Peptide conjugates of camptothecin analogs and uses thereof

Abstract

DNA-damaging agents such as topoisomerase I inhibitors have taken on added importance as agents that enhance DNA damage response (DDR) defects and inhibitors in tumors. Limitations of camptothecin analogs include their side effects such as myelosuppression and diarrhea, their relatively short half-lives, and the fact that they are substrates of efflux pump proteins. There is thus a need for novel approaches for the use of camptothecin analogs in the treatment of cancers. The present application relates to conjugates of camptothecin analogs comprising a sortilin-targeting peptide and a cleavable linker, and salts thereof. These conjugates were shown to exhibit more potent antitumor effects than corresponding unconjugated camptothecin analogs in a mouse model of Sortilin-expressing colorectal cancer. The present application also relates to pharmaceutical compositions comprising the conjugates or salts thereof, as well as to methods and uses of the conjugates, salts thereof or pharmaceutical compositions for the treatment of Sortilin-expressing cancers.

Description

[0001] PEPTIDE CONJUGATES OF CAMPTOTHECIN ANALOGS AND USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the benefit of U.S. provisional patent application No. 63 / 581 ,864 filed on September 11 , 2023, U.S. provisional patent application No. 63 / 598,353 filed on November 13, 2023, and U.S. provisional patent application No. 63 / 564,151 filed on March 12, 2024, which are incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing in computer readable form entitled “G11718- 00480_SeqList.xml”, created on September 10, 2024, and having a size of about 11401 bytes. The computer readable form is incorporated herein by reference.

[0006] TECHNICAL FIELD

[0007] The present invention generally relates to the field of oncology, and more particularly to the treatment of cancers using topoisomerase inhibitors.

[0008] BACKGROUND ART

[0009] DNA-damaging agents, long-standing mainstays of cancer chemotherapy, have taken on added importance as agents that enhance DNA damage response (DDR) defects and inhibitors in tumors. DNA damage in the face of impaired ability to repair damaged DNA can result in selective toxicity and synthetic lethality (Lord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature 2012;481 :287-94). An important target to cause-specific DNA damage is the enzyme topoisomerase I (TOP1), which is inhibited by camptothecin and its analogs (Pommier, Y. DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition. Chem Rev 2009;109:2894-902; Thomas, A., Pommier, Y. Targeting topoisomerase I in the era of precision medicine. Clin Cancer Res 2019;25:6581-9).

[0010] The camptothecin analog irinotecan is used in the treatment of colon cancer, and to a lesser extent lung cancer. Administration of irinotecan is limited by its severe side effects including myelosuppression and diarrhea.

[0011] 7-Ethyl-10-hydroxy-camptothecin (SN-38) is the active metabolite of the camptothecin analog irinotecan. SN-38 has been found to be about 100-1000 times more cytotoxic than irinotecan, but it has not been used as an anticancer drug due to its poor solubility in water (< 40 pg / mL) and pharmaceutically acceptable solvents and its low affinity to lipid membranes; it is also extensively metabolized in the liver. Bioconversion of irinotecan to SN-38 is slow and limited (~5%), and about 33-66% of irinotecan remained unhydrolyzed at the end of a 24-hour infusion (Rowinsky et al., Cancer Research 1994;54:427-436). Another camptothecin analog, exatecan (Exa), is even more potent than SN-38. As a single agent, Exa progressed as far as phase III clinical trials before its development was discontinued because of dose-limiting side effects.

[0012] An exatecan derivative, deruxtecan (Dxd), has been developed, which represents a conjugate with trastuzumab, a Her2-targeting antibody.

[0013] A limitation of camptothecin analogs is their relatively short half-lives. For example, SN-38 formed from CPT-11 has an apparent ti / 2 of approximately 12 hours (Mathijssen, RH., et al. Clinical pharmacokinetics and metabolism of irinotecan (CPT-11). Clin Cancer Res 2001 ;7:2182- 94) and Exa has a ti / 2of only approximately 10 hours in humans (Braybrooke, JP., et al. Phase I and pharmacokinetic study of the topoisomerase I inhibitor, exatecan mesylate (DX-8951f), using a weekly 30-minute intravenous infusion, in patients with advanced solid malignancies. Ann Oncol 2003;14:913-21).

[0014] Also, irinotecan, SN-38 and exatecan have been reported to be substrates of the efflux pump protein Breast Cancer Resistance Protein (BCRP / ABCG2), which is associated with reduction of antitumor activity.

[0015] There is thus a need for novel approach for the use of camptothecin analogs in the treatment of cancers.

[0016] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0017] SUMMARY

[0018] In various aspects and embodiments, the present disclosure provides the following items 1 to 52:

[0019] 1. A conjugate comprising the following structure I, or a pharmaceutically acceptable salt thereof:

[0020] A — L — B (I); wherein

[0021] A is a camptothecin analog.

[0022] B is a peptide binding to a Sortilin receptor, wherein the peptide comprises an amino acid sequence having at least 60% identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2:

[0023] X1GVRAKAGVRN(Nle)FKSESYX2(SEQ ID NO:1) X1YKSLRRKAPRWDAPLRDPALRQLLX2(SEQ ID NO:2); wherein X1is cysteine (C) or is absent, and X2is cysteine (C) or is absent;

[0024] L is a linker, wherein a first end of the linker is attached to a side chain of one or more of the amino acid or modified side chain residues of the peptide, and / or to the N- and / or C- terminal end of the peptide, and a second end of the linker is attached to the camptothecin analog.

[0025] 2. The conjugate of item 1 , wherein L is a cleavable linker, wherein a first end of the linker is attached to a side chain of one or more of the lysine (K) and / or cysteine (C) residues of the peptide, and / or to the N- and / or C-terminal end of the peptide, and a second end of the linker is attached to an oxygen or nitrogen atom of the camptothecin analog.

[0026] 3. The conjugate or pharmaceutically acceptable salt thereof of item 1 or 2, wherein the conjugate or salt thereof is of one of the following formulas la, lb, Ic or Id:

[0027] A1 — L1 — B (la);

[0028] A1 — L1 — B — L2 — A2 (lb); wherein L1 , L2, L3 and L4 are linkers which may be the same or different, and A1 , A2, A3 and A4 are camptothecin analogs which may be the same or different.

[0029] 4. The conjugate or pharmaceutically acceptable salt thereof of any one of items 1 to 3, wherein the camptothecin analog is irinotecan, SN-38, exatecan or deruxtecan.

[0030] 5. The conjugate or pharmaceutically acceptable salt thereof of item 4, wherein the camptothecin analog is SN-38.

[0031] 6. The conjugate or pharmaceutically acceptable salt thereof of item 4, wherein the camptothecin analog is exatecan.

[0032] 7. The conjugate or pharmaceutically acceptable salt thereof of item 4, wherein the camptothecin analog is deruxtecan.

[0033] 8. The conjugate or pharmaceutically acceptable salt thereof of any one of items 1 to 7, wherein the peptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0034] 9. The conjugate or pharmaceutically acceptable salt thereof of item 8, wherein the peptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2. 10. The conjugate or pharmaceutically acceptable salt thereof of item 9, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0035] 11 . The conjugate or pharmaceutically acceptable salt thereof of item 9, wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0036] 12. The conjugate or pharmaceutically acceptable salt thereof of any one of items 1 to 11 , wherein X1and / or X2is C, and wherein the first end of the linker is attached to the side chain of X1and / or X2.

[0037] 13. The conjugate or pharmaceutically acceptable salt thereof of item 12, wherein X1is absent and X2is C, and wherein the first end of the linker is attached to the side chain of X2.

[0038] 14. The conjugate or pharmaceutically acceptable salt thereof of any one of items 1 to 11 , wherein X1and X2are absent.

[0039] 15. The conjugate or pharmaceutically acceptable salt thereof of any one of items 1 to 14, wherein the linker comprises at least one amino acid.

[0040] 16. The conjugate or pharmaceutically acceptable salt thereof of item 15, wherein the at least one amino acid comprises glycine, valine and / or citrulline.

[0041] 17. The conjugate or pharmaceutically acceptable salt thereof of item 15 or 16, wherein the cleavable linker is a protease-cleavable linker.

[0042] 18. The conjugate or pharmaceutically acceptable salt thereof of item 17, wherein the protease is a cysteine protease.

[0043] 19. The conjugate or pharmaceutically acceptable salt thereof of item 18, wherein the cysteine protease is cathepsin B.

[0044] 20. The conjugate or pharmaceutically acceptable salt thereof of any one of items 1 to 19, wherein the linker comprises polyethylene glycol (PEG).

[0045] 21. The conjugate or pharmaceutically acceptable salt thereof of item 20, wherein the linker comprises from 2 to 10 PEG units.

[0046] 22. The conjugate or pharmaceutically acceptable salt thereof of any one of items 1 to 21 , wherein the linker is of one of the following structures:

[0047] • CO(CH2)aCONH(CH2)bCO, wherein a and b are independently 1 , 2, 3, or 4;

[0048] • CO(CH2)d(OCH2CH2)eCONH(CH2)fCO, wherein d and f are independently 1 , 2, 3, or 4, and e is an integer from 2 to 8; or 23. The conjugate or pharmaceutically acceptable salt thereof of item 1 , wherein the conjugate has one of the following structures:

[0049]

[0050]

[0051] 24. A pharmaceutical composition comprising the conjugate or salt thereof of any one of items 1 to 23, and a pharmaceutically acceptable excipient.

[0052] 25. A method for treating a Sortilin-expressing cancer in a subject in need thereof comprising administering to the subject an effective amount of the conjugate or salt thereof of any one of items 1 to 23, or the pharmaceutical composition of item 24.

[0053] 26. The method of item 25, wherein the Sortilin-expressing cancer is a hematological cancer, ovarian cancer, endometrial cancer, cervix cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small intestine cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testis cancer, bladder cancer, kidney cancer, or pancreatic cancer.

[0054] 27. The method of item 25 or 26, wherein the Sortilin-expressing cancer is a poor prognosis cancer.

[0055] 28. The method of any one of items 25 to 27, wherein the Sortilin-expressing cancer is an immunologically cold cancer.

[0056] 29. The method of any one of items 25 to 28, wherein the conjugate, salt thereof or composition is administered in combination with one or more additional active agents or therapies for cancer.

[0057] 30. The method of item 29, wherein the one or more additional active agents or therapies for cancer comprise an immunotherapy. 31. The method of item 30, wherein the immunotherapy comprises an immune checkpoint inhibitor.

[0058] 32. The method of item 31 , wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

[0059] 33. The method of item 32, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0060] 34. Use of the conjugate or salt thereof of any one of items 1 to 23, or the pharmaceutical composition of item 24, for treating a Sortilin-expressing cancer in a subject.

[0061] 35. Use of the conjugate or salt thereof of any one of items 1 to 23, or the pharmaceutical composition of item 24, for the manufacture of a medicament for treating a Sortilin-expressing cancer in a subject.

[0062] 36. The use of item 34 or 35, wherein the Sortilin-expressing cancer is a hematological cancer, ovarian cancer, endometrial cancer, cervix cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small intestine cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testis cancer, bladder cancer, kidney cancer, or pancreatic cancer.

[0063] 37. The use of any one of items 34 to 36, wherein the Sortilin-expressing cancer is a poor prognosis cancer.

[0064] 38. The use of any one of items 34 to 37, wherein the Sortilin-expressing cancer is an immunologically cold cancer.

[0065] 39. The use of any one of items 34 to 38, wherein the conjugate, salt thereof, composition or medicament is for administration in combination with one or more additional active agents or therapies for cancer.

[0066] 40. The use of item 39, wherein the one or more additional active agents or therapies for cancer comprise an immunotherapy.

[0067] 41 . The use of item 40, wherein the immunotherapy comprises an immune checkpoint inhibitor.

[0068] 42. The use of item 41 , wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

[0069] 43. The use of item 42, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0070] 44. The conjugate or salt thereof of any one of items 1 to 23, or the pharmaceutical composition of item 24, for use in the treatment of a Sortilin-expressing cancer in a subject.

[0071] 45. The conjugate, salt thereof or pharmaceutical composition for use according to item 44, wherein the Sortilin-expressing cancer is a hematological cancer, ovarian cancer, endometrial cancer, cervix cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small intestine cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testis cancer, bladder cancer, kidney cancer, or pancreatic cancer. 46. The conjugate, salt thereof or pharmaceutical composition for use according to item 45, wherein the Sortilin-expressing cancer is a poor prognosis cancer.

[0072] 47. The conjugate, salt thereof or pharmaceutical composition for use according to item 45 or 46, wherein the Sortilin-expressing cancer is an immunologically cold cancer.

[0073] 48. The conjugate, salt thereof or pharmaceutical composition for use according to any one of items 45 to 47, wherein the conjugate, salt thereof or composition is for administration in combination with one or more additional active agents or therapies for cancer.

[0074] 49. The conjugate, salt thereof or pharmaceutical composition for use according to item 48, wherein the one or more additional active agents or therapies for cancer comprise an immunotherapy.

[0075] 50. The conjugate, salt thereof or pharmaceutical composition for use according to item 49, wherein the immunotherapy comprises an immune checkpoint inhibitor.

[0076] 51. The conjugate, salt thereof or pharmaceutical composition for use according to item 50, wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

[0077] 52. The conjugate, salt thereof or pharmaceutical composition for use according to item 51 , wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

[0078] Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0079] BRIEF DESCRIPTION OF DRAWINGS

[0080] In the appended drawings:

[0081] FIGs. 1A-1D depict the structure of peptide drug conjugates TH2101 (FIG. 1A), TH2102 (FIG. 1B), TH2204 (FIG. 1 C), and TH2205 (FIG. 1D).

[0082] FIGs. 2A-2D depict the structure of peptide drug conjugates TH2301 (FIG. 2A), TH2303 (FIG. 2B), TH2304 (FIG. 2C), and TH2207 (FIG. 2D).

[0083] FIG. 3 depicts the structure of peptide drug conjugate TH2302.

[0084] FIG. 4 depicts the synthetic scheme for the TH2101 conjugate (TH19P01-Osu-SN-38).

[0085] FIG. 5 depicts the synthetic scheme for the TH2102 conjugate (TH19P01-Osu-lrinotecan).

[0086] FIG. 6 depicts the synthetic scheme for the TH2205 conjugate (TH19P01-Gly-(PEG)3-SN- 38).

[0087] FIGs. 7A-B depict the synthetic scheme for the TH2302 conjugate (KBP201-Gly-(PEG)3- SN-38).

[0088] FIG. 8 depicts the synthetic scheme for the TH2301 conjugate (TH19P01-Osu-Exatecan).

[0089] FIGs. 9A-C depicts the synthetic scheme for the TH2303 conjugate (TH19P01-Glyc-Osu- Exatecan). FIG. 10 depicts the synthetic scheme for the TH2304 conjugate (TH19P01-Osu- Deruxtecan).

[0090] FIG. 11A shows the detection of sortilin, P-gp, BCRB and EGFR in human colon cancer cell lines. Western blots were performed with cells lysates obtained from different colorectal cancer cell lines as well as in TNBC MDA-MB231 cells (positive control for sortilin) and in MDCK cells transfected with human MDR1 (MDCK-MDR1).

[0091] FIGs. 11B-11C show the detection of Topoisomerase 1 (TOPO1) in human colorectal cancer (CRC, FIG. 11B) and breast cancer (FIG. 11C) cell lines. Western blots were performed with cells lysates obtained from different CRC cell lines as well as in TNBC cell lines.

[0092] FIGs. 12A and B show the anti-proliferative activity of PDCs and unconjugated drugs on HT-29, HCT-116 and LoVo cancer cells. Cancer cells were incubated with increasing concentrations of PDC and unconjugated drugs as indicated in Example 1 . The concentration at which they exert half of their maximal inhibitory effect estimated by the MTT assay and calculated using the GraphPad Prism software is indicated.

[0093] FIGs. 13A-13B show that the uptake of the fluorescent peptide TH19P01-Alexa488 is sortilin-dependent in HT-29 cancer cells. Uptake of the fluorescent peptide TH19P01-Alexa488 was measured in cells transfected with either the control scrambled siRNA(siScr) or siRNA against sortilin (siSortl) as described in Example 1. FIG. 13A: Sortilin was detected by Western Blot in cells transfected with either siScr or siSortl siRNAs. Results show sortilin detection when siSortl were used. FIG. 13B: The uptake of the fluorescent TH19P01-Alexa488 was reduced in cells transfected with specific siRNA against sortlin (siSortl) relative to the uptake measured in cells transfected with scramble siRNA (siScr) (n = 2).

[0094] FIGs. 14A-14C show that the uptake the TH2101 conjugate is sortilin-dependent in HT-29 cancer cells. Uptake of the fluorescent peptide TH2101 was measured in HT-29 cancer cells transfected with either the control scrambled siRNA(siScr) or siRNA against sortilin (siSortl). Fluorescence signal intensity was acquired by confocal microscopy as described in the Examples. FIG. 14A: Sortilin was detected by Western Blot in cells transfected with either siScr or siSortl siRNAs. Results show that sortilin expression was reduced by 64% when siSortl was used. FIG. 14B: Images obtained by confocal microscopy showed that the uptake of the fluorescent TH2101 was reduced in cells transfected with siSortl when compared to the uptake measured in cells transfected with siScr. FIG. 14C: Quantification of the fluorescence levels indicated that the uptake of TH2101 in cells transfected with siSortl was almost reduced to control levels (n = 5).

[0095] FIGs. 15A-15C show the in vivo efficacy of unconjugated drugs in the HT-29 xenograft model. HT-29 cells were subcutaneously implanted in the flanks of mice. The animals were treated weekly with either irinotecan or exatecan at the indicated doses when tumor reached about 100-150 mm3. FIGs. 15A-15B show the tumor volume in mice treated with irinotecan at doses of 12.5 mg / kg, 25 mg / kg, or 90-100 mg / kg (FIG. 15A), or exatecan at a dose of 9.2 mg / kg (FIG. 15B). FIG. 15C: Tumor growth inhibition in percentage (TGI%) for each unconjugated drug at different doses are shown.

[0096] FIGs. 16A-16C show the in vivo efficacy of the peptide-irinotecan conjugate TH2102 in the HT-29 xenograft model. The animals were treated weekly with either increased doses of irinotecan or different formulations of TH2102. Treatments were initiated when tumor reached about 100-150 mm3. FIG. 16A shows the tumor volume in mice treated with different doses of unconjugated irinotecan and different formulations of the peptide-irinotecan conjugate TH2102. FIG. 16B: Tumor growth inhibition in percentage (TGI%) for irinotecan and TH2102 are shown. FIG. 16C: Mouse body weight was also monitored for the different groups and remained within the range established for the endpoint limits.

[0097] FIGs. 17A-17C show the in vivo efficacy of the peptide-irinotecan-PEG-conjugate (TH2204) and peptide-SN-38-conjugate (TH2101) on HT-29 xenograft model. HT-29 cells were subcutaneously implanted in the flanks of mice. The animals were treated weekly with either increased doses of irinotecan, or TH2204 and TH2101 at the indicated doses when tumor reached about 100-150 mm3. FIG. 17A shows the tumor volume in mice treated with unconjugated irinotecan, TH2204 or TH2101. FIG. 17B: Tumor growth inhibition in percentage (TGI%) at different dose of irinotecan, TH2204 or TH2101. FIG. 17C: Mouse body weight was also monitored for the different groups and remained within the range established for the endpoint limits.

[0098] FIGs. 18A-18E show the in vivo efficacy of peptide-SN-38 conjugates (TH2101 , TH2205) and peptide-exatecan-conjugates (TH2301 and TH2303) on HT-29 xenograft model. HT-29 cells were subcutaneously implanted in the flanks of mice. The animals were treated weekly with exatecan, TH2101 , TH2205 or TH2301 at the indicated doses when tumor reached about 100- 150 mm3. FIG. 18A shows the tumor volume in mice treated with exatecan, TH2101 , TH2205 or TH2301. FIG. 18B: Tumor growth inhibition in percentage (TGI%) for exatecan, TH2101 , TH2205 and TH2301 at an exatecan equivalent dose (9.2 mg / kg). FIG. 18C shows the body weight in mice treated with exatecan, TH2101 , TH2205 or TH2301 . Mouse body weight was monitored for the different groups and remained within the range established for the endpoint limits. FIG. 18D shows the tumor volume in mice treated with exatecan (9.2 or 18.4 mg / kg) or peptide-exatecan- conjugate TH2303 (32.4 mg / kg, 48.6 mg / kg or 64.8 mg / kg). FIG. 18E shows the body weight in mice treated with exatecan (9.2 or 18.4 mg / kg) or peptide-exatecan-conjugate TH2303 (32.4 mg / kg, 48.6 mg / kg or 64.8 mg / kg). Mouse body weight was monitored for the different groups and remained within the range established for the endpoint limits.

[0099] FIGs. 19A-B show the antitumor activity for a peptide-exatecan conjugate (TH2303) in the HCT-116 CRC xenograft model. HCT-116 cells were subcutaneously implanted in the flanks of mice. The animals were treated weekly with either vehicle, irinotecan, exatecan or TH2303 at the indicated doses. Treatments were initiated when tumor reached about 100-150 mm3. FIG. 19A: Tumor volume was measured using a caliper as described in Example 1 . FIG. 19B: Mouse body weight was also monitored for the different groups and remained within the range established for the endpoint limits.

[0100] FIGs. 20A-B show the antitumor activity for the peptide-exatecan conjugate (TH2303) in the MDA-MB-231 TNBC xenograft model. MDA-MB-231 cells were subcutaneously implanted in the flanks of mice. The animals were treated weekly with either vehicle, irinotecan, exatecan or TH2303 at the indicated doses. Treatments were initiated when tumor reached about 100-150 mm3. FIG. 20A: Tumor volume was measured using a caliper as described in Example 1. FIG. 20B: Mouse body weight was also monitored for the different groups and remained within the range established for the endpoint limits.

[0101] FIGs. 21A-B show the antitumor activity for the peptide-exatecan conjugate (TH2303) in the ovarian cancer SKOV3 xenograft model (FIG. 21A) or a colorectal cancer patient-derived xenograft (PDX) model (FIG. 21 B). SKVO3 cells were subcutaneously implanted in the flanks of mice for the ovarian xenograft tumor model whereas small tumor pieces are implanted in the flanks of mice in the case of the colorectal PDX model. The animals were treated weekly with either vehicle, exatecan or TH2303 at the indicated doses. Treatments were initiated when tumor reached about 100-150 mm3.

[0102] DETAILED DISCLOSURE

[0103] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0104] The terms "comprising", "having", "including", and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0105] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0106] The use of any and all examples, or exemplary language (“e.g.”, "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

[0107] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0108] Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

[0109] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0110] Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.

[0111] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0112] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0113] The present disclosure provides a conjugate comprising the following structure I, or a pharmaceutically acceptable salt thereof:

[0114] A — L — B (I); wherein

[0115] A is a camptothecin analog;

[0116] B is a peptide binding to a Sortilin receptor, wherein the peptide comprises an amino acid sequence having at least 60% identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2:

[0117] X1GVRAKAGVRN(Nle)FKSESYX2(SEQ ID NO:1) X1YKSLRRKAPRWDAPLRDPALRQLLX2(SEQ ID NO:2); wherein X1is cysteine (C) or is absent, and X2is cysteine (C) or is absent;

[0118] L is a linker, wherein a first end of the linker is attached to a side chain of one or more of the amino acid or modified side chain residues of the peptide (such as, azido, double bond, triple bond and others), and / or to the N- and / or C-terminal end of the peptide, and a second end of the linker is attached to a hydroxy, carboxy or nitrogen atom of the camptothecin analog.

[0119] The term “Sortilin” or “Sortilin receptor” as used herein refers to a neuronal type-1 membrane glycoprotein, encoded by the SORT 1 gene, belonging to the Vacuolar Protein Sorting 10 protein (Vps10) family of receptors. Sortilin (also known as the neurotensin receptor 3; UniProtKB Accession number Q99523) is expressed or overexpressed in a number of cancers including for example ovarian, breast, colon and prostate cancer. The encoded preproprotein (residues 34-831 , residues 1-33 corresponding to the signal peptide) is proteolytically processed after amino acid 77 by furin (or other homologous proteases) to generate the mature receptor with a molecular weight of about 100-110 kDa (residues 78-831). Amino acid residues of sortilin referenced herein correspond to positions in the full-length form ( / .e., UniProtKB Accession number Q99523).

[0120] The term “amino acid” refers to the common natural (genetically encoded) or synthetic amino acids and common derivatives thereof, known to those skilled in the art. When applied to amino acids, “standard” or “proteinogenic” refers to the genetically encoded 20 amino acids in their natural configuration. Similarly, when applied to amino acids, “non-standard,” “unnatural” or “unusual” refers to the wide selection of non-natural, rare or synthetic amino acids such as those described by Hunt, S. in Chemistry and Biochemistry of the Amino Acids, Barrett, G.C., ed., Chapman and Hall: New York, 1985. Some examples of non-standard amino acids include nonalpha amino acids and D-amino acids. In an embodiment, the peptide compound comprises only natural amino acids. In another embodiment, the peptide compound comprises one or more nonnatural or synthetic amino acids, such as D-amino acids.

[0121] The expression "sequence identity" as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, over their full length ( / .e., % identity=number of identical overlapping positions / total number of positions x 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0122] In embodiments, the peptide comprises or consists of an amino acid sequence having at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 1 , wherein the peptide binds to Sortilin.

[0123] In an embodiment, the peptide comprises 25, 24, 23, 22, 21 , 20, or 19 residues or less and comprises the sequence GVRAKAGVRN(Nle)FKSESY (SEQ ID NO:3). In another embodiment, the peptide compound comprises 25, 24, 23, 22, 21 , 20, or 19 residues or less and comprises the sequence GVRAKAGVRN(Nle)FKSESYC (SEQ ID NO:4).

[0124] In embodiments, the peptide has an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least

[0125] 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least

[0126] 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least

[0127] 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0128] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

[0129] 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 2, wherein the peptide binds to Sortilin. In an embodiment, the peptide comprises at least 15, 16 or 17 contiguous residues from SEQ ID NO: 2, wherein the peptide binds to Sortilin.

[0130] In an embodiment, the peptide comprises 30, 29, 28, 27, or 26 residues or less and comprises the sequence YKSLRRKAPRWDAPLRDPALRQLL (SEQ ID NO:5). In another embodiment, the peptide compound comprises 30, 29, 28, 27, or 26 residues or less and comprises the sequence YKSLRRKAPRWDAPLRDPALRQLLC (SEQ ID NO:6). The term “camptothecin analog” as used herein refers to compounds having a structure similar to that of camptothecin (a planar pentacyclic ring structure) and having the ability to inhibit Topoisomerase 1 (Topi) activity. Examples of camptothecin analogs include Belotecan, Topotecan, 9-Aminocamptothecin (9-AC), Irinotecan, Rubitecan, Lurtotecan, SN-38, Exatecan, Deruxtecan, ZD06519 (FD1) (Petersen et al., Mol Cancer Ther. 2024 May 2; 23(5): 606-618), hTop1 inhibitor 12 (Khodair etal., European Journal of Medicinal Chemistry, Volume 265, 116049 (2024)), and FLQY2 (Wang et al., Bioorganic Chemistry, Volume 148, 107436 (2024)). Camptothecin analogs are also described in Venditto and Simanek Mol Pharm. 2010 April 5; 7(2): 307-349) and Chen et al. (J. Med. Chem. 2024, 67, 5, 3244-3273). In an embodiment, the camptothecin analog is Irinotecan. In an embodiment, the camptothecin analog is SN-38. In an embodiment, the camptothecin analog is Exatecan. In an embodiment, the camptothecin analog is Deruxtecan.

[0131] The term “linker” as used herein means a chemical structure connecting a peptide compound herein disclosed to the camptothecin analog. The linker can be connected to the peptide compound at different functional groups on the peptide compound. For example, the linker can be connected to the peptide compound at one or moreprimary amines (amines (- NH2): this group exists at the N-terminus of each polypeptide chain (called the alpha-amine) and in the side chain of lysine (Lys, K) residues (called the epsilon-amine). For example, the linker can be connected to the peptide compound at one or more carboxyl groups (- COOH): this group exists at the C-terminus of each polypeptide chain and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E). For example, the linker can be connected to the peptide compound at one or more sulfhydryl groups (-SH): This group exists in the side chain of cysteine (Cys, C). Often, as part of a protein's secondary or tertiary structure, cysteines are joined together between their side chains via disulfide bonds (-S-S-). These must be reduced to sulfhydryls to make them available for crosslinking by most types of reactive groups. For example, the linker can be connected to the peptide compound at one or more a carbonyl groups (-CHO): Ketone or aldehyde groups can be created in glycoproteins for example by oxidizing polysaccharide post-translational modifications (glycosylation) with sodium meta-periodate. The linker may also be attached to suitable groups / moieties on modified side chain residues of the peptide (such as azido, double bond, and triple bond).

[0132] In an embodiment, L is a cleavable linker, wherein a first end of the linker is attached to a side chain of one or more of the lysine (K) and / or cysteine (C) residues of the peptide, and / or to the N- and / or C-terminal end of the peptide, and a second end of the linker is attached to an oxygen or nitrogen atom of the camptothecin analog. Through the use of cleavable linkers, the camptothecin analog can be cleaved from the peptide at the surface of a Sortilin receptor-expressing cell, inside the cell, or inside a cell organelle, e.g., by an enzyme (e.g., a protease), thereby releasing the camptothecin analog, e.g., in the vicinity of the cell or inside the cell or cell organelle). The linkers described herein are used to attach the camptothecin analog to the peptides that binds to a cancer cell (e.g., binds to cancer cells expressing a Sortilin receptor) and gets internalized into the cell (e.g., through the endosome and / or lysosomal compartment(s)). Once internalized, the linkers are cleaved or degraded to release the camptothecin analogs.

[0133] Various types of cleavable linkers may be used in the conjugates described herein. For example, the linker may be an acid-cleavable linker that exploits the natural acidity of endosomes and lysosomes (pH 4.5-6.2) which contrasts with the neutral pH (7.4) of the plasma. The most commonly used acid-cleavable linkers are hydrazones and carbonates. The cleavable linker may also be a reducible or disulfide linker. Such linkers are stable at physiological pH but susceptible to nucleophilic attacks from thiols. Glutathione (GSH) that contains exposed and highly reactive thiol groups is present in the cytosol of the cells, especially in cells under oxidative stress such as tumor cells. Other types of cleavable linkers include phosphatase cleavable linkers (cleaved by phosphatase and pyrophosphates in lysosomes) and sulfatase cleavable linkers (cleaved by sulfatases in lysosomes). The cleavable linker may also be an enzyme-cleavable linker, i.e., a linker that is cleaved by the action of intracellular and / or extracellular enzymes expressed by the target cells. Examples of enzyme-cleavable linkers include glycosidase-cleavable linkers (e.g., |3- glucuronidase or p-galactosidase cleavable linker) as well as protease cleavable linkers.

[0134] In an embodiment, the cleavable linker is a protease-cleavable linker. The protease- cleavable linker can release the camptothecin analog under the action of proteases such as cathepsin (e.g., cathepsin A. cathepsin B), trypsin or other proteases expressed by the cell (e.g., tripeptidyl-peptidase I), for example expressed in the endosomal and / or lysosomal compartment(s) of the cell.

[0135] In an embodiment, the cleavable linker comprises one or more amino acid residues, e.g., natural and / or or non-natural amino acid residues. In an embodiment, the cleavable linker comprises from 2 to 4 amino acid residues. In an embodiment, the cleavable linker comprises one of the following dipeptides: Val-Lys, Val-Cit, Phe-Lys, Trp-Lys, Asp-Lys, Val-Arg, or Val-Ala. In other embodiments, the cleavable linker comprises one of the following tetrapeptides: Val-Phe- Gly-Sar, Val-Cit-Gly-Sar, Val-Lys-Gly-Sar, Val-Ala-Gly-Sar, Val-Phe-Gly-Pro (SEQ ID NO:7), Val- Cit-Gly-Pro, Val-Lys-Gly-Pro (SEQ ID NO:8), or Val-Ala-Gly-Pro (SEQ ID NO:9).

[0136] The linkers may be prepared from linker precursors that contain reactive groups at one or both ends of the molecule. The reactive groups can be selected to allow conjugation to camptothecin analog at one end, and also facilitate conjugation to the peptide at the other end. It is desirable for the camptothecin analog to contain an amine, a hydroxyl, hydrazone, hydrazide or a sulfhydryl group in order to facilitate conjugation to the linker. For example, camptothecin analogs comprise primary amine groups and / or hydroxyl groups that can facilitate their conjugation to the linker. In an embodiment, the linker is conjugated to an amino group of the camptothecin analog. In another embodiment, the linker is conjugated to a hydroxyl group of the camptothecin analog.

[0137] The linker may further comprise additional groups, e.g., spacer groups, to increase the distance between the peptide and the camptothecin analog. Suitable spacer groups may comprise an alkylene, alkenylene, alkynylene, heteroalkylene (e.g., polyethylene glycol, PEG), carbocyclyl, heterocyclyl, aryl, heteroaryl, or a combination thereof. For example, the spacer group may comprise a PEG group, an alkylene group, or a combination thereof. The spacer group may be substituted or unsubstituted, e.g., the spacer group may comprise a substituted alkylene, substituted heteroalkylene, or a combination thereof. For example, the spacer group may comprise a PEG group (or PEG spacer), an alkylene group (or alkylene spacer), one or more heteroatoms, and / or one or more or cyclic groups. In an embodiment, the spacer group comprises 1 to 20 PEG groups. In further embodiments, the spacer group comprises 1 to 10, 1 to 8, 1 to 6, 1 to 5, or 2 to 4 PEG groups. In a further embodiment, the spacer group comprises 3 PEG groups.

[0138] In an embodiment, the linker is of one of the following structures:

[0139] • CO(CH2)aCONH(CH2)bCO, wherein a and b are independently 1 , 2, 3, or 4;

[0140] • CO(CH2)d(OCH2CH2)eCONH(CH2)fCO, wherein d and f are independently 1 , 2, 3, or 4, and e is an integer from 2 to 8; or

[0141] It is to be understood that several camptothecin analog molecules A (which may be the same or different) may be attached to a single peptide molecule B through several linkers L (which may be the same or different). In an embodiment, 1 , 2, 3 or 4 camptothecin analog molecules are attached to a single peptide molecule through linkers.

[0142] Thus, in an embodiment, the conjugate or salt thereof is of one of the following formulas la, lb, Ic or Id:

[0143] A1 — L1 — B (la);

[0144] A1 — L1 — B — L2 — A2 (lb); wherein L1 , L2, L3 and L4 are linkers (same or different), and A1 , A2, A3 and A4 are camptothecin analog molecules (same or different). In an embodiment, A1 , A2, A3 and A4 are the same camptothecin analog molecules. In an embodiment, L1 , L2, L3 and L4 are the same cleavable linkers.

[0145] In an embodiment, X1and / or X2is C, and wherein the first end of one of the linkers is attached to the side chain of X1and / or X2. In a further embodiment, X2is C, and wherein the first end of one of the linkers is attached to the side chain of X2.

[0146] In an embodiment, the first end of one or more of the linkers is attached to the side chain of one or more lysine (K) residues of the peptide B. In a further embodiment, the first ends of two of the linkers are attached to the side chain of two lysine (K) residues of the peptide B.

[0147] In an embodiment, the conjugate has one of the following structures:

[0148]

[0149]

[0150] 5 or

[0151]

[0152] In an embodiment, the conjugate compound or pharmaceutically acceptable salt thereof disclosed herein is formulated into a pharmaceutical composition. In an embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0153] Such compositions may be prepared in a manner well known in the pharmaceutical art by mixing the conjugate compound having a suitable degree of purity with one or more optional pharmaceutically acceptable carriers or excipients (see Remington: The Science and Practice of Pharmacy, by Loyd Allen, Jr, 2012, 22ndedition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe et al., 2012, 7thedition, Pharmaceutical Press). The carrier / excipient can be suitable for administration of the conjugate compound by any conventional administration route, for example, for oral, intratumoral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration. In an embodiment, the carrier / excipient is adapted for administration of the conjugate compound or salt thereof by the intravenous or subcutaneous route. In an embodiment, the carriers / excipients are adapted for administration of the conjugate compound by the intravenous route. In another embodiment, the carriers / excipients are adapted for administration of the conjugate compound or salt thereof by the subcutaneous route. In another embodiment, the carriers / excipients are adapted for administration of the conjugate compound or salt thereof by the oral route.

[0154] An "excipient" as used herein has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient" as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients and that is not toxic to the subject, i.e., is a type of excipient and / or is for use in an amount which is not toxic to the subject. Excipients are well known in the art, and the present system is not limited in these respects. In certain embodiments, the composition may include excipients such as one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants / dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof. As those of skill would recognize, a single excipient can fulfill more than two functions at once, e.g., can act as both a binding agent and a thickening agent. As those of skill will also recognize, these terms are not necessarily mutually exclusive. Examples of commonly used excipients for injectable formulations include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifying agents, preservatives, or buffers, which increase the shelf life or effectiveness.

[0155] In another aspect, the present disclosure provides a method for treating a Sortilin- expressing cancer in a subject in need thereof comprising administering to the subject an effective amount of the conjugate compound, salt thereof or composition disclosed herein. The present disclosure also provides the use of the conjugate compound, salt thereof or composition disclosed herein for treating a Sortilin-expressing cancer, or for the manufacture of a medicament for treating a Sortilin-expressing cancer, in a subject. The present disclosure also provides the conjugate compound, salt thereof or composition disclosed herein for use in treating a Sortilin- expressing cancer in a subject.

[0156] The Sortilin-expressing cancer may be any type of cancer, including a primary (or original) cancer, a relapsing cancer or a metastatic cancer, in which at least a subset of the tumor cells express Sortilin. Examples of cancers include heart sarcoma, lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma (e.g., Ewing’s sarcoma, Karposi's sarcoma), lymphoma, chondromatous hamartoma, mesothelioma; cancer of the gastrointestinal system, for example, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), gastric, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); cancer of the genitourinary tract, for example, kidney cancer (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and / or urethra cancer (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate cancer (adenocarcinoma, sarcoma), testis cancer (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver cancer, for example, hepatoma (hepatocellular carcinoma, HCC), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma); bone cancer, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; cancer of the nervous system, for example, neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); cancer of the reproductive system, for example, gynecological cancer, uterine cancer (endometrial carcinoma), cervical cancer (cervical carcinoma, pre-tumor cervical dysplasia), ovarian cancer (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulvar cancer (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vaginal cancer (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tube cancer (carcinoma); placenta cancer, penile cancer, prostate cancer, testicular cancer; cancer of the hematologic system, for example, blood cancer (acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; cancer of the oral cavity, for example, lip cancer, tongue cancer, gum cancer, palate cancer, oropharynx cancer, nasopharynx cancer, sinus cancer; skin cancer, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids; adrenal gland cancer: neuroblastoma; and cancers of other tissues including connective and soft tissue, retroperitoneum and peritoneum, eye cancer, intraocular melanoma, and adnexa, breast cancer (e.g., ductal breast cancer), head or / and neck cancer (head and neck squamous cell carcinoma), anal cancer, thyroid cancer, parathyroid cancer; secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites. In an embodiment, the Sortilin-expressing cancer is a hematological cancer (e.g., AML), ovarian cancer, endometrial cancer, cervix cancer, skin cancer (e.g., melanoma, squamous cell skin cancer), brain cancer (e.g., glioma, glioblastoma), breast cancer (e.g., triple-negative breast cancer), colorectal cancer, small intestine cancer, liver cancer, lung cancer (e.g., small cell lung carcinoma, non small cell lung carcinoma), eye cancer (e.g., ocular melanoma), prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testis cancer, bladder cancer, kidney cancer, or pancreatic cancer.

[0157] In an embodiment, the cancer is a poor prognosis cancer. The term “poor prognosis cancer” as used herein refers to a subtype of a given cancer that is associated with lower survival rate (e.g., 5-year or 10-year survival rate) relative to other subtype(s) of the same cancer. Poor prognosis cancer is generally associated with specific characteristics of the cancer subtype, for example the presence of certain mutations, chromosomal abnormalities, etc., that renders them more resistant to treatment. Poor prognosis is also associated with cancers diagnosed at a later stage (e.g., with distant metastasis). For example, for breast cancer, triple-negative breast cancer (TNBC) is considered a poor prognosis breast cancer as it is associated with a lower 5-year relative survival rate relative to other breast cancer subtypes. For ovarian cancer, invasive epithelial ovarian cancer and fallopian tube cancer are generally associated with a lower 5-year relative survival rate relative to ovarian stromal tumors and germ cell tumors. The 5-year overall survival rate of pancreatic cancer is very low (about 3%), which is partly because more than half of the patients are diagnosed at an advanced stage. Diagnosis of pancreatic cancer at stage II l / IV (with distant metastasis) is associated with very poor prognosis. Similarly, for prostate cancer, diagnosis at stage IV (with distant metastasis) is associated with poor prognosis (5-year relative survival rate of less than 30% compared to at least 80-85% for diagnosis at stages l-lll). For lung cancer, small cell lung cancer is associated with particularly poor prognosis, especially when diagnosed at a later stage (e.g., with regional or distant metastasis). Non-small cell lung cancer diagnosed at a later stage (e.g., with distant metastasis) is also associated with poor prognosis. In colorectal cancer, mucinous adenocarcinomas (characterized by the presence of abundant extracellular mucin) have been associated with reduced response to chemotherapy and poor prognosis. Peritoneal involvement and BRAF mutations also constitute poor prognosis markers for colorectal cancer. For kidney cancer, clear cell RCC is associated with worse outcomes (e.g., lower 5-year relative survival rate) than papillary RCC. In skin cancer, thicker tumors, nodal involvement and diagnosis at a later stage (e.g., with regional or distant metastasis) are associated with lower survival in melanoma.

[0158] In an embodiment, the poor prognosis cancer is a stage lll / IV cancer. In an embodiment, the poor prognosis cancer is a stage III cancer. In an embodiment, the poor prognosis cancer is a stage IV cancer.

[0159] In an embodiment, the poor prognosis cancer is a cancer having a 5-year relative survival rate of less than 60%. In an embodiment, the poor prognosis cancer is a cancer having a 5-year relative survival rate of less than 50%. In an embodiment, the poor prognosis cancer is a cancer having a 5-year relative survival rate of less than 40%. In an embodiment, the poor prognosis cancer is a cancer having a 5-year relative survival rate of less than 30%. In an embodiment, the poor prognosis cancer is a cancer having a 5-year relative survival rate of less than 20%. In an embodiment, the poor prognosis cancer is a cancer having a 5-year relative survival rate of less than 10%. In an embodiment, the poor prognosis cancer is a cancer having a 5-year relative survival rate of less than 5%.

[0160] In an embodiment, the cancer is an immunologically cold (or ignorant) cancer. The term “immunologically cold cancer” or “cold cancer” refers to a cancer that does not trigger an antitumor immune response in the patient and / or that does not respond to cancer immunotherapies such as ICI therapy (see, e.g., Bonaventura et al., “Cold Tumors: A Therapeutic Challenge for Immunotherapy.” Frontiers in immunology vol. 10 168 (2019). Immunologically cold cancers are characterized by the absence of infiltration of antitumor immune cells such as TILs, TAMs and / or NK cells in the tumor and / or the presence of high levels of immunoregulatory cells (e.g., Tregs). A number of breast cancers, ovarian cancers, prostate cancers, pancreatic cancers, and glioblastomas are considered immunologically cold cancers. Subtypes of several cancers are also considered immunologically cold cancers including subtypes of lung cancers such as non-small- cell lung cancers (NSCLC) (Cascone et al., Tumor Immunology and Immunotherapy of Non- Small-Cell Lung Cancer, Cold Spring Harb Perspect Med. 2022 May 27;12(5):a037895), renal cancers such as chromophobe renal cell carcinoma (non-clear cell renal cell carcinoma, nccRCC) (Zarrabi et al., Immune Checkpoint Inhibition in Advanced Non-Clear Cell Renal Cell Carcinoma: Leveraging Success from Clear Cell Histology into New Opportunities, Cancers 2021 vol. 13(15):3652), high tumor mutational burden RCC (Yakirevich etal., Tumor mutational burden and immune signatures interplay in renal cell carcinoma. Ann Transl Med. 2020;8(6):269), colorectal cancers (CRC) such as consensus molecular subtype (CMS) 2 and CMS3 CRC (Roelands et al., Immunogenomic Classification of Colorectal Cancer and Therapeutic Implications, Int J Mol Sci. 2017;18(10):2229), head and neck squamous cell carcinoma (Ribbat-ldel et al., Immunologic “Cold” Squamous Cell Carcinomas of the Head and Neck Are Associated With an Unfavorable Prognosis, Frontiers in medicine 2021 ;8:622330), oesophageal cancer (Puhr et al., Immunotherapy for Esophageal Cancers: What Is Practice Changing in 2021?, Cancers 2021 ; vol. 13(18):4632), liver cancer such as stage II hepatocellular carcinoma (Nguyen et al., Nature Communications volume 13, Article number: 1441 (2022)). Homozygous deletion of 9p21.3, one of the most frequent genomic defects occurring in ~13% of all cancers including melanoma (SKCM), bladder (BLCA), pancreatic cancer (pancreatic adenocarcinoma), gastric cancer (stomach adenocarcinoma), lung adeno- (LUAD) and squamous-cell carcinoma (LUSC)], has been shown to be associated with an immunologically cold phenotype.

[0161] The exact amount / dosage of conjugate to be administered will vary according to factors such as the specific cancer cell involved, and the specific cancer disease; the degree of or involvement or the severity of the cancer disease; the size, age, and general health of the cancer patient; the response of the individual patient; the particular compound administered; the bioavailability characteristics of the preparation administered; the dose regimen selected; whether the conjugate is administered alone or in combination with other agents; pharmacodynamic characteristics of the conjugate and their mode and route of administration; and other relevant characteristics that the physician or as one skilled in the art, will readily determine by the use of known techniques and by observing results obtained under analogous circumstances. The conjugate / composition is suitably administered to the patient at one time or over a series of treatments. Preferably, it is desirable to determine the dose-response curve in vitro, and then in useful animal models prior to testing in humans. The present disclosure provides dosages for the conjugates and compositions comprising same. For example, depending on the type and severity of the disease, about 1 pg / kg to 1000 mg per kg (mg / kg) of body weight per day. Further, the effective dose may be 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg / 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, and may increase by 25 mg / kg increments up to 1000 mg / kg, or may range between any two of the foregoing values. A typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0162] The conjugate compound or salt thereof or composition comprising same described herein may be used in combination with one or more additional active agents or therapies (radiotherapy, surgery, vaccines, etc.) for the treatment the targeted disease / condition or for the management of one or more symptoms of the targeted disease / condition (e.g., pain killers, anti-nausea agents, etc.). In an embodiment, the conjugate compound described herein is used in combination with one or more chemotherapeutic agents, immunotherapies, checkpoint inhibitors, cell-based therapies, etc. Examples of chemotherapeutic agents suitable for use in combination with the conjugate described herein include, but are not limited to, vinca alkaloids, agents that disrupt microtubule formation (such as colchicines and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agent (such as tyrosine kinase inhibitors), transitional metal complexes, proteasome inhibitors, antimetabolites (such as nucleoside analogs), alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acids or a derivatives thereof); geldanamycin or a derivative thereof (such as 17-AAG), and other cancer therapeutic agents recognized in the art. In some embodiments, chemotherapeutic agents for use in combination with the conjugate described herein comprise one or more of adriamycin, colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferons, camptothecin and derivatives thereof, phenesterine, taxanes and derivatives thereof (e.g., taxol, paclitaxel and derivatives thereof, taxotere and derivatives thereof, and the like), topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab- 5801 , Irinotecan, HKP, Ortataxel, gemcitabine, Oxaliplatin, Herceptin®, vinorelbine, Doxil®, capecitabine, Alimta®, Avastin®, Velcade®, Tarceva®, Neulasta®, lapatinib, sorafenib, erlotinib, erbitux, derivatives thereof, and the like. In an embodiment, the conjugate compound or composition comprising same described herein is used in combination with an EGFR or tyrosine kinase targeting agent, for example an EGFR inhibitor (RTK inhibitor). The conjugate compound or salt thereof or composition comprising same described herein may also be used in combination with one or more therapeutic antibodies or antibody fragments, e.g., therapeutic antibodies or antibody fragments used for the treatment of tumors. Examples of antibodies used for the treatment of cancers include antibodies targeting CD52 (e.g., Alemtuzumab), VEGFA / EGFR (e.g., Bevacizumab, Ramucirumab), EGFR (e.g., Cetuximab, Necitumumab, Panitumumab), CD38 (e.g., Daratumumab, Isatuximab), RANKL (e.g., Denosumab), GD2 (e.g., Dinutuximab, Naxitamab-gqgk), SLAMF7 (e.g., Elotuzumab), HER2 (e.g., Margetuximab-cmkb, Pertuzumab), CCR4 (e.g., Mogamulizumab), CD20 (Obinutuzumab, Ofatumumab, Rituximab), BCMA (e.g., Teclistamab), CD19 (e.g., Tafasitamab), CTLA-4 (e.g., Tremelimumab), LAG-3 (e.g., Relatlimab), PD-1 (e.g., Tislelizumab, Penpulimab, Sintilimab, Toripalimab, Retifanlimab, Dostarlimab), PD- L1 (e.g., Durvalumab, Avelumab, Atezolizumab), EpCAM (e.g., Oportuzumab, Edrecolomab), Nectin-4 (e.g., Enfortumab), CD79b (e.g., Polatuzumab).

[0163] The combination of active agents and / or compositions comprising same may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present invention refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent (e.g., the conjugate compound described herein) may be administered to a patient before, concomitantly, before and after, or after a second active agent (e.g., a chemotherapeutic agent or an immunotherapy) is administered. The agents may in an embodiment be combined / formulated in a single composition and thus administered at the same time.

[0164] In an embodiment, the cancer is resistant to an immunotherapy, i.e., a cancer in which the immunotherapy does not lead to inhibition of tumor growth in the patient. A cancer resistant to immunotherapy may be a cancer that has never responded to immunotherapy (primary resistance) or that has developed resistance to immunotherapy treatment after a period of (responsive) treatment (acquired resistance).

[0165] In a further embodiment, the cancer is resistant to PD-1 or PD-L1 inhibitor-based therapy (anti-PD-1 / PD-L1 therapy). In a further embodiment, the cancer is resistant to PD-1 or PD-L1 inhibitor-based therapy is melanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colon cancer, liver cancer, stomach cancer, squamous cell skin cancer or myeloma.

[0166] Immune checkpoint inhibitors have been approved or are currently being tested in phase III and IV clinical trials for several cancers including lung cancer (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer, squamous cell lung carcinoma), head and neck cancer (e.g., head and neck squamous cell carcinoma, renal cell carcinoma, gastric adenocarcinoma, nasopharyngeal neoplasms, urothelial carcinoma, colorectal cancer, mesothelioma (e.g., pleural mesothelioma), breast cancer (e.g., triple-negative breast cancer, TNBC), esophageal neoplasms, multiple myeloma, gastric and gastroesophageal junction cancer, gastric adenocarcinoma, melanoma, Merkel-cell carcinoma (MCC), lymphoma (e.g., Hodgkin and nonHodgkin lymphoma, diffuse Large B-cell lymphoma), liver cancer (e.g., hepatocellular carcinoma), melanoma, ovarian cancer, fallopian tube cancer, peritoneal neoplasms, bladder cancer, transitional cell carcinoma, prostatic neoplasms and biliary tract neoplasms (see, e.g., Darvin et al., Experimental & Molecular Medicine volume 50, Article number: 165 (2018)). Thus, in an embodiment, the cancer is one of the above-noted cancer for which immune checkpoint inhibitors have been approved or are currently being tested in phase III and IV clinical trials.

[0167] Currently approved immune checkpoint inhibitors include the anti-CTLA-4 Ipilimumab (melanoma and lung cancer), the anti-PD-1 Nivolumab (melanoma, lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, colon cancer, and liver cancer), Pembrolizumab (melanoma, lung cancer, head and neck cancer, Hodgkin lymphoma, renal cell carcinoma and stomach cancer), and Cemiplimab (squamous cell skin cancer, myeloma, and lung cancer), and the anti-PD-L1 atezolizumab (NSCLC, small cell lung cancer, TNBC), Avelumab (NSCLC, MCC) and Durvalumab (urothelial carcinoma, lung cancer). Thus, in an embodiment, the cancer is one of the above-noted cancer for which immune checkpoint inhibitors have been approved.

[0168] As used herein, the term "subject" or “patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject or patient according to the disclosure is a human.

[0169] EXAMPLES

[0170] The present disclosure is illustrated in further details by the following non-limiting examples.

[0171] Example 1 : Materials and Methods

[0172] Cell lines and cell culture

[0173] Human colorectal cancer cell lines LS123 (#CCL-255), HCT-116 (#CCL-247), SK-CO-1 (#HTB-39), T-84 (#CCL-248), LoVo (# CCL-229), HT-29 (# HTB-38) and RKO (#CRL-2577) were purchased from ATCC. Human triple-negative breast cancer (TNBC)-derived MDA-MB-231 / Luc cells were from Cell Biolabs Inc. (San Diego, CA, #AKR-231) and MDA-MB-468 (#HTB-132), MDA-MB-157 (#HTB-24), DU4475 # (HTB-123) and HCC70 (#CRL-2315) cells were from ATCC. Canine renal epithelial MDCK-MDR1 cells were provided by Dr. Amanda Yancy (AstraZeneca Pharmaceuticals, LP, Wilmington DE, USA).

[0174] All cell lines were grown as adherent monolayers at 37°C in a humidified atmosphere (5% CO2) using media described in Table 1.

[0175] Table 1 : Cell Culture Conditions Used for Cell Lines

[0176] 1: MDA-MB-157 cells were cultured in a free gas exchange with atmospheric air due to the use of the L-15 medium formulation.

[0177] For experimental use, cells were detached from the culture flask by a 5-10-minute treatment with trypsin (Wisent), then were 10-fold diluted and neutralized by addition of complete culture media. Cell counts and cell viability were assessed with a TC20 automated cell counter (BioRad) following 0.4% trypan blue (Thermo Fisher Scientific) exclusion staining of cells.

[0178] Western blotting

[0179] Cells were homogenized in 1% SDS lysis buffer supplemented with a complete protease inhibitor cocktail from Calbiochem (San Diego, CA). Cells were incubated for 30 min at room temperature (RT) with vortexing every 5 min, sonicated and centrifuged at 15,000g for 10 min at RT. Equal amounts of protein (20 pg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were then electrotransferred to a polyvinylidene fluoride (PVDF) membrane and blocked for 1 h at room temperature using 5% non-fat dry milk in Tris-buffered saline (150 mM NaCI, 20 mM Tris-HCI, pH 7.5) containing 0.1% Tween™-20 (TBST). Membranes were washed in TBST and incubated overnight with primary antibodies against SORT 1 (1 / 1 ,000 dilution), P-gp (1 / 100 dilution), BCRB (1 / 500 dilution), EGFR (1 / 1 ,000), Topoisomerase 1 (1 / 500 dilution), GAPDH (1 / 40,000 dilution) or [3-actin (1 / 50,000 dilution) diluted in TBST containing 3% BSA and 0.05% NaNs. Membranes were washed in TBST and incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (1 / 5000 dilution) in TBST containing 5% non-fat dry milk. Membranes were washed again in TBST and signals were detected using chemiluminescence (Amersham Biosciences, Baie d’Urfe, QC).

[0180] Animals

[0181] Female (HT-29) or male (HCT-116) athymic nude (Crl:NU(NCr)-Foxn7nu) and female CD1 nude (Crl:NU-Foxn1 nu; MDA-MB-231) mice aged between six and eight weeks were obtained from Charles River Laboratories, Inc. (St-Constant, QC). Animals were allowed to acclimate for 5 days before experiments. All mice were maintained in a pathogen-free environment and handled in accordance with the Guidelines of the Canadian Council on Animal Care (CCAC) for care and use of experimental animals.

[0182] Preparation of compounds for injection

[0183] Preparation of the peptide-drug conjugates is described in Example 2 below. Docetaxel (Tecoland, Irvine, CA), irinotecan (MedChemExpress, Monmouth Junction, NJ) or exatecan (MedChemExpress, Monmouth Junction, NJ) was prepared the same day as animal dosing. Docetaxel was solubilized to 50 mg / ml with injectable EtOH, diluted to 25 mg / ml with polysorbate- 80, then diluted with sterile 5% Dextrose 5% in water (D5W) to the desired concentration for injection (i.e., 2.5 mg / ml). Irinotecan was solubilized to 250 mg / ml with DMSO, then diluted with sterile 5% D5W to the desired concentration for injection (i.e., 13 mg / ml or lower). Exatecan was solubilized to 20 mg / ml with HCI-acidified DMSO, then diluted with HCI-acidified sterile 5% D5W to the desired concentration for injection (i.e., 1.4 mg / ml). TH2101 was solubilized to 80 mg / ml with formic acid-acidified DMSO and Solutol (1 :2 ratio), then diluted with sterile formic acid- acidified 5% D5Wwith to the desired concentration for injection (i.e., 5.4 mg / ml or lower). TH2102 was solubilized to 50 mg / ml with DMSO, then diluted with sterile 5% D5W to the desired concentration for injection (i.e., 5.5 mg / ml). TH2204 was solubilized to 115 mg / ml with DMSO, then diluted with sterile 5% D5W to the desired concentration for injection (i.e., 5.85 mg / ml). TH2205 was solubilized to 80 mg / ml with formic acid-acidified DMSO and Solutol (1 :2 ratio), then diluted with sterile formic acid-acidified 5% D5W with to the desired concentration for injection (i.e., 5.85 mg / ml). TH2301 was solubilized to 65 mg / ml with HCI-acidified DMSO, then diluted with HCI-acidified sterile 5% D5W to the desired concentration for injection (i.e., 4.7 mg / ml). TH2303 was solubilized to 33.3 mg / ml with HCI-acidified DMSO and Solutol (1 :2 ratio), then diluted with HCI-acidified sterile 5% D5W to the desired concentration for injection (i.e., 9.8 mg / ml). All diluted solutions were filtered before animal administrations (DMSO-safe 0.2 m syringe filter, Nylon membrane, PALL Corporation).

[0184] In vivo efficacy assessment of peptide drug conjugates using the CRC HT-29 xenograft model

[0185] Tumors were established by subcutaneous inoculation of 7x106HT-29 cells in 100 pl of HBSS (Sigma #H6648). All cells were injected into the right flank of immunocompetent mice under light isoflurane anesthesia. For all HT-29 xenograft studies, mice were treated weekly, with either vehicle, docetaxel, irinotecan or exatecan, TH2101 , TH2102, TH2204, TH2205 and TH2301 via intravenous (IV) tail vein injection at doses indicated in the respective figures. For all indicated studies, tumor growth was monitored by two-dimensional measurements taken with an electronic caliper and tumor volume was calculated according to the following formula: tumor volume (mm3) = TT / 6 x length x width2. Animal weights were measured with a precision of ±10 mg. T umors were collected when mice in the vehicle group reached approximately 1000 mm3in size.

[0186] In vivo efficacy assessment of peptide drug conjugates using the CRC HCT-116 xenograft model

[0187] Tumors were established by subcutaneous inoculation of 3x106HCT-116 cells in 100 pl of HBSS (Sigma #H6648). All cells were injected into the right flank of immunocompetent male mice under light isoflurane anesthesia. Mice were treated weekly, with either vehicle, irinotecan, exatecan or TH2303 via intravenous (IV) tail vein injection at doses indicated in the respective figures. For all indicated studies, tumor growth was monitored by two-dimensional measurements taken with an electronic caliper and tumor volume was calculated according to the following formula: tumor volume (mm3) = TT / 6 X length x width2. Animal weights were measured with a precision of ±10 mg. Tumors were collected when mice in the vehicle group reached approximately 1000 mm3in size. In vivo efficacy assessment of peptide drug conjugates using the TNBC MDA-MB-231 xenograft model

[0188] Tumors were established by subcutaneous inoculation of 7x106MDA-MB-231 cells in 100 l of HBSS (Sigma #H6648). Cells were injected into the right flank of immunocompetent female mice under light isoflurane anesthesia. Mice were treated weekly, with either vehicle, irinotecan, exatecan or TH2303 via intravenous (IV) tail vein injection at doses indicated in the respective figures. For all indicated studies, tumor growth was monitored by two-dimensional measurements taken with an electronic caliper and tumor volume was calculated according to the following formula: tumor volume (mm3) = TT / 6 X length x width2. Animal weights were measured with a precision of ±10 mg. Tumors were collected when mice in the vehicle group reached approximately 1000 mm3in size.

[0189] Sortilin RNA interference

[0190] HT-29 cells were transiently transfected for 24 hours with 100 nM of a siRNA scrambled sequence (AllStar Negative Control siRNA, 1027281) or a human siRNA generated against sortilin (Hs_SORT_5 FlexiTube siRNA: SI03115168; Qiagen, Valencia, CA) using Lipofectamine™ 2000 (ThermoFisher Scientific, Burlington, ON).

[0191] Fluorescent TH19P01 peptide uptake assay

[0192] SiScrambled- or siSORTI -transfected HT-29 cells were washed twice with HBSS and incubated in HBSS in the presence or absence of 200 nM Alexa488-labeled TH19P01. After 1 hour of incubation at 37°C, cells were washed with HBSS, trypsinized, washed again and fluorescence assessed in the FL1 channel using a C6 Accuri™ flow cytometer (BD Biosciences, San Jose, CA).

[0193] Uptake Assays of TH2101 by confocal microscopy

[0194] HT-29 cells were plated for 24 hours on glass coverslips and grown to 80% confluency. Cells were then transfected for 24h with a scrambled siRNA or siSORT 1 as described above. Cells were washed with HBSS then incubated in HBSS in the presence or absence of 200 nM TH2101 fluorescent conjugate. After 1 or 2 hours of incubation at 37°C, cells were washed with HBSS, fixed for 15 min at RT in 4% paraformaldehyde (PFA), washed again in PBS and mounted onto slides using Prolong Gold antifade reagent. Cells were finally digitalized by confocal microscopy (Nikon A1) using excitation and emission wavelengths of 405 nm and 525 nm, respectively. Fluorescence levels were quantified using the NIH Imaged Version 1.4.21 software.

[0195] Cell proliferation assay To assess the effects of free drugs (irinotecan, SN-38 and exatecan) and conjugates on HT-29, HCT-116 or LoVo cell proliferation, cells (6,000 cells / well) were first seeded for 24 hours in 96-well plates (Perkin Elmer) in complete growth medium. Cells were then treated with various concentrations of drugs in complete cell culture medium. After 72-96 hours of incubation, viable cells were estimated by MTT (Sigma-Aldrich) assay. GraphPad Prism software was used to calculate IC5o values.

[0196] Statistical analysis

[0197] Data are expressed as means ± standard error of the mean (SEM) or standard deviation (SD) as indicated in the figure legends. Statistical analysis was done using t-test for comparing two samples, whereas analysis by one-way ANOVA followed by Bonferroni’s, Dunnett’s or Tukey’s multiple comparisons was employed for three or more samples. A value of p < 0.05 was considered significant and an asterisk (*) identifies the level of such significance in the figures.

[0198] Example 2: Synthesis of peptide-drug conjugates

[0199] General Analytical Methods and Methodology for purification and Characterization of

[0200] As used herein the term RT (min) refers to the LCMS retention time, in minutes, associated with the compound. Unless otherwise indicated, the instruments and columns used to obtain the desired compounds. Instrument and column: Waters Acquity UPLC BEH C18 1.7 mm, 2.1 x 50 mm. Agilent 1290 Infinity II LUNA C185 mm, 4.6 x 150 mm. AKTA pure protein purification system using 30 RPC resign.

[0201] The following abbreviations may be used as follows: aq = aqueous cone = concentrate

[0202] DCM = Dichloromethane

[0203] DIPEA = Diisopropylethylamine

[0204] DMF = dimethylformamide

[0205] DMSO = Dimethylsulfoxide

[0206] EtOAc = Ethyl acetate

[0207] Mol = molar

[0208] MeOH = Methanol

[0209] RT = room temperature

[0210] TEA = Triethylamine

[0211] THF = Tetra hydrofuran

[0212] Synthesis of TH2102 (see FIG. 5) Step 1 : Synthesis of (S)-4-(((tert-butoxycarbonyl)glycyl)oxy)-4,11 -diethyl-3, 14-dioxo-3,4, 12, 14- tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl [1 ,4'-bipiperidine]-1 '-carboxylate.

[0213] Irinotecan (1.00 g, 1.70 mmol) and Boc-Gly-OH (717 mg, 4.09 mmol) were dissolved in CH2CI2(50.0 mL) and the mixture was treated with DCC (848 mg, 4.11 mmol) followed by DMAP (292 mg, 2.39 mmol). The mixture was stirred at room temperature for 18 h after which time, the TLC indicated a clean reaction. The white solid was filtered over a DCM-washed filter paper and the solid was washed with several portions of CH2CI2. The filtrate was evaporated, and the residue was dissolved in a little CH2CI2 and purified on the Biotage (40-g column Solvent A: CH2CI2, solvent B: 20% MeOH / CH2CI2, 0% B to 65 % B in 16 min). Pure fractions were evaporated and dried under vacuum on rotary evaporator to afford Irinotecan Gly boc (1 .20 g, 95 %) as pale yellow solid. High Pressure liquid chromatography / mass spectrometry (HPLC / MS) analysis showed a purity >95%. MH+ 744.4.

[0214] Step 2: Synthesis of (S)-4,11-diethyl-4-(glycyloxy)-3,14-dioxo-3,4,12,14-tetrahydro-1 H- pyrano[3',4':6,7]indolizino[1 ,2-b] q u inoli n-9-y I [1 ,4'-bi piperid ine]- 1 '-carboxylate.

[0215] To solid (S)-4-(((tert-butoxycarbonyl)glycyl)oxy)-4,11 -diethyl-3, 14-dioxo-3,4, 12, 14- tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl [1 ,4'-bipiperidine]-1 '-carboxylate (1.20 g, 1.61 mmol) was added a mixture of 45% TFA, 5% H2O, 50% CH2CI2(32.1 mL, 189 mmol). The compound dissolved rapidly to give a yellow-green, fluorescent solution. After 30 minutes, the volatiles were removed on the rotavap and the residue was co-evaporated several times with CH2CI2. The residue was dissolved in 1-ml of MeOH and 60-ml of CH2CI2. This solution was transferred in a 125-ml separatory funnel and was washed with sodium carbonate 5% in water (3.42 g, 1.61 mmol) (3 x 15 ml, pH 10 on the third wash). All washes were combined, the pH was adjusted to 9-10 with sodium carbonate solution and the resulting mixture was extracted with CH2CI2(3 x 20 ml). These extracts were combined with the initial organic layer and was washed with water (2 x 15 ml) and saturated brine (15 ml). The organic phase was separated and was dried (Na2SO4). After evaporation of the solvent and co-evaporation with CH2CI2, a light yellow solid was obtained which was dried under high vacuum. Product was used as such in the next step. Product (MH+644) appears in two peaks in LCMS analysis, but it was used as such in the next step.

[0216] Step 3: Synthesis of (S)-4-((2-((9-(([1 ,4'-bipiperidine]-1'-carbonyl)oxy)-4,11-diethyl-3,14-dioxo- 3,4,12,14-tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)-2-oxoethyl)amino)-4- oxobutanoic acid.

[0217] (S)-4, 11 -diethyl-4-(glycyloxy)-3, 14-dioxo-3,4, 12, 14-tetrahydro-1 H- pyrano[3',4':6,7]indolizino [1 ,2-b]quinolin-9-yl[1 ,4'-bipiperidine]-1 '-carboxylate (800 mg, 1.24 mmol) was dissolved in THF anhydrous (62.0 mL) and was treated with triethylamine (520 pL, 3.73 mmol) followed by succinic anhydride (187 mg, 1.86 mmol) Note: the solid went in solution upon the addition of the base. The reaction was left stirring overnight and acetic acid (285 pL, 4.97 mmol) was added (pH 5) and the THF was removed on the rotavap and residue was dissolved in 3 ml DMSO using 40 g C-18 column (using water: acetonitrile 0-80%) as gradient and pure fractions were freeze dried and product was used as such in the next step. LCMS: MH+ 744 is observed at 2.89 min with >99% purity.

[0218] Step 4:

[0219] In a 25-ml RBF, (S)-4-((2-((9-(([1 ,4'-bipiperidine]-1 '-carbonyl)oxy)-4, 11 -diethyl-3, 14-dioxo- 3,4,12,14-tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)-2-oxoethyl)amino)-4- oxobutanoic acid (220 mg, 0.296 mmol) was dissolved in DMSO (3.00 ml_). TBTU (95.0 mg, 0.296 mmol) was added followed by DIPEA (129 pL, 0.739 mmol). The mixture was stirred for 10 minutes at room temp (pH ~7). In a separate 25-ml RBF TH19P01 (256 mg, 0.133 mmol) was dissolved in DMSO (1.00 ml_). After 10 min. of preactivation, the solution of TH19P01 was rapidly added to the solution of pre-activated ester and the flask was washed with two portions of DMSO (0.5 ml_). As the pH of the reaction mixture was slightly acidic (pH 5 immediately after the addition), more DIPEA (64.4 pL, 0.370 mmol) was added, which brought the pH back to 7-8 and pH remained at 7-8. An LCMS of the reaction at t=10 min (after the addition of DIPEA) indicated a clean, complete reaction. After stirring for 20 minutes (total time from DIPEA addition), the reaction was quenched by the addition of acetic acid (102 pL, 1.77 mmol), which caused the pH to drop to pH ~ 4. Final dilution for Prep purification: The crude solution (~5.8 ml) was further diluted with DMSO (2.1 ml). It was cooled to 10 °C and diluted with water (100 pl) and formic acid (20 pl), total volume of solution: ~7.0 ml).

[0220] Agilent Prep LCMS purification: The purification was performed on an Agilent Prep-C18 50 x 21.2 mm, 5 pm column. Injection volume: 75 pl (~ 93 injections) to afford 120 mg of TH2102. MH+3375.74 (1126.5, charge +3) > 95% purity.

[0221] Synthesis of TH2301 (see FIG. 8):

[0222] Step 1 : To the solution of triethylamine (524 pL, 3.76 mmol) in DCM (50.0 mL) at 0°C, Boc anhydride (657 mg, 3.01 mmol) and Exatecan mesylate (1.00 g, 1.88 mmol) were added and reaction was allowed to warm to room temperature. The resultant reaction mixture was stirred at RT for 4h. The progress of the reaction was monitored by LCMS After completion of starting material, reaction mixture was loaded directly on to 80 g normal phase column and purified using DCM:methanol as eluent (0-10%) and pure fractions were evaporated to afford N-Boc-Exatecan (950 mg, 94 %). MH+ 535.96.

[0223] Step 2:

[0224] N-Boc-Exatecan (950 mg, 1.77 mmol), succinic anhydride (599 pL, 5.32 mmol), 4- dimethylaminopyridine (217 mg, 1.77 mmol) were taken in DCM (50.0 mL) and to that triethylamine (1 .28 mL, 8.87 mmol) was added and reaction was stirred at RT for 24 hours. After 24 hours there was still some starting material and again 3 equivalent succinic anhydride and triethylamine were added and reaction was stirred for another 24 hours. Most of the starting material was consumed and it was purified by normal phase chromatography after adsorbing on silica gel on 80 g column using 0-20% (DCM-Methanol) as eluent and pure fractions were evaporated to afford N-Boc-Exatecan-Su (850 mg, 75 %). MH+ 635.92

[0225] Step 3:

[0226] In a 25-ml RBF, N-Boc-Exatecan-Su (93.3 mg, 0.142 mmol), TBTU (33.4 mg, 0.142 mmol) were dissolved in DMSO (1.45 mL). N,N-diisopropylethylamine; DIEA; Hunig's base (62.0 pL, 0.356 mmol) was added. The mixture was stirred for 10 minutes at room temp (pH ~7). In a separate 25-ml RBF TH19P01 (123 mg, 0.0641 mmol) was dissolved in DMSO (482 pL). After 10 min. of preactivation, the solution of TH19P01 was rapidly added to the solution of pre-activated ester and the flask was washed with 1 portion of DMSO (241 pL). As the pH of the reaction mixture was slightly acidic (pH 5 immediately after the addition), N,N-diisopropylethylamine; DIEA; Hunig's base (31.0 pL, 0.178 mmol) was added, which brought the pH back to 7-8. A LCMS of the reaction at t=10 min (after the addition of DIPEA) indicated a clean, complete reaction. After stirring for 20 minutes (total time from DIPEA addition), the reaction mixture was purified using HPLC (Akta) using 20-80% acetonitrile (0.1% FA) and water (0.1% FA) to afford N-Boc-Exatecan- Su-TH19P01 obtained. N-Boc-Exatecan-Su-TH19P01 (100.0 mg, 0.00317 mmol) was dissolved in DCM (6.00 mL) in 100 ml RBF and to that HCI (4N 1 ,4 dioxane) (39.6 pL, 0.158 mmol) was added at once there was colour change in the reaction mixture to yellow. Reaction was monitored with LCMS and once all the SM was consumed, reaction mixture was evaporated by blowing nitrogen gas over the RBF.

[0227] The crude was kept over high vacuum for 30 minutes and it was dissolved in water and injected in HPLC (Akta) using 10-80% acetonitrile (0.1% FA) and water (0.1% FA) and pure fractions were freeze dried to afford TH2301. MH+2959.18 (986.58, charge +3) > 95% purity.

[0228] Synthesis of TH2304 (see FIG. 10):

[0229] Step 1 : Synthesis of 2-((tert-butyldimethylsilyl)oxy)acetic acid:

[0230] Ethyl [(tert-butyldimethylsilyl)oxy]acetate (1.00 g, 2.99 mmol) in ethanol (20.0 mL) and KOH (252 mg, 4.49 mmol) was added and reaction was stirred at RT for 12 hours at room temperature. After 12 hours at 23°C, the reaction mixture was concentrated, and the residue was dissolved in water (100 mL). Concentrated HCI was added carefully to the mixture to pH 4 at 0 °C and the reaction mixture was extracted with EtOAc (20 mL X 3). The organic layer was washed with H2O (30 mL) and aqueous saturated NaCI (30 mL). Organic layer was dried by passing over sodium sulphate and concentrated under vacuum to afford 2-((tert-butyldimethylsilyl)oxy)acetic acid (500 mg, 55 %) as semisolid.1HNMR-DMSO-d6: 4.07 (s, 2H), 0.82 (s, 9H), 0.77 (s, 6H).

[0231] Step 2: Synthesis of 2-((tert-butyldimethylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4- methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro- 1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2- b]quinolin-1-yl)acetamide: To the solution of AN-01-0125 (250 mg, 1.31 mmol) in DMF (8.33 mL) at 0°C, HATU (599 mg, 1.58 mmol) and DIPEA (686 pL, 3.94 mmol) were added and reaction was stirred at 0°C for 10 minutes and then Exatecan mesylate (698 mg, 1.31 mmol) dissolved in 0.3 ml DMF was added, and reaction was allowed to warm to room temperature. The resultant reaction mixture was stirred at RT for 4h. The progress of the reaction was monitored by LCMS. After completion of starting material, reaction mixture was loaded directly on to 80 g normal phase column and purified using DCM: methanol as eluent to afford 2-((tert-butyldimethylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9- hydroxy-4-methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro- 1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1-yl)acetamide (450 mg, 56 %). MH+ 608.14 for the product is observed at 2.68 min in 4-minute run.

[0232] Step 3: Synthesis of 4-(((1S,9S)-1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4- methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro- 1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2- b]quinolin-9-yl)oxy)-4-oxobutanoic acid:

[0233] N-((1 S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9, 10,13,15-hexahydro- 1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-1-yl)acetamide (450 mg, 0.740 mmol) was taken in DCM (100 mL) and to that succinic anhydride (222 mg, 2.22 mmol), Et3N (321 pL, 2.22 mmol) and 4-dimethylaminopyridine (90.5 mg, 0.740 mmol) were added. Reaction was stirred at RT for 24 hours. Reaction was sluggish and 1 extra equivalent of the reagent was added. Once most of the starting material was consumed, reaction mixture was purified using normal phase chromatography using (DCM: methanol) 0-10% eluent and pure fractions were evaporated under vacuum to afford 4-(((1S,9S)-1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro- 4-methyl- 10,13-dioxo-2,3,9, 10,13,15-hexahydro-1 H, 12H- benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl)oxy)-4-oxobutanoic acid (150 mg, 29 %). MH+ (708.01) for the product was observed at 2.60 min on 4 min run with >95% purity.

[0234] Step 4: Synthesis of TH2304

[0235] 4-(((1S,9S)-1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9- yl)oxy)-4-oxobutanoic acid (120 mg, 0.170 mmol) was taken in dry DCM and to that N- hydroxysuccinimide (23.4 mg, 0.203 mmol) and DCC (42.8 pL, 0.186 mmol) were added and reaction was stirred at RT for 2 hours. Reaction was monitored with LCMS. Once all the starting material was consumed, reaction mixture was adsorbed on silica gel and purified using normal phase chromatography using DCM: methanol (0-10%) eluent and pure fractions were concentrated to afford (1S,9S)-1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4- methyl- 10, 13-dioxo-2,3,9, 10,13,15-hexahydro- 1 H, 12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2- b]quinolin-9-yl (2,5-dioxopyrrolidin- 1 -yl) succinate (85.0 mg, 62 %) as colourless solid. MH+ (805.13) is observed at 2.72 min on 4 min run on UPLC.

[0236] (1S,9S)-1-(2-((tert-butyldimethylsilyl)oxy)acetamido)-9-ethyl-5-fluoro-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl (2,5-dioxopyrrolidin-1 -yl) (28.0 mg, 0.0303 mmol), N,N-diisopropylethylamine; DIEA; Hunig's base (15.8 pL, 0.0908 mmol) were dissolved in DMSO (307 pL) followed by addition of TH19P01 (26.2 mg, 0.0136 mmol) in DMSO (102 pL). The reaction mixture was monitored by LCMS for completion. Once all the starting material was consumed, the reaction mixture was purified by HPLC (Akta) using 10-80% acetonitrile (0.1% FA) and pure fractions were freeze dried to afford TH19P01-Osu-Exatecan-OTBS (67.0 mg, 67 %) 3303.77 (1101.81 , charge +3) is observed at 2.28 minutes on 4 min run >94% purity which was used as such in the next step.

[0237] TH19P01-Osu-Exatecan: TH19P01-Osu-Exatecan-OTBS (23.0 mg, 0.00557 mmol) was taken in DCM (3.07 mL) and to that TBAF (223 pL, 0.0223 mmol) and acetic acid (1 .28 pL, 0.0223 mmol) were added and reaction was monitored for the completion. Once reaction was complete it was dried by flushing nitrogen gas over it and crude was dissolved in 1 ml DMSO and purified using HPLC (Akta) using 30-80% acetonitrile (0.1% FA) to afford TH19P01-Osu-Exatecan (12.5 mg, 73 %) as final product. 3075.25 (1015.73, charge +3) is observed at 1.49 minutes on 4 min run >95% purity.

[0238] Synthesis of TH2101 (see FIG. 4):

[0239] Step 1 : (S)-9-((tert-butyldiphenylsilyl)oxy)-4, 11 -diethyl-4-hydroxy- 1 , 12-dihydro- 14H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinoline-3,14(4H)-dione (TBDPS-SN-38)

[0240] To a suspension of SN-38 (1 .0 g, 2.55 mmol) in 51 mL of anhydrous DCM were added Et3N (1.6 mL, 11.5 mmol) and t-butyldiphenylchlorosilane (TBDPSCI, 2.65 mL, 10.2 mmol). The reaction mixture was stirring overnight at RT and then was washed with 0.2 N HCI, saturated NaHCO3, and saturated NaCI. The organic layer was dried over MgSCU, filtered, and evaporated under vacuum. The residue was dissolved in DCM and precipitated by addition of hexane. The solids were filtered and dried under vacuum to give compound TBDPS-SN-38 (1 .54 g, 96% yield) as a white solid.

[0241] Step 2: (S)-9-((tert-butyldiphenylsilyl)oxy)-4, 11 -diethyl-3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl glycinate hydrochloride (TBDPS-SN-38-Gly)

[0242] To a solution of TBDPS-SN-38 (1.61 g, 2.55 mmol) and Boc-Gly-OH (1.07 g, 6.12 mmol) in 75 mL of anhydrous DCM were added DCC (1.26 g, 6.12 mmol) and DMAP (436 mg, 3.57 mmol). The reaction mixture was stirred at RT for 20h. The mixture was filtered. After filtration, the solvent was evaporated under vacuum to give the crude which was purified by column (normal phase - Biotage) with the gradient from 0-70% EtOAc in DCM to give desired product (1 .78 g, 89 % yield). TBDPS-SN-38-Gly(Boc) (1.78 g, 2.25 mmol) was dissolved in 8.8 mL dioxane, then 8.8 mL HCI / dioxane 4M was added to the solution. The mixture was stirred in 20 min. The reaction was monitored by LC-MS. The solvent was removed by flush air, then ether was added. The precipitate was filtered, washed with ether, and evaporated under vacuum to give product (1.58 mg, 97% yield) as a yellow solid.

[0243] Steps 3 and 4: (S)-4-((2-((9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14- tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)-2-oxoethyl)amino)-4- oxobutanoic acid (

[0244] TBDPS-SN-38-Gly (780 mg, 1.08 mmol) was dissolved in THF anhydrous (53.8 mL) and treated with triethylamine (750 pL, 5.38 mmol) followed by succinic anhydride (323 mg, 3.23 mmol). The reaction followed by LC-MS. After stirring overnight, solvent was evaporated by rotavap. The residue was dissolved in 3 ml DMSO and purified by Biotage (reverse phase) using C-18 column with water: acetonitrile 10-80% as gradient. The pure fractions were freeze dried and product TBDPS-SN-38-Gly-OSu (536 mg, 63% yield) as a colorless solid. Purity of the product was evaluated by HPLC-MS and was > 99% with a m / z mass of 788.0 (M+H).

[0245] Step 4: Synthesis of TH2201 :

[0246] TBDPS-SN-38-Gly (243 mg, 308 pmol) was dissolved in DMSO (3.1 ml_). TBTU (98.9 mg, 308 pmol) was added followed by DIPEA (134 pL, 770 pmol). The mixture was stirred for 10 minutes at room temp (pH ~7). After 10 min of preactivation, the solution of TH19P01 (267 mg, 139 pmol) in DMSO (1 .04 mL) was rapidly added to the solution of pre-activated ester. As the pH of the reaction mixture was slightly acidic (pH 5 immediately afterthe addition), more DIPEA (67.1 pL, 385 pmol) was added, which brought the pH back to 8-9 and remained there. The reaction was followed by LC-MS. After stirring for 20 minutes, the reaction was quenched by the addition of water. The precipitated was filtered and washed with water giving protected conjugate (463 mg).

[0247] Step 5:

[0248] To the suspension of the solid obtained (344 mg, 99.4 pmol) in DCM anhydrous (34 mL), and TBAF (1 M in THF) (149 pL, 149 pmol) was added. The reaction was followed by LC-MS. After 45 min, the solid was filtrated and washed with DCM and water (0.1% FA) to remove TBAF. Then, it was purified by HPLC (Akta) with gradient from 10 - 40% acetonitrile in water giving conjugate TH2101 (87.7 mg, 30% yield) as a colorless solid. Purity of the conjugate was evaluated by HPLC-MS and was >98% with a m / z mass of 2985 (996.5, charge +3).

[0249] Synthesis of TH2205 (see FIG. 6):

[0250] Steps 1-3 are similar to those described above for TH2101.

[0251] Step 4: Synthesis of (S)-1-((9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo-3,4,12,14- tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)-1 ,4-dioxo-7,10,13-trioxa-3- azahexadecan-16-oic acid (TBDPS-SN-38-Gly-PEG3)

[0252]

[0253] To a solution of bis-PEG3-acid (72.6 mg, 290 pmol) in DMF (0.7 mL) and DCM (10 mL) was added NMM (79.8 pL, 726 pmol), TBDPS-SN-38-Gly (105 mg, 145 pmol) in 4.5 mL DCM, 6-CI HOBt (29.5 mg, 174 pmol), and EDCI (33.4 mg, 174 pmol) at 0°C. The resulting reaction solution was stirred at 0°C for 4h. The progress of the reaction was monitored by LC-MS. After starting material was totally consumed, the reaction was concentrated. The resulting residue was purified by reverse-phase chromatography (Biotage) using 10-80% acetonitrile (0.1% FA) in water (0.1% FA) as gradient to yield the desired product TBDPS-SN-38-Gly-PEG3(90.8 mg, 68% yield) as a white solid. Purity of the product was evaluated by HPLC-MS and was > 99% with a m / z mass of 920.3 (M+H).

[0254] Step 5:

[0255] To a solution of TBDPS-SN-38-Gly-PEG3(233 mg, 253 pmol) in DMSO (4 mL) was added HATU (96.3 mg, 253 pmol) and DIEA (132 pL, 760 pmol). The mixture was stirred at room temperature in 15 min. Then the solution of TH19P01 (219 mg, 114 pmol) in DMSO (1.5 mL) was added to the reaction mixture. After stirring in 15 min, the reaction was stopped, and the mixture was purified by HPLC (Akta) using 30-80% acetonitrile (0.1% FA) in water (0.1% FA) as gradient. The collected fractions were lyophilized to give protected conjugate (162 mg, 23% yield). To the suspension of the solid obtained (162 mg, 43.5 pmol) in DCM anhydrous (15 mL), and TBAF (1M in THF) (86.9 pL, 86.9 pmol) was added. The reaction was followed by LC-MS. After 45 min, solvent was removed in vacuo. Solid was dissolved in DMSO, then purified by HPLC (Akta) using 30-80% acetonitrile (0.1% FA) in water (0.1% FA) as gradient giving conjugate TH2205 (57 mg, 40 % yield) as a colorless solid. Purity of the conjugate was evaluated by HPLC-MS and was > 98% with a m / z mass of 3249 (1084.8, charge +3).

[0256] Synthesis of TH2302 (see FIGs. 7A-B):

[0257] Steps 1-4 are similar to those described above for TH2205. Step 5:

[0258] TBDPS-SN-38-Gly-PEG3 (105 mg, 114 pmol) in DMSO (2.5 mL) was treated with TBTU (36.6 mg, 114 pmol), DIEA (59.6 pL, 342 pmol) and KBP-201 (144 mg, 48.8 pmol). The mixture was purified by HPLC (Akta) using 30-80% acetonitrile (0.1% FA) in water (0.1% FA) as gradient giving protected conjugate (87.3 mg, 18% yield). Then, the product obtained (87.3 mg, 18.3 pmol) was deprotected by TBAF (36.6 pmol, 36.6 pL). After worked up and purified as described above to give TH2302 (49 mg, 62% yield) as a white solid. Purity of the conjugate was evaluated by UPLC-MS and was > 95% with a m / z mass of 4287 (1072, charge +4).

[0259] Synthesis of TH2303 (see FIG. 9A):

[0260] Step 1 : Synthesis of (1 S,9S)-1-((tert-butoxycarbonyl)amino)-9-ethyl-5-fluoro-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-9-yl glycinate (Boc-Exatecan-Gly)

[0261] To a solution of Boc-Exatecan (958 mg, 1 .79 mmol) and |Z-Gly-OH (900 mg, 4.30 mmol) in 53 mL of anhydrous DCM were added DCC (887 mg, 4.30 mmol) and DMAP (307 mg, 2.51 mmol). The reaction mixture was stirred at room temperature in 4h. The mixture was filtered. After filtration, the solvent was evaporated under vacuum to give the crude which was purified by column (normal phase - Biotage) with the gradient from 0-60% EtOAc in DCM to give compound Boc-Exatecan-Gly(Z) (1.09 g, 84% yield), which was dissolved in 30 mL MeOH / DCM (ratio 1 / 1), treated with palladium-on-carbon (150 mg, 10 wt. %) followed by H2bubbles for 1.5 hours. The resulting mixture was filtered through celite and washed with MeOH and DCM. The filtrate and washing were concentrated giving Boc-Exatecan-Gly (705 mg, 79% yield) as a white solid. Purity of the conjugate was evaluated by UPLC-MS and was > 95% with a m / z mass of 593 (M+H).

[0262] Step 2: Synthesis of 4-((2-(((1 S,9S)-1-((tert-butoxycarbonyl)amino)-9-ethyl-5-fluoro-4-methyl- 10,13-dioxo-2,3,9,10,13,15-hexahydro-1 H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1 ,2- b]quinolin-9-yl)oxy)-2-oxoethyl)amino)-4-oxobutanoic acid (Boc-Exatecan-Gly-OSu)

[0263] Employing the protocol for the synthesis of TBDPS-SN-38-Gly-OSu, Boc-Exatecan-Gly (56 mg, 94.7 pmol) in THF (6 mL) was treated with triethylamine (47,9 pL, 473 pmol) and succinic anhydride (28.4 mg, 284 mmol), worked up, and purified as described above to give Boc- Exatecan-Gly-OSu (31.4 mg, 48% yield). Purity of the conjugate was evaluated by UPLC-MS and was > 95% with a m / z mass of 693.06 (M+H).

[0264] Steps 3-4

[0265] In a 25-ml RBF, TBTU (5.73 mg, 0.0244 mmol) was dissolved in DMSO (248 pL). N,N- Diisopropylethylamine; DIEA; Hunig's base (10.6 pL, 0.0611 mmol) was added followed by AN- 01-123-1 (19.2 mg, 0.0244 mmol). The mixture was stirred for 10 minutes at room temp (pH ~7). In a separate 25-ml RBF TH19P01 (21.2 mg, 0.0110 mmol) was dissolved in DMSO (82.7 pL). After 10 min. of preactivation, the solution of TH19P01 was rapidly added to the solution of preactivated ester and the flask was washed with 1 portion of DMSO (41.3 pL). As the pH of the reaction mixture was slightly acidic (pH 5 immediately after the addition), N,N- diisopropylethylamine; DIEA; Hunig's base (5.32 pL, 0.0305 mmol) was added, which brought the pH back to 7-8. A LCMS of the reaction at t= 10 min (after the addition of DIPEA) indicated a clean, complete reaction. After stirring for 20 minutes (total time from DIPEA addition), the reaction was quenched by the addition of water. The precipitated was filtered and washed with water to afford Boc protected exatecan attached to TH19P01 and used for next step without purification. The next step was deprotection of the Boc group using 4N HCI in 1 ,4 dioxane. The product was purified using HPLC (Akta) using 10-80% acetonitrile (0.1% FA) in water (0.1% FA) and pure fractions were freeze dried to afford TH2303 as white solid. UPLC-MS and was > 95% with a m / z mass of 3073.28 (1024.41 , charge +3).

[0266] Synthesis of TH2310 (see FIGs. 9B-9C):

[0267] Step 1 : (S)-9-((tert-butyldiphenylsilyl)oxy)-4, 11 -diethyl-4-hydroxy- 1 , 12-dihydro- 14H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinoline-3,14(4H)-dione (TBDPS-SN-38)

[0268] TBDPS-SN-38 was prepared as described in step 1 of the synthesis of TH2101

[0269] Step 2 and 3:

[0270] Step 2:

[0271] TBDPS-SN-38 (3.00 g, 4.19 mmol) was taken in DCM (300 mL) and to that, DCC (2.07 g, 10.0 mmol), dimethylaminopyridine (716 mg, 5.86 mmol) and Z-L-LYS(BOC)-OH (3.82 g, 10.0 mmol) were added. Reaction was monitored with LC-MS. The reaction stopped at 70 % and it was brought to completion by addition of DCC and Z-L-LYS(BOC)-OH (1 equivalent each). Reaction mixture was loaded directly on to the dry silica gel (30g) and purified using 80 g column using ethyl acetate: hexanes (0-100%) solvent for elution and pure fractions were evaporated to afford (S)-9-((tert-butyldiphenylsilyl)oxy)-4, 11 -diethyl-3, 14-dioxo-3,4, 12,14-tetrahydro-1 H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl N2-((benzyloxy)carbonyl)-N6-(tert-butoxycarbonyl)- L-lysinate (3.20 g, 77 %) as final product. MH+ 910.1 is observed (because no ionisation was observed) at 1 .87 min on 4 min run with > 95% purity which was used as such in the next step.

[0272] Step 3:

[0273] (S)-9-((tert-butyldiphenylsilyl)oxy)-4, 11 -diethyl-3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl N2-((benzyloxy)carbonyl)-N6-(tert-butoxycarbonyl)- L-lysinate ( 3.20 g, 3.32 mmol) was dissolved in the mixture of DCM (50.0 mL) and methanol (150 mL) and nitrogen was bubbled through reaction mixture for 10 minutes and Pd on carbon 10% (1.00 g) was added at once. The reaction mixture was bubbled with nitrogen again and hydrogen gas was introduced with the help of balloon. Reaction mixture was stirred at RT and monitored with LCMS for completion. Reaction was almost complete after bubbling hydrogen for 20 hours. The reaction mixture was filtered through Celite® and solvent was removed under vacuum to afford (S)-9-((tert-butyldiphenylsilyl)oxy)-4,11 -diethyl-3, 14-dioxo-3,4, 12,14-tetrahydro- 1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl N6-(tert-butoxycarbonyl)-L-lysinate (4) (2.00 g, 70 %) as white solid. MH+ is 859 and is observed as 2 peaks. Product was used as such in the next step.

[0274] Steps 4 and 5: (S)-9-((tert-butyldiphenylsilyl)oxy)-4,11 -diethyl-3, 14-dioxo-3,4, 12,14-tetrahydro- 1 H-pyrano[3',4':6,7]indolizino[1 ,2- b]q u inoli n-4-y I N6-(tert-butoxycarbonyl)-N2-(4-((2,5- dioxopyrrolidin-1-yl)oxy)-4-oxobutanoyl)-L-lysinate (TBDPS-SN-38-Lys(Boc)-OSu)

[0275] Step 4:

[0276] (S)-9-((tert-butyldiphenylsilyl)oxy)-4, 11 -diethyl-3, 14-dioxo-3,4, 12,14-tetrahydro- 1 H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl N6-(tert-butoxycarbonyl)-N2-(4-((2,5- dioxopyrrolidin-1-yl)oxy)-4-oxobutanoyl)-L-lysinate (4) (2g, 2.33 mmol), Succinic anhydride (699 mg, 6.98 mmol) , dimethyl amino pyridine (322 uL, 2.33 mmol) and triethylamine (973 uL, 6.98 mmol) were taken in DCM (150 mL) and reaction was stirred at RT and monitored for the completion. Once starting material was totally consumed, reaction mixture was absorbed on silica gel and purified using DCM: Methanol as eluent using 40g column. Pure fractions were pooled together and evaporated under vacuum to afford 4-(((S)-6-((tert-butoxycarbonyl)amino)-1-(((S)- 9- ((tert- b uty Id i p he ny Isi lyl) oxy)-4 , 11 -diethyl-3, 14-dioxo-3,4, 12,14-tetrahydro- 1 H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)-1-oxohexan-2-yl)amino)-4-oxobutanoic acid (5)(1.18 g, 53 %) as off white solid. MH+ 953.47 was observed at 3.21 min on 4 min run with > 90% purity. Product was used as such in the next step.

[0277] Step 5:

[0278] 4-(((S)-6-((tert-butoxycarbonyl)amino)-1-(((S)-9-((tert-butyldiphenylsilyl)oxy)-4,11 -diethyl- 3,14-dioxo-3,4, 12, 14-tetrahydro- 1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)- 1 - oxohexan-2-yl)amino)-4-oxobutanoic acid (5) (1.18 g, 1.08 mmol) and N-hydroxysuccinimide (150 mg, 1.30 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCI (249 mg, 1.30 mmol) were taken in DMF (3.00 mL) and reaction was stirred at RT and monitored for completion. Once reaction was complete, water was added to the reaction mixture and it was filtered with sintered funnel to collect precipitates as (S)-9-((tert-butyldiphenylsilyl)oxy)-4, 11- diethyl-3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H-pyrano[3',4':6,7]indolizino[1 , 2- b] q u i no I in-4-y I N6-

[0279] (tert-butoxycarbonyl)-N2-(4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutanoyl)-L-lysinate (6) (1.03 g, 90 %) as off white solid. MH+ 1056.45 is observed at 3.30 minutes on 4 minutes run with > 95% purity.

[0280] Step 6:

[0281] (S)-9-((tert-butyldiphenylsilyl)oxy)-4, 11 -diethyl-3, 14-dioxo-3,4, 12,14-tetrahydro- 1 H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl N6-(tert-butoxycarbonyl)-N2-(4-((2,5- dioxopyrrolidin-1-yl)oxy)-4-oxobutanoyl)-L-lysinate (6) (500 mg, 0.473 mmol) was taken in DMSO (5.74 mL) and to that TH19P01 (424 mg, 0.220 mmol) was added. To the reaction mixture diisopropyl ethyl amine (182 uL, 1.42 mmol) was added and reaction was stirred at RT till all the starting material in the form of NH-ester was consumed. Reaction mixture was quenched by addition of water and precipitates were collected by filtration to afford (2S,5S,8S, 11 S, 14S, 17S,20S,23S,26S,29S,35S,38S,41 S,44S,47S)-23-(2-amino-2-oxoethyl)- 17- benzyl-20-butyl-14,38-bis((S)-10-((((S)-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo- 3,4,12,14-tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)carbonyl)-2,2- dimethyl-4, 12, 15-trioxo-3-oxa-5, 11 ,16-triazaicosan-20-yl)-8-(2-carboxyethyl)-26,44-bis(3- guanidinopropyl)-2-(4-hydroxybenzyl)-5,11-bis(hydroxymethyl)-29,47-diisopropyl-35,41- dimethyl-4,7,10,13,16,19,22,25,28,31 ,34,37,40,43,46,49,52-heptadecaoxo- 3,6,9,12,15,18,21 ,24,27,30,33,36,39,42,45,48,51-heptadecaazatripentacontanoic acid (7)(710 mg, crude) and dried under vacuum and used as such in the next step. Two peaks at 2.31 and 2.77 min are observed corresponding to partial deprotected (one of the TBS group cleaved during the reaction) product and desired product correspondingly.

[0282]

[0283] (2S,5S,8S, 11 S, 14S, 17S,20S,23S,26S,29S,35S,38S,41 S,44S,47S)-23-(2-amino-2-oxoethyl)- 17- benzyl-20-butyl-14,38-bis((S)-10-((((S)-9-((tert-butyldiphenylsilyl)oxy)-4,11-diethyl-3,14-dioxo- 3,4,12,14-tetrahydro-1 H-pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)carbonyl)-2,2- dimethyl-4, 12, 15-trioxo-3-oxa-5, 11 ,16-triazaicosan-20-yl)-8-(2-carboxyethyl)-26,44-bis(3- guanidinopropyl)-2-(4-hydroxybenzyl)-5, 11 -bis(hydroxymethyl)-29,47-diisopropyl-35,41- dimethyl-4,7,10,13,16,19,22,25,28,31 ,34,37,40,43,46,49,52-heptadecaoxo- 3,6,9,12,15,18,21 ,24,27,30,33,36,39,42,45,48,51-heptadecaazatripentacontanoic acid (7 ) (710 mg, 0.187 mmol) crude was dissolved in DCM and to that TBAF (280 pL, 0.280 mmol) was added and reaction was monitored for complete deprotection of TBDPS using LCMS. Once deprotection was complete, 4N HCI in dioxan (1.87 ml_, 7.46 mmol) was added and reaction was stirred for further 1 hour. Once all the deprotection was complete, solvent was removed by blowing nitrogen gas and residue was dissolved in 3 ml DMSO and purified using ACTA using Water: Acetonitrile as eluent (0-80%, 0.1 % formic acid). Pure fractions were pooled together to afford (2S,5S,8S,11S,14S,17S,20S,23S,26S,29S,35S,38S,41S,44S,47S)-14,38-bis(4-(4-(((S)-6- amino- 1 -(((S)-4, 11 -diethyl-9-hydroxy-3, 14-dioxo-3,4, 12, 14-tetrahydro- 1 H- pyrano[3',4':6,7]indolizino[1 ,2-b]quinolin-4-yl)oxy)-1-oxohexan-2-yl)amino)-4- oxobutanamido)butyl)-23-(2-amino-2-oxoethyl)-17-benzyl-20-butyl-8-(2-carboxyethyl)-26,44- bis(3-guanidinopropyl)-2-(4-hydroxybenzyl)-5,11-bis(hydroxymethyl)-29,47-diisopropyl-35,41- dimethyl-4,7,10,13,16,19,22,25,28,31 ,34,37,40,43,46,49,52-heptadecaoxo- 3,6,9,12,15,18,21 ,24,27,30,33,36,39,42,45,48,51-heptadecaazatripentacontanoic acid (262 mg, 45 %)

[0284] MH+3 (1044.15) for the conjugate was observed at 1.16 min on 4 min run > 98% purity.

[0285] Example 3: Analysis of the expression of efflux pumps, Sortilin and Topoisomerase 1 in cancer cell lines

[0286] The expression of the efflux pumps for camptothecin analogs P-glycoprotein (P-gp or MDR1) and breast cancer resistance protein (BCRB or ABCG2), as well as that of Sortilin (SORT1), was assessed in several human colorectal cancer cell lines. The expression of the epidermal growth factor receptor (EGFR), for potential combination with the EGFR inhibitor Cetuximab (Erbitux®) used for the treatment of metastatic colorectal cancer, was also tested. Because of its expression of SORT1 , BCRB and EGFR, the HT-29 cell lines was selected for studying the effects of the conjugates (FIG. 11 A). As shown in FIGs. 11B-C, all colorectal cancer and breast cancer cell lines tested, including HT-29, express Topoisomerase 1 (TOPO1), the target of camptothecin analogs.

[0287] Example 4: Effect of camptothecin analogs and peptide-camptothecin analog conjugates on the growth inhibition of HT-29 cells in vitro

[0288] FIGs. 12A-B show the anti-proliferative activity of PDCs and unconjugated drugs on HT-29, HCT-116 and LoVo cancer cells. SN-38 and Exatecan IC5o values were lowerthan those obtained for Irinotecan. Peptide-camptothecin analog conjugates were able to inhibit HT-29, HCT-116 and / or LoVo tumor cell growth, confirming that the conjugation of the camptothecin analogs to the peptides does not inhibit their anti-proliferative activity.

[0289] Example 5: Internalization of peptides and peptide conjugates is sortilin-dependent

[0290] It was next assessed whether down-regulation of the expression of sortilin in HT-29 cancer cells could affect the internalization of the TH19P01 peptide (SEQ ID NO:3, present in several of the conjugates described herein) and of the TH2101 conjugate (that comprises the TH19P01 peptide). The results reported in FIGs. 13A-13B and 14A-14C show that treatment with an siRNA against sortilin reduces sortilin expression in HT-29 cancer cells (FIGs. 13A and 14A), and that the reduced sortilin expression is associated with a reduction of the uptake of the fluorescently- tagged TH19P01 peptide and TH2101 conjugate (FIGs. 13B and 14B-C). These results demonstrate that the TH19P01 peptide specifically binds to sortilin at the surface of the cells, and thus that it may be used in conjugates to deliver camptothecin analogs to sortilin-expressing cells.

[0291] Example 6: Effects of camptothecin analogs and camptothecin analog conjugates in colorectal, ovarian and breast cancer mouse models

[0292] The antitumor effects of unconjugated camptothecin analogs and camptothecin analog conjugates was tested in vivo in the HT-29, HCT-116 and patient-derived xenograft colorectal cancer mouse models, the MDA-MB-231 triple-negative breast cancer (TNBC) xenograft model, and the SKOV3 ovarian cancer xenograft model. The results reported in FIGs. 15A-C show a weak inhibition of tumor growth in mice treated with the unconjugated camptothecin analogs irinotecan and exatecan. For irinotecan, administration of a dose corresponding to 1 / 8 of the maximum tolerated dose (MTD) did not have any effect on tumor growth. In contrast, administration of this dose of irinotecan (1 / 8 MTD) but as part of a conjugate with the sortilin- targeting peptide of SEQ ID NO:1 (TH2102) almost completed inhibited HT-29 tumor growth (FIGs. 16A-B). A similar effect was obtained with the other irinotecan conjugates TH2204, the SN-38 conjugates TH2101 and TH2205, as well as the exatecan conjugates TH2301 and TH2303 (FIGs. 17A-B and 18A-B and D). The TH2303 exatecan conjugate was also able to almost completely inhibit HCT-116 tumor growth (FIG. 19A), and to completely inhibit MDA-MB-231 tumor growth (FIG. 20A). Administration of the different conjugates was not associated with significant weight loss in any of the models tested (FIGs. 16C, 17C, 18C, 18E, 19B and 20B). The results reported in FIGs. 21 A and 21 B show that the exatecan conjugate TH2303, but not unconjugated exatecan, is able to reduce the growth of ovarian tumor cells (SKOV3) and patient- derived xenograft (PDX) colorectal cancer cells, respectively.

[0293] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise.

Claims

WHAT IS CLAIMED IS:

1. A conjugate comprising the following structure I, or a pharmaceutically acceptable salt thereof:A — L — B (I); whereinA is a camptothecin analog.B is a peptide binding to a Sortilin receptor, wherein the peptide comprises an amino acid sequence having at least 60% identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2:X1GVRAKAGVRN(Nle)FKSESYX2(SEQ ID NO:1)X1YKSLRRKAPRWDAPLRDPALRQLLX2(SEQ ID NO:2); wherein X1is cysteine (C) or is absent, and X2is cysteine (C) or is absent;L is a linker, wherein a first end of the linker is attached to a side chain of one or more of the amino acid or modified side chain residues of the peptide, and / or to the N- and / or C- terminal end of the peptide, and a second end of the linker is attached to the camptothecin analog.

2. The conjugate of claim 1 , wherein L is a cleavable linker, wherein a first end of the linker is attached to a side chain of one or more of the lysine (K) and / or cysteine (C) residues of the peptide, and / or to the N- and / or C-terminal end of the peptide, and a second end of the linker is attached to an oxygen or nitrogen atom of the camptothecin analog.

3. The conjugate or pharmaceutically acceptable salt thereof of claim 1 or 2, wherein the conjugate or salt thereof is of one of the following formulas la, lb, Ic or Id:A1 — L1 — B (la);A1 — L1 — B — L2 — A2 (lb);wherein L1 , L2, L3 and L4 are linkers which may be the same or different, and A1 , A2, A3 and A4 are camptothecin analogs which may be the same or different.

4. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 3, wherein the camptothecin analog is irinotecan, SN-38, exatecan or deruxtecan.

5. The conjugate or pharmaceutically acceptable salt thereof of claim 4, wherein the camptothecin analog is SN-38.

6. The conjugate or pharmaceutically acceptable salt thereof of claim 4, wherein the camptothecin analog is exatecan.The conjugate or pharmaceutically acceptable salt thereof of claim 4, wherein the camptothecin analog is deruxtecan.

8. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 7, wherein the peptide comprises an amino acid sequence having at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2.

9. The conjugate or pharmaceutically acceptable salt thereof of claim 8, wherein the peptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence set forth in SEQ ID NO: 1 or 2.

10. The conjugate or pharmaceutically acceptable salt thereof of claim 9, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2.11 . The conjugate or pharmaceutically acceptable salt thereof of claim 9, wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or 2.

12. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 11 , wherein X1and / or X2is C, and wherein the first end of the linker is attached to the side chain of X1and / or X2.

13. The conjugate or pharmaceutically acceptable salt thereof of claim 12, wherein X1is absent and X2is C, and wherein the first end of the linker is attached to the side chain of X2.

14. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 11 , wherein X1and X2are absent.

15. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 14, wherein the linker comprises at least one amino acid.

16. The conjugate or pharmaceutically acceptable salt thereof of claim 15, wherein the at least one amino acid comprises glycine, valine and / or citrulline.

17. The conjugate or pharmaceutically acceptable salt thereof of claim 15 or 16, wherein the cleavable linker is a protease-cleavable linker.

18. The conjugate or pharmaceutically acceptable salt thereof of claim 17, wherein the protease is a cysteine protease.

19. The conjugate or pharmaceutically acceptable salt thereof of claim 18, wherein the cysteine protease is cathepsin B.

20. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 19, wherein the linker comprises polyethylene glycol (PEG).

21. The conjugate or pharmaceutically acceptable salt thereof of claim 20, wherein the linker comprises from 2 to 10 PEG units.

22. The conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 21 , wherein the linker is of one of the following structures:• CO(CH2)aCONH(CH2)bCO, wherein a and b are independently 1 , 2, 3, or 4;• CO(CH2)d(OCH2CH2)eCONH(CH2)fCO, wherein d and f are independently 1 , 2, 3, or 4, and e is an integer from 2 to 8; or23. The conjugate or pharmaceutically acceptable salt thereof of claim 1 , wherein the conjugate has one of the following structures:24. A pharmaceutical composition comprising the conjugate or salt thereof of any one of claims 1 to 23, and a pharmaceutically acceptable excipient.

25. A method for treating a Sortilin-expressing cancer in a subject in need thereof comprising administering to the subject an effective amount of the conjugate or salt thereof of any one of claims 1 to 23, or the pharmaceutical composition of claim 24.

26. The method of claim 25, wherein the Sortilin-expressing cancer is a hematological cancer, ovarian cancer, endometrial cancer, cervix cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small intestine cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testis cancer, bladder cancer, kidney cancer, or pancreatic cancer.

27. The method of claim 25 or 26, wherein the Sortilin-expressing cancer is a poor prognosis cancer.

28. The method of any one of claims 25 to 27, wherein the Sortilin-expressing cancer is an immunologically cold cancer.

29. The method of any one of claims 25 to 28, wherein the conjugate, salt thereof or composition is administered in combination with one or more additional active agents or therapies for cancer.

30. The method of claim 29, wherein the one or more additional active agents or therapies for cancer comprise an immunotherapy.

31. The method of claim 30, wherein the immunotherapy comprises an immune checkpoint inhibitor.

32. The method of claim 31 , wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

33. The method of claim 32, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

34. Use of the conjugate or salt thereof of any one of claims 1 to 23, or the pharmaceutical composition of claim 24, for treating a Sortilin-expressing cancer in a subject.

35. Use of the conjugate or salt thereof of any one of claims 1 to 23, or the pharmaceutical composition of claim 24, for the manufacture of a medicament for treating a Sortilin-expressing cancer in a subject.

36. The use of claim 34 or 35, wherein the Sortilin-expressing cancer is a hematological cancer, ovarian cancer, endometrial cancer, cervix cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small intestine cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testis cancer, bladder cancer, kidney cancer, or pancreatic cancer.

37. The use of any one of claims 34 to 36, wherein the Sortilin-expressing cancer is a poor prognosis cancer.

38. The use of any one of claims 34 to 37, wherein the Sortilin-expressing cancer is an immunologically cold cancer.

39. The use of any one of claims 34 to 38, wherein the conjugate, salt thereof, composition or medicament is for administration in combination with one or more additional active agents or therapies for cancer.

40. The use of claim 39, wherein the one or more additional active agents or therapies for cancer comprise an immunotherapy.

41. The use of claim 40, wherein the immunotherapy comprises an immune checkpoint inhibitor.

42. The use of claim 41 , wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

43. The use of claim 42, wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

44. The conjugate or salt thereof of any one of claims 1 to 23, or the pharmaceutical composition of claim 24, for use in the treatment of a Sortilin-expressing cancer in a subject.

45. The conjugate, salt thereof or pharmaceutical composition for use according to claim 44, wherein the Sortilin-expressing cancer is a hematological cancer, ovarian cancer, endometrial cancer, cervix cancer, skin cancer, brain cancer, breast cancer, colorectal cancer, small intestine cancer, liver cancer, lung cancer, eye cancer, prostate cancer, head and neck cancer, stomach cancer, bone cancer, thyroid cancer, testis cancer, bladder cancer, kidney cancer, or pancreatic cancer.

46. The conjugate, salt thereof or pharmaceutical composition for use according to claim 45, wherein the Sortilin-expressing cancer is a poor prognosis cancer.

47. The conjugate, salt thereof or pharmaceutical composition for use according to claim 45 or 46, wherein the Sortilin-expressing cancer is an immunologically cold cancer.

48. The conjugate, salt thereof or pharmaceutical composition for use according to any one of claims 45 to 47, wherein the conjugate, salt thereof or composition is for administration in combination with one or more additional active agents or therapies for cancer.

49. The conjugate, salt thereof or pharmaceutical composition for use according to claim 48, wherein the one or more additional active agents or therapies for cancer comprise an immunotherapy.

50. The conjugate, salt thereof or pharmaceutical composition for use according to claim 49, wherein the immunotherapy comprises an immune checkpoint inhibitor.

51. The conjugate, salt thereof or pharmaceutical composition for use according to claim 50, wherein the immune checkpoint inhibitor is a PD1 or PD-L1 inhibitor.

52. The conjugate, salt thereof or pharmaceutical composition for use according to claim 51 , wherein the PD1 or PD-L1 inhibitor is an anti-PD1 or anti-PD-L1 antibody.

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