrence of the cancer.

[0118] Alternatively, individual doses and dosage regimens may be selected to provide, for example, a total dose of about 15 mg administered over a one-month period. The conjugate may be administered as a single daily dose 5 days per week in weeks 1, 2, and 3, with no administration in week 4, in a four-week cycle. In yet another alternative, the conjugate may be administered as a single daily dose 3 days per week in weeks 1 and 3, with no administration in weeks 2 and 4, in each four-week cycle. In yet another alternative, the conjugate may be administered 2 days per week in weeks 1 and 2 (i.e., days 1, 4, 8, and 11 of a three-week cycle). As a further alternative, the conjugate may be administered once per week in weeks 1 and 2 (i.e., days 1 and 8 of a three-week cycle).

[0119] A "subject" may be a human patient; a laboratory animal such as a rodent (e.g., a mouse, rat, or hamster), rabbit, monkey, chimpanzee, etc.; a domestic animal such as a dog, cat, or rabbit; an agricultural animal such as a cow, horse, pig, sheep, or goat; or a captive wild animal such as a bear, panda, lion, tiger, leopard, elephant, zebra, giraffe, gorilla, dolphin, or whale.

[0120] Cancer may be a population of cancer cells that is tumorigenic or non-tumorigenic, including benign and malignant tumors. Cancer can arise spontaneously by germline or somatic mutations, or cancer can be chemically, virally, or radiation induced. Cancer includes, but is not limited to, carcinoma, sarcoma, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, leukemia, adenocarcinoma, and myeloma. Cancers include lung cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, cutaneous melanoma, intraocular melanoma, uterine cancer, ovarian cancer, endometrial cancer, leiomyosarcoma, rectal cancer, stomach cancer, colon cancer, breast cancer, triple-negative breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, non-small cell lung cancer, small cell lung cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic leukemia, acute leukemia, lymphocytic lymphoma, pleural mesothelioma, bladder cancer, Burkitt's lymphoma, ureter cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, and central nervous system (CNS) cancer. The tumor may be a neoplasm of the gastroesophageal junction (GEJ), a primary CNS lymphoma, a spinal axis tumor, a brain stem glioma, a pituitary adenoma, a cholangiocarcinoma, a Hürthle cell thyroid carcinoma, or an adenocarcinoma of the gastroesophageal junction.

[0121] Other definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of chemistry and biology.In addition, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.Thus, for example, if a compound / composition is substituted with "an" alkyl or aryl, the compound / composition may be substituted with at least one alkyl and / or at least one aryl.

[0122] "Oxo" refers to the radical =O.

[0123] "Alkyl" generally has 1 to 15 carbon atoms (e.g., C1-C 15"Alkyl" refers to a straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms, such as aryl, ... 13 The alkyl may contain 1 to 8 carbon atoms (e.g., C1-C8 alkyl). The alkyl may contain 1 to 5 carbon atoms (e.g., C1-C5 alkyl). The alkyl may contain 1 to 4 carbon atoms (e.g., C1-C4 alkyl). The alkyl may contain 1 to 3 carbon atoms (e.g., C1-C3 alkyl). The alkyl may contain 1 to 2 carbon atoms (e.g., C1-C2 alkyl). The alkyl may contain 1 carbon atom (e.g., C1 alkyl). The alkyl may contain 5 to 15 carbon atoms (e.g., C5-C 15 alkyl). The alkyl may contain 5 to 8 carbon atoms (e.g., C5-C8 alkyl). The alkyl may contain 2 to 5 carbon atoms (e.g., C2-C5 alkyl). The alkyl may contain 3 to 5 carbon atoms (e.g., C3-C5 alkyl). In other embodiments, the alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), 1-pentyl (n-pentyl). The alkyl is attached to the remainder of the molecule by a single bond.

[0124] "Alkoxy" refers to a radical attached via an oxygen atom of the formula --O-alkyl, where alkyl is an alkyl chain as defined above.

[0125] "Alkylene" or "alkylene chain" generally refers to a straight or branched divalent alkyl group having from 1 to 12 carbon atoms, e.g., methylene, ethylene, propylene, i-propylene, n-butylene, etc., linking the remainder of the molecule to a radical group.

[0126] "Aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon from 5 to 18 carbon atoms, and at least one of the rings in the ring system is fully unsaturated, i.e., it contains a ring with a delocalized (4n+2) π-electron system according to the Hückel theory. Ring systems from which the aryl group is derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin, and naphthalene.

[0127] An “aralkyl” or “aryl-alkyl” is an alkyl group of the formula —R c -aryl radical, where R c is an alkylene chain as defined above, e.g., methylene, ethylene, etc. The alkylene chain part of the aralkyl radical may be optionally substituted as described above for an alkylene chain.

[0128] "About" may allow for a degree of variation within a value or range, for example, within 10%, within 5%, or within 1% of the stated value or stated limit of the range.

[0129] The term "comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is not intended to exclude embodiments of any compounds, compositions, methods, processes, etc. that may "consist of" or "consist essentially of" the described features. The invention(s) illustratively described herein may suitably be practiced in the absence of any element or elements or limitation not specifically disclosed herein.

[0130] The term "heteroalkyl" refers to an alkyl group as defined above in which one or more skeletal carbon atoms of the alkyl are replaced with a heteroatom (with the appropriate number of substituents or valences - e.g., -CH2- can be replaced with -NH- or -O-). For example, each substituted carbon atom is independently replaced with a heteroatom, for example, carbon is replaced with nitrogen, oxygen, selenium, or other suitable heteroatom. In some cases, each substituted carbon atom is independently substituted for oxygen, nitrogen (e.g., -NH-, -N(alkyl)-, or -N(aryl)-, or another substituent contemplated herein), or sulfur (e.g., -S-, -S(=O)-, or -S(=O)2-). The heteroalkyl is bonded to the remainder of the molecule at a carbon atom of the heteroalkyl. The heteroalkyl is bonded to the remainder of the molecule at a heteroatom of the heteroalkyl. Heteroalkyl is a C1-C 18 Heteroalkyl is a C1-C 12 Heteroalkyl is a C1-C6 heteroalkyl. Heteroalkyl is a C1-C4 heteroalkyl. Heteroalkyl may include alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl, and heterocycloalkylalkyl, as defined herein.

[0131] The term "radical" as used herein refers to a fragment of a molecule, which fragment has an open valence for bond formation. A monovalent radical has one open valence such that it can form one bond with another chemical group. Unless otherwise specified, a radical of a molecule is created by the removal of one hydrogen atom from the molecule to create a monovalent radical with one open valence at the position where the hydrogen atom was removed. Where appropriate, radicals may be divalent, trivalent, etc., where two, three, four or more hydrogen atoms or other groups have been removed to create a radical that can bond with two, three, four or more chemical groups. Where appropriate, radical open valences may be created by the removal of a non-hydrogen atom (e.g., a halogen) or by the removal of two or more atoms (e.g., a hydroxyl group), so long as the removed atoms are a small fraction (20% or less of the atom count) of the total atoms in the molecule forming the radical.

[0132] "Substantially" may allow for a degree of variation within a value or range, for example, within 90%, within 95%, or within 99% of a stated value or stated limit of a range.

[0133] The concepts of the present disclosure have been illustrated and described in detail in the figures and descriptions herein, and the results in the figures and descriptions should be considered as exemplary, not limiting in character, with the understanding that only illustrative embodiments have been shown and described, and that all changes and modifications that fall within the spirit of the present disclosure are desired to be protected. Indeed, numerous specific details have been provided to provide a thorough understanding of the present disclosure. It should be understood that certain examples may be implemented without some or all of these specific details, and that the present disclosure is not limited to a particular biological system, a particular cancer, or a particular organ or tissue, which may of course vary, but remain applicable in light of the data provided herein.

[0134] The entire contents of any and all patent publications, non-patent publications, and reference texts cited herein are incorporated herein by reference, except that in the event of any disclosure or definition that contradicts this specification, the disclosure or definition in this specification shall be deemed to control.

[0135] Various techniques and mechanisms of the present disclosure will sometimes describe a connection or coupling between two components. Terms such as coupled, connected, coupled, connected, and similar terms using their inflections are used interchangeably unless a distinction is noted or otherwise made clear from the context. These words and expressions do not necessarily convey a direct connection, but include a connection via an intermediate component. It should be noted that a connection between two components of interest does not necessarily mean a direct and uninterrupted connection, since there may be various other components between the two components of interest. As a result, a connection does not necessarily mean a direct and uninterrupted connection, unless otherwise noted.

[0136] The use of headings and subheadings is for ease of reference only and is not intended to limit the scope of the disclosure under a given heading or subheading to the subject matter set forth therein. Rather, the disclosure under any heading or subheading is applicable to all subject matter herein unless expressly indicated otherwise or contradicted by context. [Example]

[0137] The following examples serve to illustrate the present disclosure and are not intended to limit its scope in any way. EXAMPLES

[0138] Synthesis of folate ligands with the brush border membrane (BBM) linker MVKC

[0139] [ka]

[0140] The 2-chlorotrityl resin was swelled in 10 mL of DCM per gram of resin and then filtered. 2.0 equivalents of the first amino acid were dissolved in 10 mL of DCM per gram of resin with 4.0 equivalents of DIPEA and then added to the resin. The mixture was stirred for 2 hours and then capped by the addition of 0.8 mL of MeOH per gram of resin over 30 minutes. The resin was washed twice with DMF, then DCM, then MeOH.

[0141] Additional amino acids and peptidomimetics were conjugated to the peptides as follows: Fmoc was first deprotected by suspending the resin 3x in 20% piperidine / DMF solution (v / v) for 20 min per suspension, washing 5 times with DMF, then DCM, then DMF between each suspension. Next, 2 equivalents of the appropriate amino were dissolved in 20 mL of DMF per gram of resin along with 2 equivalents of HATU and 4 equivalents of DIPEA. The solution was incubated for 5 min, then added to the resin and bubbled with inert gas for 2-3 h.

[0142] The coupling step was repeated under the same conditions before proceeding to the next amino acid. 3 equivalents of DOTA-tris(t-Bu ester), HATU and 6 equivalents of DIPEA were used for coupling. Alloc was deprotected by adding 1 equivalent of Pd(PPh3)4 and 20 equivalents of phenylsilane, freshly rinsed with MeOH, to the resin in DCM at a concentration of about 0.05 M under inert gas for 1 h. The resin was washed three times with DMF, sodium N,N-diethyldithiocarbamate (0.03 M in DMF) and DCM. Pteoric acid protected with COCF3 was deprotected with 40% NH4OH / DMF solution (v / v) for 6 h. The final peptide conjugate was cleaved by incubating 95% TFA / 2.5% TIPS / 2.5% H2O solution with 5 mM TCEP with the resin for 1 h. The cleavage products were precipitated into ice-cold diethyl ether and coupled to maleimide-bearing dyes without further purification.

[0143] The final product was purified using RP-HPLC (Agilent Technologies; Santa Clara, Calif.; C18 10 μm; 19 mm×250 mm). EXAMPLES

[0144] Synthesis of S0456-maleimide and coupling of folate-MVKC-SH with the BBM linker (BBM1(MVKC))

[0145] [ka]

[0146] S0456 maleimide and Evans Blue maleimide (International Publication No. WO / 2019 / 165200) were synthesized according to previously published protocols. Final products were purified using RP-HPLC (Agilent Technologies; Santa Clara, CA; C18 10 μm; 19 mm×250 mm). EXAMPLES

[0147] Synthesis of BBM3(MVK)

[0148] [ka] EXAMPLES

[0149] Folate conjugate binding curve KB cells were incubated with increasing concentrations of OTL38, BBM1, BBM2 or BBM3 in staining buffer for 1 hour at 4° C., washed three times with staining buffer and then analyzed via flow cytometry.

[0150] OTL38 was synthesized as described in Mahalingam et al., Evaluation of Novel Tumor-Targeted Near-Infrared Probe for Fluorescence-Guided Surgery of Cancer, Journal of Medicinal Chemistry 2018 61(21), 9637-9646. BBM2 was synthesized according to the scheme set forth herein and is a peptide with the sequence MVKGYGKC. EXAMPLES

[0151] Synthesis of folate BBM radioligand conjugate precursors

[0152] [ka]

[0153] Synthesis of folate-BBM-DOTA precursor for albumin binder coupling: Reagents and conditions: a) 2-chlorotrityl resin, DIPEA, DCM, rt, room temperature, 2 hrs; b) i. 20% piperidine DMF, Ar, 20 min (x3); ii. Fmoc-Val-OH, HATU, DIPEA, DMF, Ar, 3 hrs (x2); iii. 20% piperidine DMF, Ar, 20 min (x3); iv. Fmoc-Met-OH, HATU, DIPEA, DMF, Ar, 3 hrs (x2); c) i. 20% piperidine DMF, Ar, 20 min (x3); ii. DOTA-tris(t-Bu ester), HATU, DIPEA, DMF, Ar, 3 hrs; d) Pd(PPh3)4, phenylsilane, DCM, Ar, 1 hr; e) i. Fmoc-Cys-OH, HATU, DIPEA, DMF, Ar, 3 hrs (x2); ii. 20% piperidine DMF, Ar, 20 min (x3); iii. Fmoc-Glu-OtBu, HATU, DIPEA, DMF, Ar, 3 hrs; f) i. 20% piperidine DMF, Ar, 20 min (x3); ii. N10-TFA-Pteoric acid, HATU, DIPEA, DMF; g) i. 50% NH4OH DMF, Ar; ii. TCEP, 95 / 2.5 / 2.5 TFA / TIPS / H2O, 1 hr. EXAMPLES

[0154] Synthesis of BBM4

[0155] [ka]

[0156] Synthesis of DOTA-MVK(EB-folate)-OH. Reagents and conditions: a) Tolidine, HATU, DIPEA, DMF, overnight; b) i. HCl, NaNO2, ACN, H2O, 0℃, 30 min; ii. 1-amino-8-naphthol-2,4-disulfonic acid, NaHCO3, H2O, 0℃ to room temperature, 1.5 hours; c) DOTA-MVK(Cys-folate)-OH, DMSO, Ar, room temperature, 4 hours. EXAMPLES

[0157] Synthesis of BBM5

[0158] [ka]

[0159] Synthesis of DOTA-MVK(IP-folate)-OH. Reagents and conditions: a) 4-iodophenyl-butyric acid, HATU, DIPEA, DMF, room temperature, 3 hours; b) DOTA-MVK(Cys-folate)-OH, DMSO, Ar, room temperature, 4 hours. EXAMPLES

[0160] Validation of folate targeting via flow cytometry Each of OTL38, BBM1 and BBM2 was incubated with KB cells pre-cultured in folate-deficient medium in the absence or presence of 100-fold excess of glucosamine folate for 1 hour at 4°C. The cells were washed three times and then analyzed via flow cytometry. The results are shown in Figures 1A-1C. EXAMPLES

[0161] Folate conjugate binding curve KB cells were incubated with increasing concentrations of OTL38, BBM1, BBM2 or BBM3 in staining buffer for 1 hour at 4°C, washed three times with staining buffer, suspended in phosphate buffered saline, and then analyzed via flow cytometry. Results are shown in Figures 2A-2D. Concentrations of all conjugates were normalized via fluorescence. All conjugates at 50 nM showed competition with glucosamine folate. EXAMPLES

[0162] In vivo studies in mice Swiss Webster mice were maintained on a folate deficient diet for over 3 weeks. Mice were then tail vein injected with 1 nmol of OTL38, BBM1 or BBM2 (1-2 mice / conjugate) and sacrificed 24, 1, 3 or 6 hours after injection. Mice were dissected and their hearts, lungs, livers, spleens and kidneys were scanned for fluorescence. The results are shown in Figures 3A and 3B, 4A and 4B, 5A and 5B, and 6A and 6B, respectively.

[0163] Separately, other Swiss Webster mice were maintained on a folate deficient diet for over 3 weeks and tail vein injected with 1 nmol of OTL38, BBM1, BBM2 or BBM3 (1 mouse / conjugate). Mice were scanned for fluorescence 1 hour (1 h pi) and 3 hours (3 h pi). Results for OTL38 and BBM3 are shown in Figure 7. At 3 hours post-injection, mice were dissected and their heart, lungs, liver, spleen and kidneys were scanned for fluorescence. Results for OTL38, BBM1, BBM2 and BBM3 are shown in Figures 8A and 8B.

[0164] In yet another experiment, Swiss Webster mice were maintained on a folate deficient diet for over 3 weeks and tail vein injected with 5 nmol of OTL38 or BBM3 (1 mouse / conjugate). Mice were scanned for fluorescence 1 hour (1 h pi), 3 hours (h pi), 6 hours (6 h pi) and 24 hours (24 h pi). Results are shown in Figures 9A and 9B. 24 hours post-injection, mice were dissected and their heart, lungs, liver, spleen and kidneys were scanned for fluorescence. Results are shown in Figure 10.

[0165] In yet another experiment, athymic nude mice that had been fed a folate-deficient diet for over 3 weeks were tail vein injected with 5 nmol of OTL38, BBM1 or BBM2 (1 mouse / conjugate). 24 hours after injection, the mice were dissected and their tumors (KB), hearts, lungs, livers and kidneys were scanned for fluorescence. The results are shown in FIG. 11.

[0166] Athymic nude mice maintained on a folate deficient diet for over 3 weeks were tail vein injected with 5 nmol of OTL38 or BBM3 (1-2 mice / conjugate). Mice were scanned for fluorescence in the tumor (MDA-MB-231) versus kidney at 1 hour (1 hour pi), 3 hours (3 hours pi), 6 hours (6 hours pi), and 24 hours (24 hours pi) post-injection. Results are shown in Figure 12.

[0167] Athymic nude mice were maintained on a folate deficient diet for over 3 weeks and then injected with 5 nmol of OTL38 or BBM3 via the tail vein (1-2 mice / conjugate). Mice were scanned for fluorescence in the tumor (MDA-MB-231) versus kidney at 1 hour (1 hour pi), 3 hours (3 hours pi), 6 hours (6 hours pi), and 24 hours (24 hours pi) post-injection. The results are shown in Figure 13.

[0168] Athymic nude mice were maintained on a folate-deficient diet for over 3 weeks and then injected via the tail vein with 5 nmol of OTL38 or BBM3 (1-2 mice / conjugate). 24 hours after injection, the mice were dissected and their tumors (MDA-MB-231), heart, lungs, liver, spleen, and kidneys were scanned for fluorescence. The results are shown in Figure 14. EXAMPLES

[0169] BBM4 and BBM5 111 Radiolabeling with In The folate-DOTA conjugate was diluted with ammonium acetate (0.5 M, pH 8.0) to reach a final conjugate concentration of 0.5 mM. 111InCl3) was added to obtain a specific activity of 1 MBq / nmol and then heated to 90°C for 10 min. Sodium-diethylenetriaminepentaacetic acid solution (5 mM, pH 7.0) was added to complex any unreacted traces of radioisotope. Radiochemical purity was analyzed by radio-HPLC on an Agilent 1260 Infinity II with a Flow-RAM detector and a reversed-phase XBridge Shield RP18 column (3.0 x 50 mm, 3.5 μm) purchased from LabLogic Systems Ltd. (Brandon, FL). The mobile phase consisted of 20 mM ammonium acetate aqueous buffer (pH 7) (A) and acetonitrile (B) with a linear gradient from 5% B to 95% B over 15 min. Radiochemical purity was determined by: 111 The results were greater than 95% for the In-radiolabelled folate-DOTA conjugate. 111 FIG. 16 is a radiochromatogram of In-BBM4. 111 This is a radiochromatogram of In-BBM5. EXAMPLES

[0170] In KB cells 111 In-BBM4 and 111 In-BBM5 binding KB cells (200,000) were seeded in 24-well amine-coated plates and allowed to adhere overnight. When the cells reached confluency, they were incubated with increasing concentrations of 111 In-BBM4 or 111Incubation with In-BBM5 in the absence or presence of 100-fold excess of glucosamine folate was performed for 1 hour at room temperature. After incubation, cells were washed 3 times with PBS to remove unbound radioactivity and dissolved in 1.0 M NaOH. Samples were transferred to tubes and cell-bound radioactivity was measured using a Packard Cobra gamma counter. Specific binding constants were calculated using a one-site specific nonlinear fit. Figure 17A is a graph of concentration (nM) vs. CPM showing the binding of Indium-111 radiolabeled BBM4 conjugate in KB cells. Figure 17B is a graph of concentration (nM) vs. CPM showing the binding of Indium-111 radiolabeled BBM5 conjugate in KB cells. EXAMPLES

[0171] In vivo 111 In-BBM4 and 111 In-BBM5 binding Radioactive scanning was performed on a VECTor / CT system with a clustered multi-pinhole high-energy collimator (MILabs BV, Utrecht, The Netherlands) at the Bindley Bioscience Center of Purdue University. 12-week-old female ND4 Swiss Webster mice and 12-week-old female athymic nu / nu mice were purchased from Envigo (Indianapolis, IN). All mice were given a folate-deficient diet and free access to water. Mice were housed on a standard 12-hour light / dark cycle. 5 × 10 6 MDA-MB-231 tumors grew to approximately 1 cm by inoculating MDA-MB-231 cells. 3 After growth to 100 μg / ml, SPECT / CT scans were performed. Each mouse received up to 5 nmol of 111 In-BBM4 or 111In-BBM5 was injected intravenously. Mice (n=1 per group) were anesthetized with 3% isoflurane in oxygen and whole-body scans were performed at multiple time points. Luminescence scans were performed over 15-60 min. CT scans were acquired with the X-ray source set at 60 kV and 615 μA. SPECT images were acquired using U-SPECT II software and 171 and 241 keV X-rays. 111 Reconstruction was performed with an In γ-energy window. The POS-EM algorithm was used with 16 subsets and 4 iterations on a 0.8 mm voxel grid. CT images were reconstructed using NRecon software. Data sets were fused and filtered using PMOD software (version 3.2).

[0172] FIG. 18 shows the effect of Indium-111 radiolabeled BBM4 conjugate ( 111 Figure 19 shows the SPECT / CT images of Indium-111 radiolabeled BBM5 conjugate (In-BBM4) in healthy mice. 1 nmol of In-111 radiolabeled was injected. The yellow arrow indicates the kidney. 111 Figure 20 shows the SPECT / CT images of Indium-111 radiolabeled BBM4 conjugate (In-BBM5) in healthy mice. 1 nmol was injected with approximately 250 uCi of In-111 radiolabeled. The yellow arrow indicates the kidney. Figure 21 shows the SPECT / CT images of Indium-111 radiolabeled BBM4 conjugate (In-BBM5) in healthy mice. 111 SPECT / CT image of In-BBM4) is shown. Approximately 250uCi of In-111 radiolabeled material was injected at 5nmol. The yellow arrow indicates the kidney. EXAMPLES

[0173] BBM4 and BBM5 177 Radiolabeling with Lu The folate-DOTA conjugate was diluted with ammonium acetate (0.5 M, pH 8.0) to reach a final conjugate concentration of 0.5 mM. 177LuCl3) was added to obtain a specific activity of 4 MBq / nmol and then heated to 90°C for 10 min. Sodium-diethylenetriaminepentaacetic acid solution (5 mM, pH 7.0) was added to complex any unreacted traces of radioisotope. Radiochemical purity was analyzed by radio-HPLC on an Agilent 1260 Infinity II with a Flow-RAM detector and a reversed-phase XBridge Shield RP18 column (3.0 x 50 mm, 3.5 μm) purchased from LabLogic (Brandon, FL). The mobile phase consisted of 20 mM ammonium acetate aqueous buffer (pH 7) (A) and acetonitrile (B) with a linear gradient from 5% B to 95% B over 15 min. Radiochemical purity was determined by: 177 The results were greater than 95% for the 111 Lu radiolabeled folate-DOTA conjugate. 177 FIG. 22 is a radiochromatogram of Lu-BBM4. 177 This is a radiochromatogram of Lu-BBM5. EXAMPLES

[0174] In vivo 177 Lu-BBM4 and 177 Lu-BBM5 binding Twelve-week-old female athymic nu / nu mice were purchased from Envigo and given free access to folate-deficient diet and water. Mice were housed on a standard 12-hour light / dark cycle. 5×10 6 By inoculating KB cells, KB tumors were grown to approximately 300 mm 3 Tumors were allowed to grow until 0.5×L×W. Prior to the start of the therapy study, mice were randomized into control or treatment groups. On day 0, mice received a single intravenous dose of sterile saline or 5 nmol of BBM4 or BBM5 radiolabeled with 18 MBq of lutetium-177 via the tail vein. Tumors were measured in two perpendicular directions every other day during therapy, and their volumes were calculated as 0.5×L×W. 2where L is the longest axis (in millimeters) and W is the axis perpendicular to L (in millimeters). Humane endpoint criteria were weight loss of more than 20% of initial body weight, 1,800 mm 3 End-point disease was defined as tumor volume exceeding 100 μg / kg or open ulcer formation. Mice were euthanized upon reaching one of the predefined endpoint criteria.

[0175] FIG. 23A is a 177 Figure 1 shows the total volume of KB tumors in mice treated with Lu radiolabeled BBM conjugate versus days in tumor volume (mm 3 ) is a graph of FIG. 177 FIG. 23C is a graph of relative tumor size versus days showing the relative size of KB tumors in mice treated with Lu radiolabeled BBM conjugate. 177 FIG. 1 is a graph of days versus relative body weight (%) showing relative body weight of mice treated with Lu-radiolabelled BBM conjugate.

[0176] Radiation scans were performed on a VECTor / CT system with a clustered multi-pinhole high-energy collimator (MILabs BV, Utrecht, The Netherlands) at the Bindley Bioscience Center of Purdue University. Mice scanned were selected from the radiotherapy treatment groups in Figures 23A-23C. Mice (n=1 per group) were anesthetized with 3% isoflurane in oxygen and whole-body scans were performed at multiple time points. Luminescence scans were performed over a period of 15-60 minutes. CT scans were acquired with the X-ray source set at 60 kV and 615 μA. SPECT images were acquired using U-SPECT II software and a 208 keV CT scanner. 177 Reconstruction was performed with the Lu γ-energy window. The POS-EM algorithm was used with 16 subsets and 4 iterations on a 0.8 mm voxel grid. CT images were reconstructed using NRecon software. Data sets were fused and filtered using PMOD software (version 3.2). Figure 24 shows the 177Figure 25 shows SPECT / CT images of a mouse treated with Lu radiolabeled BBM4 conjugate. 5 nmol of approximately 500 uCi of Lu-177 radiolabeled was injected. The upper arrow indicates the tumor and the lower arrow indicates the kidney. 177 1 shows a SPECT / CT image of a mouse treated with Lu radiolabeled BBM5 conjugate, radiolabeled with approximately 500 uCi of Lu-177, and 5 nmol was injected. The upper arrow indicates the tumor and the lower arrow indicates the kidney. EXAMPLES

[0177] Folate dye conjugate structures with BBM enzyme-labile linkers [ka] EXAMPLES

[0178] Folate radioconjugate structures with BBM enzyme-labile linkers [ka]

[0179] (References) Further references 1. Weitman, SD; Lark, RH; Coney, LR; Fort, DW; Frasca, V.; Zurawski, VR; Kamen, BA, Distribution of the Folate Receptor GP38 in Normal and Malignant Cell Lines and Tissues. Cancer Research 1992, 52 (12), 3396. 2. Coney, L. R.; Tomassetti, A.; Carayannopoulos, L.; Frasca, V.; Kamen, B. A.; Colnaghi, M. I.; Zurawski, V. R., Cloning of a Tumor-associated Antigen: MOv18 and MOv19 Antibodies Recognize a Folate-binding Protein. Cancer Research 1991, 51 (22), 6125. 3. Garin-Chesa, P.; Campbell, I.; Saigo, P. E.; Lewis, J. L., Jr.; Old, L. J.; Rettig, W. J., Trophoblast and ovarian cancer antigen LK26. Sensitivity and specificity in immunopathology and molecular identification as a folate-binding protein. The American journal of pathology 1993, 142 (2), 557-567. 4. Parker, N.; Turk, M. J.; Westrick, E.; Lewis, J. D.; Low, P. S.; Leamon, C. P., Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Analytical Biochemistry 2005, 338 (2), 284-293. 5. Low, P. S.; Henne, W. A.; Doorneweerd, D. D., Discovery and Development of Folic-Acid-Based Receptor Targeting for Imaging and Therapy of Cancer and Inflammatory Diseases. Accounts of Chemical Research 2008, 41 (1), 120-129. 6. Low, P. S.; Kularatne, S. A., Folate-targeted therapeutic and imaging agents for cancer. Current Opinion in Chemical Biology 2009, 13 (3), 256-262. 7. Holm, J.; Hansen, S. I.; Hoier-Madsen, M.; Bostad, L., A high-affinity folate binding protein in proximal tubule cells of human kidney. Kidney International 1992, 41 (1), 50-55. 8. Birn, H.; Spiegelstein, O.; Christensen, E. I.; Finnell, R. H., Renal Tubular Reabsorption of Folate Mediated by Folate Binding Protein 1. Journal of the American Society of Nephrology 2005, 16 (3), 608. 9. Sandoval, R. M.; Kennedy, M. D.; Low, P. S.; Molitoris, B. A., Uptake and trafficking of fluorescent conjugates of folic acid in intact kidney determined using intravital two-photon microscopy. American Journal of Physiology-Cell Physiology 2004, 287 (2), C517-C526. 10. Zhou, P.; Sun, X.; Zhang, Z., Kidney-targeted drug delivery systems. Acta Pharmaceutica Sinica B 2014, 4 (1), 37-42. 11. Muller, C.; Schibli, R., Prospects in Folate Receptor-Targeted Radionuclide Therapy. Frontiers in Oncology 2013, 3 (249). 12. Muller, C.; Struthers, H.; Winiger, C.; Zhernosekov, K.; Schibli, R., DOTA Conjugate with an Albumin-Binding Entity Enables the First Folic Acid-Targeted 177Lu-Radionuclide Tumor Therapy in Mice. Journal of Nuclear Medicine 2013, 54 (1), 124-131. 13. Farkas, R.; Siwowska, K.; Ametamey, S. M.; Schibli, R.; van der Meulen, N. P.; Muller, C., 64Cu- and 68Ga-Based PET Imaging of Folate Receptor-Positive Tumors: Development and Evaluation of an Albumin-Binding NODAGA-Folate. Molecular Pharmaceutics 2016, 13 (6), 1979-1987. 14. Radford, L. L.; Fernandez, S.; Beacham, R.; El Sayed, R.; Farkas, R.; Benesova, M.; Muller, C.; Lapi, S. E., New 55Co-labeled Albumin-Binding Folate Derivatives as Potential PET Agents for Folate Receptor Imaging. Pharmaceuticals 2019, 12 (4). 15. Arano, Y.; Fujioka, Y.; Akizawa, H.; Ono, M.; Uehara, T.; Wakisaka, K.; Nakayama, M.; Sakahara, H.; Konishi, J.; Saji, H., Chemical Design of Radiolabeled Antibody Fragments for Low Renal Radioactivity Levels. Cancer Research 1999, 59 (1), 128-134. 16. Uehara, T.; Koike, M.; Nakata, H.; Hanaoka, H.; Iida, Y.; Hashimoto, K.; Akizawa, H.; Endo, K.; Arano, Y., Design, Synthesis, and Evaluation of [188Re]Organorhenium-Labeled Antibody Fragments with Renal Enzyme-Cleavable Linkage for Low Renal Radioactivity Levels. Bioconjugate Chemistry 2007, 18 (1), 190-198. 17. Akizawa, H.; Uehara, T.; Arano, Y., Renal uptake and metabolism of radiopharmaceuticals derived from peptides and proteins. Advanced Drug Delivery Reviews 2008, 60 (12), 1319-1328. 18. Akizawa, H.; Imajima, M.; Hanaoka, H.; Uehara, T.; Satake, S.; Arano, Y., Renal Brush Border Enzyme-Cleavable Linkages for Low Renal Radioactivity Levels of Radiolabeled Antibody Fragments. Bioconjugate Chemistry 2013, 24 (2), 291-299. 19. Suzuki, C.; Uehara, T.; Kanazawa, N.; Wada, S.; Suzuki, H.; Arano, Y., Preferential Cleavage of a Tripeptide Linkage by Enzymes on Renal Brush Border Membrane To Reduce Renal Radioactivity Levels of Radiolabeled Antibody Fragments. Journal of Medicinal Chemistry 2018, 61 (12), 5257-5268. 20. Uehara, T.; Yokoyama, M.; Suzuki, H.; Hanaoka, H.; Arano, Y., A Gallium-67 / 68-Labeled Antibody Fragment for Immuno-SPECT / PET Shows Low Renal Radioactivity Without Loss of Tumor Uptake. Clinical Cancer Research 2018, 24 (14), 3309-3316. 21. Uehara, T.; Kanazawa, N.; Suzuki, C.; Mizuno, Y.; Suzuki, H.; Hanaoka, H.; Arano, Y., Renal Handling of 99mTc-Labeled Antibody Fab Fragments with a Linkage Cleavable by Enzymes on Brush Border Membrane. Bioconjugate Chemistry 2020, 31 (11), 2618-2627. 22. Bendre, S.; Zhang, Z.; Kuo, H.-T.; Rousseau, J.; Zhang, C.; Merkens, H.; Roxin, A.; Peptidomimetics. Molecules 2020, 25 (17), 3854. 23. Vaidyanathan, G.; Kang, CM; McDougald, D.; Minn, I.; Brummet, M.; Pomper, MG; Zalutsky, MR, Brush border enzyme-cleavable linkers: Evaluation for reducing renal uptake of radiolabeled prostate-specific membrane antigen inhibitors. Nuclear Medicine and Biology 2018, 62-63, 18-30. 24. Radiopharmaceutical Agents (China Patent Application Publication No. CN111065646A, published on April 24, 2020). 25. Novel radiometal-binding compounds for diagnosis or treatment of prostate specific membrane antigen-expressing cancer (Canadian Patent Application Publication No. CA3079906A1, published April 25, 2019). 26. Trifunctional constructs with tunable pharmacokinetics useful in imaging and anti-tumor therapies (U.S. Patent Application Publication No. 2019 / 0015531 A1, published September 18, 2018). 27. Folate conjugates of albumin-binding entities (U.S. Patent No. 9,295,739 B2, filed August 10, 2012).

Claims

1. A conjugate of Formula I or Formula II. FRTL-BBMecL-AA (Formula I) or FRTL-Alb-BBMecL-AA (Formula II) [In the formula, FRTL is folate receptor targeting ligand; BBMecL is a renal brush border membrane (BBM) enzyme cleavable linker; Alb is the albumin binding moiety; AA is an activator.

2. 2. The conjugate of claim 1 , wherein the FRTL has the following structure: 【Chemistry 1】 [In the formula, T is S, O, NR ４ｂ and -HC=CH-; U, V and W are each -(R ６ａ )C=, -N=, -(R ６ａ ) C(R ７ａ )- and -N(R ４ａ )-(wherein, R ６ａ and R ７ａ is hydrogen, halo and C １ -C １２ alkoxy, or R ６ａ and R ７ａ represent a divalent moiety independently selected from the group consisting of: X and Y are halo, R ２ , OR ２ , S.R. ３ and N.R. ４ R ５ are each independently selected from the group consisting of: Q is C or CH; A １ and A ２ is oxygen, sulfur, -C(Z)-, -C(Z)O-, -OC(Z)-, -N(R ４ｂ )-, -C(Z)N(R ４ｂ ) -, -N(R ４ｂ )C(Z)-,-OC(Z)N(R ４ｂ ) -, -N(R ４ｂ )C(Z)O-,-N(R ４ｂ ) C(Z)N(R ５ｂ )-, -S(O)-, -S(O) ２ -, -N(R ４ｂ ) S (O) ２ -, -C(R ６ｂ ) (R ７ｂ )-, -N(C≡CH)-,-N(CH ２ C≡CH)-, C １ -C １２ Alkylene and C １ -C １２ Alkyneoxy wherein Z is oxygen or sulfur; R ２ , R ３ , R ４ , R ４ａ , R ４ｂ , R ５ , R ５ｂ , R ６ｂ and R ７ｂ is hydrogen, halo, C １ -C １２ Alkyl, C １ -C １２ Alkoxy, C １ -C １２ Alkanoyl, C １ -C １２ Alkenyl, C １ -C １２ Alkynyl, (C １ -C １２ alkoxy)carbonyl and (C １ -C １２ each independently selected from the group consisting of: A ３ is an amino acid, R １ is hydrogen, halo, C １ -C １２ Heteroalkyl and C １ -C １２ alkoxy; R ６ and R ７ is hydrogen, halo, C １ -C １２ Alkyl and C １ -C １２ alkoxy, or R ６ and R ７ together form a carbonyl group, q is an integer from 1 to 3; p, r, s and t are each independently 0 or 1; ＊ indicates the point of attachment to the BBMecL or Alb.

3. 3. The conjugate of claim 2, wherein Q is CH and / or X is OH and Y is NH2.

4. W and U are -N(R ４ａ )-, Q is CH and V is CH ２ And A １ -N(R ４ｂ )-, s is 1, p is 1, and t is 0.

5. W and U are -N(R ４ａ )-, Q is CH and V is CH ２ And A １ -N(R ４ｂ 4. The conjugate of claim 3, wherein s is 1, p is 1, and t is 0.

6. R ４ａ and R ４ｂ is independently alkyl or heteroalkyl; X is OH, Y is NH 2 , and R 4a and R 4b are independently alkyl or heteroalkyl; or W and U are -N(R 4a )-, Q is CH, V is CH 2 , A 1 is -N(R 4b )-, s is 1, p is 1, t is 0, and R 4a and R 4b are independently alkyl or heteroalkyl; The conjugate of claim 2.

7. R ４ａ and R ４ｂ is methyl, The conjugate of claim 6.

8. 3. The conjugate of claim 2, wherein the FRTL has the following structure: 【Chemistry 2】 [In the formula, 【Chemistry 3】 indicates the attachment point of the FRTL to Alb or BBMecL.

9. 2. The conjugate of claim 1, wherein Alb has the following structure: 【Chemistry 4】 [In the formula, 【Chemistry 5】 indicates the binding point of Alb to FRTL, R １２－１９ are independently -H, -C １ -C ６ Alkyl, -F, -Cl, -Br, -I, -CN, -CHO, -B(OH) ２ , -C(O)alkyl, -C(O)aryl-, -C=C-C(O)aryl, -C=C-S(O) ２ Aryl, —CO ２ H, -SO ３ H, -SO ２ N.H. ２ , -PO ３ H ２ Or -SO ２ F, R ２０ and R ２１ are independently -H, -C １ -C ６ Alkyl, -F, -Cl, -Br, -I, -O-C １－６ Alkyl, -CN, -CHO, -B(OH) ２ , -C=C-C(O)aryl, -C=C-S(O) ２ Aryl, —CO ２ H, -SO ３ H, -SO ２ N.H. ２ , -PO ３ H ２ , -SO ２ F or CF ３ , 【Chemistry 6】 【change】 , ABD035, ABDCon, designed ankyrin repeat proteins (DARPins), dsFV CA645, nanobodies, or novel variable domains of antigen receptors (VNARs) fused to anti-human serum albumin domain clone E06.

10. BBMecL, (i) Met-Val, (ii) X-Lys or X-Arg, where X=Gly or Arg, or a combination thereof; (iii) Gly-Tyr, (iv) Gly-Phe-(Lys), (v) Gly-Pro, (vi) Ala-X or Leu-X, where X is any amino acid, or a combination thereof; (vii) Asp-X or Glu-X, where X is any amino acid; or Combinations of these (viii) γ-Glu-X, where X is any amino acid, and an acceptor peptide; (ix) sucrose, maltose, trehalose, lactose, palatinose, or a combination of two or more of the foregoing; (x) iodoinsulin B chain, (xi) phlorizin, (xii) p-nitrophenyl phosphate, or (xiii) A combination of two or more of the above. The conjugate of claim 1 , comprising:

11. A.A. (i) an optical imaging agent; (ii) a radioactive imaging agent, or (iii) a radiotherapeutic agent; The conjugate of claim 1 ,

12. The conjugate of claim 1 , wherein AA is an optical imaging agent comprising a fluorescent dye.

13. 13. The conjugate of claim 12, wherein the fluorescent dye is selected from the group consisting of S0456, fluorescein isothiocyanate (FITC), rhodamine, LS288, heptamethine cyanine dyes (HMCD), SS180, acridine orange (AO), IRDye800CW, IR783, IR825, ZW800-1 and indocyanine green (ICG).

14. A.A. １８ F. ４４ Sc, ４７ Sc, ５２ Mn, ５５ Co, ６４ Cu, ６ 7Cu, ６７ G.A. ６８ G.A. ８６ Y. ８９ Zr, ９０ Y. ９９ｍ Tc, １１１ In, １１４ｍ In, １１７ｍ Sn, １２４ I, １２５ I, １３１ I, １４９ Tb, １５３ Sm, １５２ Tb, １５５ Tb, １６１ Tb, １７７ Lu, １８６ Re, １８８ Re, ２１２ Pb, ２１２ Bi, ２１３ Bi, ２２３ Ra, ２２４ Ra, ２２５ Ab, ２２５ Ac and ２２７ 2. The conjugate of claim 1, which is a radioactive imaging or radiotherapeutic agent comprising a radioisotope selected from the group consisting of Th.

15. A.A. ６８ G.A. １８ F. ９０ Y. ９９ｍ Tc, １１１ In, １７７ Lu, ２２５ A c, １８ P. １２４ I, １２５ I, １３１ I and ２１１ 2. The conjugate of claim 1, comprising a radiolabeled prosthetic group comprising a radioisotope selected from the group consisting of At.

16. the radiolabeled prosthetic group being 【Chemistry 7】 16. The conjugate of claim 15, comprising a structure selected from: wherein R and R' are independently hydrogen or methyl, and n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

17. AA is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid), SarAr (1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosane-1,8-diamine, NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (2,2'-(7-(4-((2-aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid), HYNIC (6-hydrazinonicotinic acid), NETA (4-[2-(bis-carboxymethylamino)- ethyl]-7-carboxymethyl-[1,4,7]triazonan-1-yl)acetic acid, TRAP (1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid), HBED (N,N-bis(2-hydroxybenzyl)-ethylenediamine-N,N-diacetic acid), 2,3-HOPO (3-hydroxypyridin-2-one), PCTA (3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid), DFO (desferrioxamine), DTPA (diethylenetriaminepentaacetic acid), OCTAPA (N,N-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N-diacetic acid), H ２ macropa (N,N'-bis[(6-carboxy-2-pyridylmethyl]-4,13-diaza-18-crown-6), H ２ 2. The conjugate of claim 1, comprising a chelator selected from the group consisting of dedpa (1,2-[[carboxy)-pyridin-2-yl]-methylamino]ethane, β-1-diaminopropionic acid, an EC20 head comprising aspartic acid and cysteine, and derivatives of any of the foregoing.

18. A.A. 【Chemistry 9】 【change】 2. The conjugate of claim 1, comprising a chelating agent selected from the group consisting of:

19. 2. The conjugate of claim 1 having the following structure: 【Chemistry 10】 (BBM1), or 【Chemistry 11】 (BBM2), or 【Chemistry 12】 (BBM3)

20. 2. The conjugate of claim 1 having the following structure: 【Chemistry 13】 BBM4 (DOTA-MVK(EB-folate)-OH), or 【Chemistry 14】 (BBM5(DOTA-MVK(IP-folic acid)-OH)

21. 2. The conjugate of claim 1 having the following structure: 【Chemistry 15】 or 【Chemistry 16】 or 【Chemistry 17】

22. 2. The conjugate of claim 1 having the following structure: 【Chemistry 18】

23. 2. The conjugate of claim 1 having the following structure: 【Chemistry 19】

24. 2. The conjugate of claim 1 having the following structure: 【Chemistry 20】

25. A composition comprising the conjugate of any one of claims 1 to 24 and a pharma- ceutically acceptable carrier.

26. 26. Use of a conjugate according to any of claims 1 to 24 or a composition according to claim 25 in the manufacture of a medicament for imaging, treating or imaging and treating cancer in a subject by targeted radioactivity to cells of a tumour, macrophages associated with said tumour or both, alone or further in combination with an optical imaging agent.

27. 27. The use of claim 26, wherein the tumor-associated macrophages comprise tumor-associated macrophages (TAMs).

28. 1. A kit for imaging, treating, or imaging and treating a tumor in a subject, comprising: At least one dosage unit of the conjugate according to any one of claims 1 to 24 or the composition according to claim 25 in a first container; at least one dosage unit of a second active agent or a composition comprising said second active agent; The kit comprising:

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