Cell targeting constructs and uses thereof

Abstract

Provided herein are cell-targeted cytotoxic compounds. Such compounds are useful, for example, for selectively binding and killing cancer cells, such as cancer stem cells expressing LGR4, LGR5 or LGR6. In some embodiments, the cytotoxic compounds comprise an LGR binding polypeptide, a cytotoxic agent (e.g., MMAE), and an Fc region (e.g., a mutant Fc domain). Also provided are methods of treating cell proliferative diseases, such as cancer, using the compounds.

Description

Background Art

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 381,050, filed Oct. 26, 2022, U.S. Provisional Patent Application No. 63 / 386,777, filed Dec. 9, 2022, and U.S. Provisional Patent Application No. 63 / 478,428, filed Jan. 4, 2023, the entire contents of which are incorporated herein by reference.

[0002] This invention was made with government support under Grant No. CA023100 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] This application contains an ST.26 XML sequence listing, which has been submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing was created on Oct. 24, 2023, is named CLFR.P0508WO_ST26, and is 100 KB. 1. Field of the Technology

[0004] The present disclosure generally relates to the fields of molecular biology and medicine. More specifically, it relates to cell-targeted cytotoxic constructs. 2. Background Art

[0005] Immunotoxins and immunoconjugates have shown clinical promise for treating diseases such as cancer, but significant clinical limitations remain. The successful development of targeted therapies (e.g., targeted therapies for cancer applications) relies on identifying ligands and antigens specific to target cells, generating molecules capable of specifically targeting those components, and ultimately using highly toxic molecules to kill the target cells. Immunoconjugates composed of antibodies and small toxic drugs or radioisotopes have been successfully tested in vitro, in animal models, and shown activity in the clinical setting. In addition to small molecules using toxin components, several highly cytotoxic protein components, such as diphtheria toxin, ricin A chain, Pseudomonas aeruginosa exotoxin, and gelonin (rGel), have been used for targeted therapy. However, problems such as capillary leak syndrome, immunogenicity, and unintended toxicity (towards non-target cells) continue to limit the implementation of successful treatment, especially for long-term or chronic applications.

[0006] The treatment of cancers that include cancer stem cells (CSCs) presents particularly challenging clinical problems. CSCs are typically a small subpopulation of cells within a tumor that, when transplanted into an animal host, have the ability to self-renew, differentiate, and be tumorigenic, and many cell surface markers such as CD44, CD24, and CD133 are commonly used to identify and enrich CSCs from tumors (Yu et al., 2012). CSCs are typically resistant to conventional chemotherapy and radiotherapy, and CSCs may be the origin that drives cancer metastasis. CSCs remain an important clinical problem in cancer treatment, and developing methods to kill CSCs is one of the major goals in the field of oncology. There remains a need for highly specific and highly active toxin molecules, as well as cell-targeting moieties that contain such molecules, which exhibit improved therapeutic efficacy and pharmacological properties. In particular, there is a need to improve the treatment of cancers whose growth is dependent on CSCs. SUMMARY OF THE INVENTION

[0007] In some aspects, the present disclosure overcomes limitations in the prior art by providing improved constructs that can be used to deliver toxins to cells (e.g., cancer cells) and exhibit improved therapeutic properties (e.g., improved efficacy) and / or improved pharmacokinetic properties (e.g., improved half-life). In some aspects, constructs or polypeptides are provided that include an LGR-targeting moiety or an R-spondin-targeting moiety (e.g., the Fu1-Fu2 domain) and a cytotoxic payload (e.g., MMAE, deruxtecan, PNU159682). A variety of cytotoxic payloads can be used (e.g., FIGS. 25 to 27, 29), and multiple cytotoxic payloads can be included in the construct (e.g., Figures 30 to 32 ). The construct can also include a mutant Fc domain (e.g., the “DHS” mutant), and the Fc can dimerize the construct (e.g., see the schematic in Figure 1 ). Since the constructs provided herein can target or selectively bind to LGRs (e.g., LGR4, LGR5, LGR6), they can be particularly useful for killing cancer stem cells and / or for treating cancers that include cancer stem cells (e.g., FIGS. 3A to E, Figure 4 ). The LGR-targeting moiety can also selectively bind to ZNRF3 and / or RNF43. As shown in the following examples, the constructs can exhibit improved efficacy and half-life, and mutant forms of the LGR-targeting moiety (e.g., R28A) and the Fc hinge region (e.g., DHS Fc) are provided, which are observed to increase the plasma half-life of the construct in vivo without altering the efficacy of the construct (e.g., Figures 28 to 29)。Using partial reduction of one or more disulfide bonds in sortase A, sortase E, and / or the LGR-binding moiety or the Fc hinge region, one or more additional cytotoxic moieties can be covalently linked to the construct such that the additional cytotoxic moieties are covalently bound to the construct. Using a variety of cancer animal models, including in vivo xenograft animal models of colorectal cancer, gastric cancer, neuroblastoma, and ovarian cancer, the anti-cancer effects of the constructs provided herein are observed in vivo (e.g., see FIGS. 6A - B, 7A - B, 8A - D, 9, 12A - B, 18A - B, and Figures 21 to 24 )。Additional copies of the LGR-targeting moiety can be used to enhance binding to cancer cells expressing LGR, such as cancer stem cells ( Figure 17 )。The constructs provided herein may or may not contain a linker (e.g., Figures 19 to 20 )。Compared to the constructs provided in Yu et al. (2021), the anti-cancer compounds provided herein are not only chemically different but also may offer advantages in terms of potency, efficacy, and / or in vivo pharmacokinetic half-life; for example, compared to R1FF-MMAE (Yu et al., 2021), the FcF2 compound provided herein unexpectedly shows a distribution half-life approximately 15 times longer than that of R1FF-MMAE, as measured in vivo using plasma pharmacokinetic analysis in mice (e.g., Figure 34 )。Methods of treating a disease (e.g., cancer) with the constructs are also provided. The therapeutic compounds provided herein can be particularly useful for treating cancers whose growth is dependent on CSCs.

[0008] In some aspects, compounds are provided that selectively target cancers expressing the R-sponin receptors LGR4, LGR5, and / or LGR6. These receptors are expressed in a variety of cells, including epithelial stem cells, normal tissues, and tumors, and play important roles in embryogenesis, tissue homeostasis, and regeneration. LGR5 and LGR6 are expressed at elevated levels in a variety of different types of cancers, cancer stem cells (CSCs), and stem cells in the ovarian surface and fallopian tube epithelium from which ovarian cancers originate. High-grade serous ovarian cancer is one of the tumors that express abnormally high levels of LGR5 and LGR6 mRNA. R-spondin is the natural ligand of LGR5 and LGR6, which binds to the LGR5 and LGR6 with nanomolar affinity. As shown in the following examples, to target cancer stem cells (e.g., cancer stem cells in ovarian cancer and other tumors), the sortase reaction was used to site-specifically conjugate the potent cytotoxin monomethyl auristatin E (MMAE) to two furin-like domains (Fu1-Fu2) of RSPO1, which mediate its binding to LGR5 and LGR6 and their coreceptors ZNRF3 and RNF43 through a protease-cleavable linker, and an immunoglobulin Fc domain was included at the N-terminus and used to dimerize the receptor-binding domain such that each molecule carried two MMAEs. The resulting molecule, FcF2-MMAE, exhibited: 1) selective LGR5-dependent low-nanomolar cytotoxicity against ovarian cancer cells in vitro; 2) selectivity dependent on binding to both LGR receptors and ubiquitin ligase coreceptors; 3) good stability and plasma pharmacokinetic properties when administered IV, with an elimination half-life of 29.7 hours; 4) selective inhibition of LGR5-rich tumors in vivo, rather than isogeneic LGR5-deficient tumors; and 5) therapeutic efficacy in two different aggressive wild-type human ovarian cancer xenograft models. FcF2-MMAE may offer benefits over the previously generated R1FF-MMAE (Yue et al., 2021), which include: a) improving folding by leveraging the chaperone function of Fc, thereby increasing the yield of protein from transiently transfected cultures; b) dimerizing the resulting molecule such that it carries two MMAE molecules rather than one MMAE molecule; c) increasing the plasma half-life by including an Fc mutant form with improved FcRn binding characteristics; and d) increasing the affinity for binding to LGR and ZNRF3 / RNF43 by including two rather than just one copy of the Fu1-Fu2 (FuFu) domain. The achievement of these goals was demonstrated by a significant increase in the yield of the FcF2-His precursor, higher potency and selectivity when tested in isogeneic OVCAR8 / EV cells and OVCAR8 / LGR5 cells, a 6-fold increase in the terminal plasma half-life, and improved efficacy in the xenograft model.Importantly, the dimerization of the two Fu1-Fu2 domains caused by the presence of the Fc domain did not impair the efficiency of the sortase reaction, which remained high. These results support the view that the Fu1-Fu2 domain of RSPO1 can function as a drug carrier for targeted delivery of therapeutic compounds, and that FcF2-MMAE can selectively target cells in tumors expressing stem cell markers. Without wishing to be bound by any theory, these results are consistent with the view that the Fu1-Fu2 portion of the compound can engage both (i) an LGR (e.g., LGR4 or LGR5 or LGR6) and (ii) ZNRF3 or RNF43 simultaneously. In e.g. Figures 13 to 16 Also shown herein are exemplary amino acid sequences and nucleotides encoding polypeptides included in the therapeutic compounds of the present disclosure. In some embodiments, compounds are provided herein that comprise a polypeptide that (in the N-terminal to C-terminal direction) comprises: an Fc domain (e.g., a mutant Fc domain, such as SEQ ID NO:13) and SEQ ID NO:75; wherein the polypeptide is covalently linked to a cytotoxic moiety (e.g., valine-citrulline-PABA-MMAE).

[0009] One aspect of the present disclosure relates to compounds that comprise one or more cytotoxic agents conjugated to a polypeptide comprising one or more LGR-binding domains, wherein (i) the polypeptide further comprises an Fc region, and / or (ii) the polypeptide comprises at least two copies of the LGR-binding domain; and wherein each LGR-binding domain comprises a polypeptide having at least 95% sequence identity to at least one of SEQ ID NO: 4, 78 to 83, 85 to 89, 90 to 96, 98, or 102. The LGR-binding domains may each independently comprise an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87. In some embodiments, the LGR-binding domain is from human R-spondin-1 (hR-spondin-1), human R-spondin-2 (hR-spondin-2), human R-spondin-3 (hR-spondin-3), or human R-spondin-4 (hR-spondin-4). The LGR-binding domain may independently comprise an amino acid sequence selected from FuFu (SEQ ID NO: 4) or FuFu N137A (SEQ ID NO: 17). The LGR-binding domain may comprise a mutation at position R28 or a substitution mutation at position R30, wherein the numbering is according to Kabat. The substitution mutation may be a substitution of arginine with alanine. In some embodiments, the substitution mutation is R28A. The LGR-binding domain may comprise a Fu1-Fu2(R30A) mutant (SEQ ID NO: 91) or a Fu1-Fu2(R30A) mutant (SEQ ID NO: 100). In some embodiments, the LGR-binding domain comprises a Fu1-Fu2(R30A) mutant (SEQ ID NO: 100). The LGR-binding domain may comprise Fu1-Fu2(R22 to R31 deletion) (SEQ ID NO: 92), Fu1-Fu2(K25 to R31 deletion) (SEQ ID NO: 93), Fu1-Fu2(R28 to R31 deletion) (SEQ ID NO: 94), or Fu1-Fu2(R22 to K27 deletion) (SEQ ID NO: 95). The polypeptide may comprise FcST4 (SEQ ID NO: 105). In some embodiments, the Fc region is located N-terminal to the LGR-binding domain, or wherein the polypeptide comprises, in the N- to C-direction: an Fc region and an LGR-binding domain. In some embodiments, the Fc region is an IgG Fc domain. The polypeptide may comprise SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.In some embodiments, the polypeptide comprises SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, or SEQ ID NO: 88; and wherein the polypeptide does not comprise SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, or SEQ ID NO: 89. In some embodiments, the polypeptide comprises SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, or SEQ ID NO: 89; and wherein the polypeptide does not comprise SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, or SEQ ID NO: 88. In some embodiments, the human IgGFc domain is capable of binding to human FcRn at acidic pH, wherein the Fc domain has the following substitution mutations: (i) an aspartic acid substitution at position 309 (L / V309D); (ii) a histidine substitution at position 311 (Q311H); and (iii) a serine substitution at position 434 (N434S) or a tyrosine substitution at position 434 (N434Y); wherein the amino acid position numbering is according to the Kabat system; wherein the Fc domain binds to FcRn at acidic pH with a higher affinity than the wild type. In some embodiments, the substitution mutation at position 434 is serine (N434S) or tyrosine (N434Y). The polypeptide may comprise SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. The polypeptide may comprise SEQ ID NO: 13. The Fc domain may be glycosylated. The Fc domain may have the same or substantially the same binding affinity for FcγR as compared to the wild type Fc domain. The Fc domain may have the same or substantially the same binding affinity for one, two, or all of FcγRI, FcγRII, and FcγRIII as compared to the wild type. In some embodiments, the Fc domain does not bind to FcRn detectably or selectively at neutral pH, and / or does not exhibit or substantially does not exhibit binding to FcRn at neutral pH. In some embodiments, as compared to the wild type, the Fc domain exhibits: (i) enhanced binding to FcRn at pH 5.8, and (ii) reduced binding to FcRn or undetectable binding to FcRn at pH 7.4. The Fc domain may be non-glycosylated. In some embodiments, the Fc domain has a glutamic acid substitution at position 264 (V264E). The IgG may be IgG1, IgG2, IgG3, or IgG4. In some embodiments, the IgG is IgG1. In some embodiments, the Fc domain comprises the following substitution mutations:. (i) IgGl-Fc EDHS (V264E; L309D; Q311H; N434S), (ii) IgG1-Fc EDHY (V264E; L309D; Q311H; N434Y), (iii) IgG1-Fc DHS (L309D; Q311H; N434S), (iv) IgGl-Fc DHY (L309D; Q311H; N434Y), (v) IgG2-DHS (V309D; Q311H; N434S), (vi) IgG3-DHS (L309D; Q311H; N434S), or (vii) IgG4-DHS (L309D; Q311H; N434S). The Fc domain may comprise or consist of IgG1-Fc DHS (L309D; Q311H; N434S). The compound may dimerize via disulfide bonds formed in the Fc domain. The Fc domain may be separated from the LGR binding domain by a linker. The linker may comprise G4S (SEQ ID NO:18) or (G4S)2 (SEQ ID NO:5). In some embodiments, the Fc domain is not separated from the LGR binding domain by a linker, or wherein the polypeptide does not comprise a linker. The polypeptide may comprise, from the N-terminus to the C-terminus: an Fc domain and an LGR binding domain; or wherein the Fc domain is closer to the N-terminus of the polypeptide than the LGR binding domain. The compound may comprise two copies of FuFu (SEQ ID NO:4) or FuFu N137A (SEQ ID NO:17). The two copies of FuFu (SEQ ID NO:4) or FuFu N137A (SEQ ID NO:17) may be separated by a linker, preferably a G4S linker (SEQ ID NO:18) or a (G4S)2 linker (SEQ ID NO:5). In some embodiments, the compound comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:19. In some embodiments, the compound comprises SEQ ID NO:1. The compound may comprise SEQ ID NO:20. In some embodiments, the polypeptide comprises a leader sequence. The leader sequence may be an endogenous leader sequence, an IgG leader sequence, or an IgK leader sequence. In some embodiments, the IgG leader sequence is an IgGk leader sequence (SEQ ID NO:8). In some embodiments, the polypeptide does not comprise a leader sequence. The cytotoxic agent may be a conjugated drug. In some embodiments, the drug is maytansine, auristatin, amanitin, calicheamycin, psymberin, duocarmycin, anthracycline, camptothecin, doxorubicin, paclitaxel, tubulysin, eribulin, or pyrrolobenzodiazepine The drug can be an auristatin, such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or PF-06380101. In some embodiments, the auristatin is monomethyl auristatin E (MMAE). The drug can be a camptothecin analogue (e.g., topotecan, irinotecan, belotecan, or deruxtecan). The drug can be an anthracycline analogue (e.g., PNU-159682; CAS No.: 202350-68-3). The conjugated drug can be linked to the polypeptide via a linker. The linker can be a protease-cleavable linker, such as citrulline-valine. In some embodiments, the compound comprises at least one spacer or linker (e.g., 1, 2, or 3 spacers or linkers) between the cytotoxic agent and the LGR-binding domain. The compound can comprise two linkers or spacers. The linker or spacer can comprise G4S (SEQ ID NO:18) or (G4S)2 (SEQ ID NO:5). The cytotoxic moiety can be a cytotoxic protein, such as a serine protease. The serine protease can be granzyme B (GrB). In some embodiments, the compound comprises SEQ ID NO:1 covalently linked to monomethyl auristatin E (MMAE). In some embodiments, the compound comprises SEQ ID NO:2 covalently linked to monomethyl auristatin E (MMAE). The compound can dimerize via a disulfide bond in the Fc domain. In some embodiments, the cytotoxic agent has been covalently bound to the polypeptide via a sortase linker. The sortase can be a sortase A linker or a sortase E linker. In some embodiments, the compound comprises a sortase linker between the LGR-binding domain and the cytotoxic agent. In some embodiments, the cytotoxic agent has been covalently bound to the polypeptide via a sortase, such as by causing the sortase to catalyze the covalent binding of the cytotoxic agent to the polypeptide. The sortase can be a sortase A linker or sortase E. The sortase linker can comprise the sequence LPXT(G) n(where n = 1 to 10 or any range derivable therefrom (e.g., n = 4 to 9)), or LAHTGG (SEQ ID NO:106). In some embodiments, the sortase linker is LPETGG (SEQ ID NO:6). The compound may further comprise a second cytotoxic agent. The first cytotoxic agent and the second cytotoxic agent may each independently be selected from the cytotoxic agents described above or herein. The first cytotoxic agent and the second cytotoxic agent may be covalently bound to the polypeptide via a sortase linker. In some embodiments, the first cytotoxic agent is covalently linked to a first sortase linker on the N-terminal side of the polypeptide, and wherein the second cytotoxic agent is covalently linked to a second sortase linker on the N-terminal side of the polypeptide. The first sortase linker comprises the sequence LPXT(G)n, where n = 1 to 10. The first sortase linker may be LPETGG (SEQ ID NO:6). The second sortase linker may be LAHTGG (SEQ ID NO:106). In some embodiments, the first cytotoxic moiety has been covalently bound to the first sortase linker using sortase A, and wherein the second cytotoxic moiety has been covalently bound to the second sortase linker using sortase E. In some embodiments, the first cytotoxic moiety and the second cytotoxic moiety are each independently a conjugated drug as described above or herein, or a cytotoxic protein as described above or herein. The first cytotoxic moiety and the second cytotoxic moiety may be different conjugated drugs. The first cytotoxic moiety and the second cytotoxic moiety may have the same structure. In some preferred embodiments, both the first cytotoxic moiety and the second cytotoxic moiety are monomethyl auristatin E (MMAE). In some embodiments, the first cytotoxic agent or the second cytotoxic agent is linked to the polypeptide via a disulfide bond, preferably wherein the disulfide bond is present in the Fc region or the LGR binding domain. The disulfide bond may be included in a maleimide group. The maleimide group may be covalently bound to a cleavable linker. The cleavable linker may comprise a valine (Val)-citrulline (Cit) bond. In some embodiments, the first cytotoxic agent is covalently bound to the polypeptide to a sortase linker that comprises the sequence LPXT(G) n or LAHTGG (SEQ ID NO:106), where n = 1 to 10; and wherein the second cytotoxic agent has been linked to the polypeptide via a disulfide bond. The disulfide bond may be present in the Fc region. The disulfide bond may be present in the LGR binding domain. The polypeptide may comprise SEQ ID NO:76. In some embodiments, the polypeptide comprises SEQ ID NO:77. In some embodiments, the polypeptide is covalently linked to -PABA-MMAE. The compound is comprised in a pharmaceutical composition. The pharmaceutical composition may be formulated for intravenous, intraperitoneal, subcutaneous, intratumoral, intrathecal, inhaled, intraarterial, or intrapleural administration.

[0010] Another aspect of the present disclosure relates to pharmaceutical compositions comprising the compounds as described above herein. The pharmaceutical composition can be formulated for intravenous, intraperitoneal, subcutaneous, intratumoral, intrathecal, inhalation, intraarterial, or intrapleural administration.

[0011] Another aspect of the present disclosure relates to nucleic acids encoding the polypeptides described above or herein.

[0012] Another aspect of the present disclosure relates to host cells comprising the nucleic acids described above or herein. The cell can be a bacterial cell. The cell can be a eukaryotic cell (e.g., a human cell, an insect cell, or a yeast cell). The human cell can be a HEK293 cell, a Chinese Hamster Ovary (CHO) cell, or a variant thereof.

[0013] Another aspect of the present disclosure relates to a method of generating a therapeutic compound that binds to an LGR receptor, wherein the method comprises: (a) expressing in a cell a polypeptide encoded by a nucleic acid as described above or herein, wherein the polypeptide comprises a sortase linker at the end of the polypeptide; (b) obtaining the polypeptide; and (c) contacting a cytotoxic agent and the polypeptide with a first transpeptidase, thereby covalently conjugating the cytotoxic compound to the polypeptide. The cell can be a bacterial cell or a eukaryotic cell (e.g., a mammalian cell or an insect cell). The mammalian cell can be a HEK293 cell, a Chinese Hamster Ovary (CHO) cell, or a variant thereof. In some embodiments, the first transpeptidase is sortase A or sortase E. In some embodiments, prior to step (c), the cytotoxic moiety comprises a C-terminal sortase donor sequence and the polypeptide comprises an N-terminal sortase acceptor sequence. The C-terminal sortase donor sequence can be LPXT(G) n , where n = 1 to 10. The C-terminal sortase donor sequence can be LPETGG (SEQ ID NO:6). The sortase linker can also comprise –(His) n –; where n = 1 to 10, preferably n = 4 to 9. The N-terminal sortase acceptor sequence can comprise 1 to 10 glycine residues. In some embodiments, the N-terminal sortase acceptor sequence is GGG. The cytotoxic agent can be a conjugated drug, such as a maytansine alkaloid, an auristatin, an amanitin, calicheamicin, psymberin, duocarmycin, an anthracycline, camptothecin, doxorubicin, paclitaxel, tubulysin, eribulin, or a pyrrolobenzodiazepine In some embodiments, the drug is an auristatin. The auristatin can be monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or PF-06380101. In some embodiments, the auristatin is monomethyl auristatin E (MMAE). The drug can be a camptothecin analogue (e.g., topotecan, irinotecan, belotecan, or derotecan). The drug can be an anthracycline analogue (e.g., PNU-159682; CAS No.: 202350-68-3). In some embodiments, the LGR receptor is LGR4, LGR5, or LGR6. Prior to step (c), the cytotoxic moiety can comprise an N-terminal sortase donor sequence, and the polypeptide can comprise a C-terminal sortase acceptor sequence. The C-terminal sortase donor sequence can be LAHTGG (SEQ ID NO: 106). The N-terminal sortase acceptor sequence can comprise from 1 to 10 glycine residues. The N-terminal sortase acceptor sequence can be, for example, GG or GGG. The cytotoxic agent can be a conjugated drug, e.g., a conjugated drug as described above or herein. The conjugated drug can be monomethyl auristatin E (MMAE), PNU-159682, topotecan, irinotecan, belotecan, or derotecan. The method can further comprise (d) covalently conjugating a second cytotoxic compound to the polypeptide by either (i) contacting the second cytotoxic agent and the polypeptide with a second transpeptidase, or (ii) covalently conjugating the second cytotoxic agent to the polypeptide by forming a disulfide bond through a partial disulfide reaction. The partial disulfide reaction can bond the second cytotoxic agent to the Fc region or the LGR binding domain. The second cytotoxic compound can comprise a linker, and wherein the partial disulfide reaction can bind the linker to the polypeptide. The linker can comprise a thiol-reactive maleimide group. The linker can further comprise a cleavable bond. The cleavable bond can comprise a valine (Val)-citrulline (Cit) bond. The second transpeptidase can preferably be sortase A or sortase E. In some preferred embodiments, wherein the first transpeptidase is sortase A and the second transpeptidase is sortase E. The second cytotoxic agent can be a cytotoxic agent as described above or herein (e.g., preferably MMAE).

[0014] Another aspect of the present disclosure relates to a method of producing a polypeptide, comprising: (a) expressing the nucleic acid as described above or herein in a cell under conditions that produce the encoded polypeptide; and (b) purifying the polypeptide from the cell.

[0015] Another aspect of the present disclosure relates to a method of treating a subject having a cell proliferative disease, which comprises administering to the subject an effective amount of a compound described above or herein and / or a pharmaceutical composition described above or herein. The cell proliferative disease can be an autoimmune disease. In some embodiments, the cell proliferative disease is cancer or a pre-cancerous condition. The cancer or pre-cancerous condition can be characterized by the presence of cancer stem cells. The cancer stem cells can present LGR on their surface. The LGR can be selected from LGR4, LGR5, and LGR6, preferably LGR5. The cancer can be ovarian cancer, myeloma, lymphoma, lung cancer, breast cancer, brain cancer, prostate cancer, spleen cancer, pancreatic cancer, cervical cancer, uterine cancer, head and neck cancer, esophageal cancer, liver cancer, skin cancer, kidney cancer, leukemia, bone cancer, testicular cancer, colon cancer, basal cell carcinoma, hepatocellular carcinoma, hepatobiliary cancer, colorectal cancer, or bladder cancer. In some embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, colon cancer, gastric cancer, cholangiocarcinoma, lung cancer, liver cancer, skin cancer, neuroblastoma, or leukemia. In some embodiments, the cancer is ovarian cancer or acute lymphoblastic leukemia. The cancer can be metastatic cancer. The method can further comprise administering to the subject at least a second anti-cancer treatment. The second anti-cancer treatment can be surgical treatment, chemotherapy, radiotherapy, gene therapy, or immunotherapy.

[0016] Another aspect of the present disclosure relates to a method for killing / treating cancer stem cells, the method comprising contacting the cancer stem cells with a compound described above or herein or a pharmaceutical composition described above or herein.

[0017] Another aspect of the present disclosure relates to a method for inhibiting the proliferation of cancer stem cells, the method comprising contacting the cancer stem cells with a compound described above or herein or a pharmaceutical composition described above or herein.

[0018] Another aspect of the present disclosure relates to a method for treating cancer, the method comprising contacting the cancer stem cells with a compound described above or herein or a pharmaceutical composition described above or herein.

[0019] Another aspect of the present disclosure relates to a method for reducing the spread of cancer cells and / or cancer stem cells, the method comprising contacting the cancer stem cells with a compound described above or herein or a pharmaceutical composition described above or herein.

[0020] As used herein, "pH selectively binds to FcRn" or "binds to FcRn in a pH-selective manner" means that a polypeptide such as an Fc domain (e.g., a mutant or variant IgG Fc domain) has the property of being able to bind to FcRn at an acidic pH (e.g., pH 5.8), and preferably, compared to a wild-type Fc domain (e.g., a wild-type FcIgG domain), the polypeptide or Fc domain has an enhanced ability to bind to FcRn at an acidic pH. In some embodiments, an Fc domain or polypeptide that pH selectively binds to FcRn also exhibits reduced FcRn binding compared to wild-type (e.g., wild-type IgG Fc domain) at physiological pH, or no detectable FcRn binding at physiological pH.

[0021] As used herein, with respect to a particular component, "substantially free of" is used herein to mean that no particular component is intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any accidental contamination of the composition is far less than 0.05%, preferably less than 0.01%. Most preferably, the composition is one in which no amount of the particular component is detected by standard analytical methods.

[0022] As used herein, "substantially the same binding affinity" means that two molecules exhibit statistically indistinguishable reversible binding to a ligand based on at least one binding assay, or where the equilibrium constants for the reversible binding of the two molecules to the ligand show a difference in K D values of less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. Using standard methods known in the art, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR), based on observing K on and K off binding characteristics, the K D values can be calculated.

[0023] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances and is expressed as K D . The affinity of a binding domain for its target can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM); alternatively, it can be from 100 nM to 1 nM or from 0.1 nM to 10 nM. Additionally, it is expected that when the affinity between two substances is within the above affinity ranges, the substances specifically bind.

[0024] As used herein, the term "LGR binding domain" refers to a polypeptide that selectively binds to an LGR receptor, preferably an LGR4, LGR5, or LGR6 receptor. The polypeptide may comprise natural and / or non-natural amino acids (e.g., D-amino acids). As used herein, "binding to LGR4 / LGR5 / LGR6" or "LGR4 / LGR5 / LGR6 binding" refers to the ability of a compound (e.g., a polypeptide) to bind or selectively bind to at least one of LGR4, LGR5, or LGR6. For example, a polypeptide or compound may bind to one, two, or all of LGR4, LGR5, or LGR6. When a polypeptide or compound binds to two or all of LGR4, LGR5, or LGR6, it is not required that the compound bind to the LGRs with equal affinity. In some aspects, polypeptides that selectively bind to LGR4, LGR5, and LGR6 are provided herein.

[0025] As used herein, the term "encoding" or variations thereof with respect to nucleic acids is used to make the invention readily understandable to those skilled in the art; however, these terms may be interchangeably used with "comprising / including" or variations thereof, respectively.

[0026] As used in the specification herein, a noun without a quantifier may mean one or more. As used in the claims herein, when used in conjunction with the word "comprising / including", a noun without a quantifier may mean one or more than one.

[0027] Unless explicitly indicated to refer only to alternatives or that the alternatives are mutually exclusive, the use of the term "or" in the claims is used to mean "and / or", but the present disclosure supports definitions that refer only to alternatives and "and / or". As used herein, "another" may mean at least second or more.

[0028] Throughout this application, the term "about" is used to indicate that a value includes the inherent variations of the device for measuring the variable, the error of the method for determining the value, or the variations that exist among the subjects being studied.

[0029] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate some preferred embodiments of the present invention, they are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following figures form a part of this specification and are included to further illustrate certain aspects of the present invention. The present invention can be better understood by referring to one or more of these figures in combination with the detailed description of the specific embodiments given herein.

[0031] This patent or patent application document contains at least one figure drawn in color. After requesting and paying the necessary fees, the official will provide a copy of the published patent or patent application with color figures.

[0032] Figure 1 : Schematic diagram of FcF2-His and its conversion to FcF2-MMAE using a sortase reaction.

[0033] Figures 2A - F: Analytical characterization of FcF2-His and FcF2-MMAE. Figures 2A - C, analysis of FcF2-His; Figures 2D - F, analysis of FcF2-MMAE. Figures 2A and 2D, reverse-phase HPLC analysis (C4 column); Figures 2B and 2E, HPLC-based size exclusion analysis (SEC300 column). Figures 2C and 2F, SDS-PAGE analysis under non-reducing and reducing conditions stained with Instant Blue.

[0034] Figures 3A - E: Cytotoxicity of FcF2-MMAE against cell lines. Potency and selective cytotoxicity of two representative batches of FcF2-MMAE in HEK293 / EV vs HEK293 / LGR5 (Figures 3A and 3B), and potency and selective cytotoxicity of two additional representative batches in the OVCAR8 / EV vs OVCAR8 / LGR5 model (Figures 3C and 3D). Figure 3E, potency of FcF2-MMAE against 8 human ovarian cancer cell lines expressing endogenous LGR levels. Each curve represents growth inhibition during 120 hours of exposure to increasing concentrations of FcF2-MMAE. Viability was determined using the CCK8 reagent.

[0035] Figure 4 : Relative contribution of the Fu1 and Fu2 domains to the selective cytotoxicity of FcF2-MMAE. Effect of the ability to disrupt the binding of Fu1 (FcF2-Q71R-MMAE), Fu2 (FcF2-F106R-F110R-MMAE), or both (FcF-Q71R-F106R-F110R-MMAE) on the selectivity of growth inhibition in the case of 120-hour exposure of OVCAR8 / EV cells and OVCAR8 / LGR5 cells. Values are the mean ± SEM of 3 independent experiments, with each experiment performed in triplicate cultures.

[0036] Figures 5A - B: Plasma pharmacokinetics of FcF2 - MMAE. Figure 5A, plasma concentration of FcF2 - MMAE determined by ELISA as a function of time after IV injection of 0.1 nmol / g (9 μg / g) in BALB / c mice. Figure 5B, concentration of FcF2 - MMAE in the plasma fraction of mouse whole blood determined by ELISA as a function of time during incubation.

[0037] Figures 6A - B: Efficacy and toxicity of FcF2 - MMAE against human ovarian cancer OVCAR8 / EV (low LGR5) and OVCAR8 / LGR5 (LGR5 - rich) xenografts. Figure 6A, tumor volume as a function of time. Figure 6B, mouse weight during and after treatment. Dose regimen: 0.5 nmol / g (42.65 mg / kg) qdx4 IP. N = 8 per group. Vertical arrows indicate dosing days. Vertical bars, ±SEM.

[0038] Figures 7A - B: Efficacy and toxicity of FcF2 - MMAE as a function of dose (total of 6 doses) administered IP every 7 days in the OVCAR8 / LGR5 xenograft model. Figure 7A, tumor growth as a function of dose. Figure 7B, mouse weight during and after treatment as a function of dose. Vertical arrows indicate dosing days. Vertical bars, ±SEM.

[0039] Figures 8A - D: Efficacy of a single dose of 1.0 nmol / g FcF2 - MMAE in CAOV3 and KF - 28 xenograft models. Mean tumor volume (Figure 8A) and animal weight (Figure 8B) of KF - 28 xenografts. B. Mean tumor volume (Figure 8C) and animal weight (Figure 8D) of CAOV3. Vertical arrows indicate dosing days. Vertical bars, ±SEM.

[0040] Figure 9 : Efficacy of FcF2 - MMAE at a dose of 0.5 nmol / g (total of 4 doses) against wild - type OVCAR8 and CAOV3 xenografts. A and C, tumor growth as a function of time. B and D, mouse weight during and after treatment. Vertical arrows indicate dosing days. Vertical bars, ±SEM.

[0041] Figures 10A - B: Documentation of different glycosylation forms of FcF2 - His. Figure 10A, FcF2 - His samples before and after treatment with PNGase were run under reducing conditions and stained with Instant Blue. Figure 10B, characterization of FcF2 - MMAE by Western blot analysis of non - reducing gels probed with anti - RSPO1 (left) and anti - MMAE (right).

[0042] Figures 11A - C: Flow cytometry recordings of differential LGR5 expression in isogenic pairs of HEK293 and OVCAR8 cells and depletion of LGR5-expressing cells mediated by FcF2-MMAE. Figure 11A, HEK293 / EV cells vs HEK293 / LGR5 cells; Figure 11B, OVCAR8 / EV cells vs OVCAR8 / LGR5 cells (EV cells: green, secondary antibody only; red, primary and secondary antibodies. LGR5 cells: purple, secondary antibody only; blue, primary and secondary antibodies). Figure 11C, flow cytometry analysis of a mixture of OVCAR8 / EV cells and OVCAR8 / LGR5 cells before and after exposure to 10 nM FcF2-MMAE for 120 hours (gray and black curves, unstained untreated and FcF2-MMAE-treated cells; green and blue curves, untreated and treated OVCAR8 / EV and OVCAR8 / LGR5 cells stained with anti-LGR5).

[0043] Figures 12A - B: Efficacy and toxicity of FcF2-MMAE in a dosing regimen of 1.5 nmol / g (128 mg / kg) q7dx4 IP against human ovarian cancer OVCAR8 / LGR5 (low LGR5) and OVCAR8 / LGR5 (LGR5-rich) xenografts. Figure 12A, tumor volume as a function of time. Figure 12B, mouse weights during and after treatment. N = 8 per group. Vertical arrows indicate dosing days. Vertical bars, ±SEM.

[0044] Figure 13 : Amino acid sequence of FcF2 containing an 8×His tag. The amino acid sequence contains an IgGκ leader sequence followed by a DHS variant of IgG1 Fc linked to the Fu1-Fu2 domain (Lee et al., 2019). The amino acids highlighted in blue in the Fc portion of the construct are used to denote the DHS mutations in the Fc portion. The purple arrow indicates the protease cleavage site at the end of the leader sequence. The blue arrow indicates the cysteine in the Fc region that can form a disulfide bond with the corresponding cysteine in another separate Fc region, thus forming a dimerized molecule.

[0045] Figure 14 : For generating Figure 13 the nucleotide sequence of the FcF2 construct in which there is no DHS mutation in the Fc portion of the construct. The nucleotide sequence shown includes codon optimization. The codons highlighted in purple in the Fc portion of the construct represent the regions that will subsequently be altered using GeneArt to contain the DHS mutant form of the Fc region. The color coding of the remaining different regions corresponds to the regions described in Figure 13

[0046] Figure 15 : The sequence with DHS mutations before codon optimization. The blue-enhanced codons in the Fc region now correspond to the DHS mutations in DHS Fc. The color coding for the remaining different regions corresponds to Figure 13 the regions described in

[0047] Figure 16 : The FcF2-8×His nucleotide (nt) sequence after codon optimization for expression in human cells. This nucleotide sequence corresponds to Figure 13 the amino acid sequence shown in

[0048] Figure 17 : The construct of FcST4. The amino acid sequence of FcST4 is shown (top). The vector construct of the FcST4 molecule is designed to contain an IgG leader sequence at the N-terminus, followed by a mutant Fc domain, which is linked to two modified receptor-binding domains (ST) in tandem, with a linker sequence between the two ST domains. A second spacer sequence is inserted exactly upstream of the LPETGG sortase recognition (donor) motif, and an 8×His tag is located at the C-terminus.

[0049] Figure 18 A to B: Analysis of the potency of FcST2-MMAE against 8 human ovarian cancer cell lines. Figure 18 A, The survival of 8 human ovarian cancer cell lines as a function of the FcST2-MMAE concentration. Figure 18 B, The IC 50 values of the 8 cell lines tested.

[0050] Figure 19 : FcF2 without a linker. The FcF2 construct without a linker is shown (bottom). In contrast to the FcF2 with a linker (top), the FcF2 without a linker does not contain a G4S linker in the polypeptide.

[0051] Figure 20 : The cytotoxicity of FcF2-MMAE and FcF2Δlinker-MMAE.

[0052] Figure 21 : In vivo FcF2-MMAE activity in a xenograft mouse model of human colorectal cancer. Mice bearing human colorectal xenografts (LoVo) were administered a dose that did not cause any observed clinical toxicity. FcF2-MMAE led to a reduction in the average weight of the tumors without changing the body weight of the mice.

[0053] Figure 22: Efficacy of FcF2-MMAE in a colon cancer LoVo xenograft model. Dosage regimen: IP injection of FcF2-MMAE, q7dx4. Each data point is the average of the sizes of all tumors in the group. Vertical bars are SEM.

[0054] Figure 23 : Efficacy of FcF2-MMAE in a gastric cancer AGS xenograft model. Dosage regimen: IP injection of FcF2-MMAE, q7dx4. Each data point is the average of the sizes of all tumors in the group. Vertical bars are SEM.

[0055] Figure 24 : Efficacy of FcF2-MMAE in a neuroblastoma SKNAS xenograft model. Dosage regimen; IP injection of FcF2-MMAE, q7dx4. Each data point is the average of the sizes of all tumors in the group. Vertical bars are SEM.

[0056] Figures 25A to C: Analysis of the structural integrity and selective cytotoxicity of FcF2-PNU159682. Figure 25A, SDS-PAGE analysis of two different batches of FcF2-PNA159682. Figure 25B, Growth inhibition of OVCAR8 / EV relative to OVCAR8 / LGR5 cells as a function of the concentration (pM) of FcF2-PNU159685. Data are the mean ± SEM of triplicate cultures. Figure 25C, Schematic diagram of the structure of PNU159683.

[0057] Figures 26A to B: Construction of the GGGC-GGFG-drutac linker. Figure 26A, Schematic diagram of the conjugation of GGGC with maleimide-GGFG-drutac. Figure 26B, Reverse-phase HPLC analysis of the GGGC-MA-GGFG-drutac linker, C4 column.

[0058] Figures 27A to B: Generation of FcF2-drutac and recording of its cytotoxicity and cytotoxic selectivity. Figure 27A, Reverse-phase HPLC (C4 column) analysis of FcF2-drutac recording purity. Figure 27B, Growth inhibition of OVCAR8 / EV cells relative to OVCAR8 / LGR5 cells as a function of the concentration of FcF2-drutac. Data are the mean ± SEM of triplicate cultures.

[0059] Figure 28: Record of the retention of potency and cytotoxic selectivity of FcF2-MMAE containing mutations R28A and R30A. Growth inhibition of OVCAR8 / EV cells relative to OVCAR8 / LGR5 cells as a function of the concentration of FcF2-MMAE (WT), FcF2-R28A-MMAE, and FcF2-R30A-MMAE. Data are mean ± SEM of triplicate cultures.

[0060] Figure 29 : Pharmacokinetics of FcF2-MMAE (WT), FcF2-R28A-MMAE, and FcF2-R30A-MMAE. A, Plasma concentration of each form as a function of time after IV injection in BALB / c mice. B, Table of area under the curve (0 to 120 hours) for each form.

[0061] Figure 30 : Schematic diagram of loading MMAE onto both ends of the FcF2 molecule using both sortase A and sortase E.

[0062] Figure 31 : Record of loading GGGC-MA-Dye650 onto FcF2-LAHTGG-His by sortase E. The figure shows SDS-PAGE analysis of the sortase E reaction product after elution from SP-agarose resin detected using Dye650 fluorescence (lanes 7 and 8).

[0063] Figure 32 : Western blot analysis of MMAE incorporation achieved by partial reduction of disulfide bonds using TCEP. Upper panel, blot probed with anti-RSPO1 antibody; lower panel, blot probed with anti-MMAE.

[0064] Figure 33 : Nucleotide and amino acid sequences of the FcF2(R28A) construct. The Kozak sequence is shown in light blue. The IgGk leader sequence is highlighted in green. The Fc sequence is highlighted in gray, with the DHS mutation highlighted in yellow underlined. The G4SG4S linker is highlighted in red. The LPETG sequence is highlighted in purple. The 8×His tag is highlighted in light blue. The stop×2 sequence is highlighted in dark blue. The Fu1-Fu2 (FuFu) sequence is highlighted in yellow. The R28A substitution mutation in FuFu is further shown in underlined white font and highlighted in black.

[0065] Figure 34: In vivo plasma distribution half-lives of R1FF-MMAE (“RSPO1-MMAE”) and FcF2-MMAE in female BALB / C mice. The FcF2-MMAE construct showed a half-life approximately 15-fold longer than that of R1FF-MMAE. The distribution half-life (T 1 / 2 ) and elimination half-life (T 1 / 2 ) values are shown. DETAILED DESCRIPTION

[0066] In some aspects, the present disclosure overcomes limitations in the prior art by providing constructs that selectively target and kill cancer cells, including cancer stem cells. The construct can include a polypeptide having a region that selectively binds to the LGR4, LGR5, and / or LGR6 receptor expressed by cancer cells or CSCs. The construct can also include a cytotoxic moiety (e.g., MMAE) linked to the polypeptide by a cleavable linker such that the construct can be endocytosed into cancer cells and subsequently released into the cell. Thus, delivery of the cytotoxic moiety to cancer cells such as cancer stem cells can result in selective killing of cancer cells. The present disclosure is based in part on the discovery that inclusion of an Fc region (e.g., a mutant Fc region, such as the DHS Fc mutant provided herein) in the polypeptide can result in improved pharmacological properties (e.g., improved in vivo half-life of the construct) and / or improved therapeutic efficacy. Inclusion of the Fc region can also produce a dimeric construct that effectively doubles both the number of LGR binding domains and the number of cytotoxic moieties in the construct. In some aspects, the present disclosure is based on the observation that inclusion of two or more LGR binding domains within the polypeptide can result in improved therapeutic properties of the construct for treating cancer (e.g., cancer including cancer stem cells). Methods for treating cancer and selectively targeting cancer stem cells are also provided herein.

[0067] In various aspects, therapeutic constructs are provided. The therapeutic construct can comprise (i) one or more cell targeting moieties, such as an LGR5 / LGR6 binding domain (e.g., the Fu1-Fu2 sequence, SEQ ID NO:4; Fu1-Fu2(N137A), SEQ ID NO:17) and (ii) a cytotoxic moiety (e.g., MMAE or deruxtecan). The therapeutic construct can, for example, comprise 1, 2, 3, or 4 copies of the cell targeting moiety (e.g., the Fu1-Fu2 sequence, SEQ ID NO:4; Fu1-Fu2(N137A), SEQ ID NO:17), which can be separated by a linker (e.g., a G4S linker). In some embodiments, the therapeutic construct comprises an Fc domain, such as a wild-type or mutant Fc domain (e.g., the DHS mutant Fc; SEQ ID NO:13). The Fc domain can cause dimerization of two copies of the therapeutic construct, which effectively doubles the number of cell targeting moieties and cytotoxic moieties in the dimeric construct. As shown in the following examples, inclusion of the DHS Fc domain results in an increase in the in vivo half-life of the construct. In some embodiments, the therapeutic construct can comprise a radioisotope, an imaging agent, or radiotherapy. The therapeutic construct can be comprised in a pharmaceutical composition (e.g., a formulation for injection (e.g., intravenous or intratumoral injection), inhalation), or comprised in a liposome or nanoparticle. Methods of using the therapeutic construct are also provided herein. In some embodiments, the therapeutic construct can be used to treat cancers that express LGR4, LGR5, or LGR6.

[0068] In some aspects, the therapeutic compounds provided herein can comprise the Fu1-Fu2 receptor-binding domain of R-spondin to target cancer cells that express LGR4, LGR5, and / or LGR6, such as ovarian cancer cells. As shown in the following examples, the Fu1-Fu2 receptor-binding domain of RSPO1 can be conjugated or covalently bonded to monomethyl auristatin E (MMAE) against ovarian cancer cells that are rich in the stem cell receptor LGR5. A modified IgG1 Fc domain with an extended half-life modification (Lee et al., 2019) is linked to the N-terminus of the Fu1-Fu2 domain, which carries a sortase recognition sequence at its C-terminus. During the synthesis of this protein in HEK293E cells, these two chains are linked by an intermolecular disulfide bond between the Fc domains, resulting in a dimeric molecule "FcF2-His", which contains two LPETGG (SEQ ID NO:6) sequences at the C-terminus, to which MMAE can be covalently linked or conjugated using a sortase reaction to produce "FcF2-MMAE" (e.g., Figure 1)。This molecule exhibits low nM cytotoxicity, as well as good stability and pharmacokinetic profiles, in a panel of human ovarian cancer cell lines. In addition, FcF2-MMAE causes selective killing of LGR5-rich tumor cells in vitro and differentially inhibits the growth of syngeneic LGR5-deficient and LGR5-rich tumors in vivo. It exhibits activity in two different human ovarian cancer xenograft models, at clinically relevant dosing regimens, and at doses that produce only transient adverse side effects. These results in the ovarian cancer models suggest that these compositions and methods can be used to selectively target and kill cancer stem cells expressing LGR5 / LGR6. The FcF2-MMAE and other therapeutic compounds provided herein can be used to reduce the growth of and / or treat a variety of different types of cancer. I. LGR-Binding Polypeptides

[0069] In some aspects, the therapeutic compounds provided herein can comprise an LGR-binding domain polypeptide that selectively binds LGR4, LGR5, and / or LGR6. LGR5 has been observed to be expressed in cancers, including basal cell carcinoma, hepatocellular carcinoma, colorectal tumors, and ovarian tumors (McClanahan et al., 2006). LGR6 is expressed in cancers, including adenocarcinoma (Cortesi et al., 2019). By selectively binding to LGR4, LGR5, and / or LGR6, the therapeutic constructs provided herein can target select cell types, such as cancer cells or stem cells. In some embodiments, the LGR-binding domain can selectively bind LGR5 and LGR6. The LGR-binding domain can be a polypeptide, which can comprise natural and / or non-natural amino acids.

[0070] In some embodiments, the LGR-binding domain comprises or consists of a polypeptide sequence. The polypeptide may comprise a Furin L (FurinL) sequence. The Furin L sequence is each of two cysteine-rich Furin-like domains in the R-spondin polypeptide (De Lau et al., 2012). In some embodiments, the polypeptide comprises a first Furin L sequence (also referred to as "Fu1" or "Furin L repeat 1") and a second Furin L sequence (also referred to as "Fu2" or "Furin L repeat 2"). The LGR-binding domain may comprise Furin L from R-spondin, such as human R-spondin-1 (hR-spondin-1), human R-spondin-2 (hR-spondin-2), human R-spondin-3 (hR-spondin-3), or human R-spondin-4 (hR-spondin-4), as described, for example, in De Lau et al. (2012) and Jin-Gen et al. (2015). The first Furin L sequence (Fu1) and the second Furin L sequence (Fu2) may comprise or consist of Furin L sequences from: human R-spondin-1 (hR-spondin-1), human R-spondin-2 (hR-spondin-2), human R-spondin-3 (hR-spondin-3), or human R-spondin-4 (hR-spondin-4). In some embodiments, the LGR-binding domain comprises or consists of the Furin region of R-spondin (e.g., the Fu1-Fu2 region), such as human R-spondin-1 (hR-spondin 1), human R-spondin-2 (hR-spondin-2), human R-spondin-3, or human R-spondin-4 (hR-spondin-4). In some embodiments, the LGR-binding domain is the Fu1-Fu2 sequence (SEQ ID NO:4). The LGR-binding domain may be included multiple times in the therapeutic compounds or polypeptides provided herein (e.g., repeated 1, 2, 3, or 4 times in the therapeutic polypeptides provided herein). In some embodiments, the LGR-binding domain comprises one or more substitution mutations compared to the LGR-binding domain in human R-spondin (e.g., Fu1-Fu2(N137A) (SEQ ID NO:17)). The LGR-binding domain may comprise a polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of the R-spondin or Furin L sequences described above or provided herein.

[0071] As used herein, "Fu1-Fu2" and "FuFu" are used interchangeably herein to refer to a polypeptide comprising both a Fu1 furin L sequence and a Fu2 furin L sequence from an R-spondin protein. In some embodiments, the Fu1 region and the Fu2 region may be derived from the same R-spondin polypeptide (e.g., from human RSPO1). However, in some embodiments, the polypeptide may comprise a Fu1 region from a first R-spondin and a Fu2 region from a second R*spondin. For example, furin L sequences from different human R*spondin proteins, such as a Fu1 region from RSPO1 and a Fu2 region from RSPO2, may be included in a single polypeptide to selectively bind to LGR receptors, such as LGR4, LGR5, and / or LGR6.

[0072] In some embodiments, the LGR binding domain comprises one or more copies of the following "Fu1*Fu2" sequence (SEQ ID NO: 4): SRGIKGKRQRRISAEGSQACAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAAAGTMECSSP; or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% sequence identity.

[0073] Other LGR binding domains may comprise a Fu1 and / or Fu2 domain from human RSPO2, RSPO3, or RSPO4; or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9% sequence identity to one of these LGR5 / LGR6 binding domains (e.g., to SEQ ID NO: 4 or to one of SEQ ID NOs: 78 to 83). Fu1 and Fu2 domains that can be used include: RSPO1 RSPO1*Fu1: RSPO1*Fu2: RSPO2 RSPO2*Fu1: RSPO2*Fu2: RSPO2-Fu1-Fu2: RSPO3 RSPO3-Fu1: RSPO3-Fu2: RSPO3-Fu1-Fu2: RSPO4 RSPO4-Fu1: RSPO4-Fu2: RSPO4-Fu1-Fu2:

[0074] In some embodiments, the Fu1 or Fu2 region may be included in the construct instead of a polypeptide comprising both the Fu1 region and the Fu2 region to selectively bind to the LGR4, LGR5, and / or LGR6 receptors. A single Fu1 or Fu2 region can be used to generate a therapeutic compound that is cytotoxic and selective for LGR4, LGR5, and / or LGR6 (e.g., Fc-Fu1-MMAE or Fc-Fu2-MMAE). A single Fu1 or Fu2 region can be included in a polypeptide as described herein in combination with another cell targeting polypeptide (such as an scFv region). For example, the Fu1 region and an scFv (e.g., selectively targeting a liver antigen) can be included in a single polypeptide covalently linked to a drug (e.g., a cytotoxic moiety such as MMAE), and can selectively direct the drug to the target tissue of the scFv (e.g., the liver), while reducing or avoiding toxicity to the intestine mediated by the Fu2 domain.

[0075] As described herein, the LGR binding domain can comprise both the Fu1 region and the Fu2 region. In some embodiments, inclusion of both the Fu1 region and the Fu2 region can result in a construct with increased cytotoxicity and / or selectivity compared to inclusion of only a single Fu1 or Fu2 region.

[0076] The above Fu1-Fu2 sequence may contain substitution mutations to remove the N-glycosylation site at position (N137). For example, in some embodiments, the N-glycosylation site in Fu1-Fu2 is removed by an asparagine-to-alanine substitution mutation at position 137 (i.e., N137A). The FuFu(N137A) sequence contains the sequence (SEQ ID NO: 17): Or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5 or 99.9 sequence identity to FuFu(N137A) (SEQ ID NO: 17).

[0077] The Fu1-Fu2 sequence may contain 1, 2, 3, 4, 5 or more substitution mutations, which may provide additional beneficial effects, such as altering the pharmacokinetics of the construct or increasing the half-life after administration to a mammalian subject. For example, the Fu1-Fu2 sequence may contain the (R28A) substitution mutation: The Fu1-Fu2 sequence may contain the (R30A) substitution mutation: As shown in Example 7 and Figure 28 as shown, neither the R28A nor the R30A mutation altered the selective potency against LGR5-rich cells; however, the R28A (but not the R30A) mutation prolonged the initial half-life of the molecule in vivo and increased the AUC 0-120 by 3.4-fold. In some preferred embodiments, the Fu1-Fu2 sequence may contain the R28A mutation, which may result in an increase in the half-life (e.g., plasma half-life) after administration to a mammalian subject.

[0078] Deletion mutants of the FuFu sequence may also be used. The following deletion mutants may increase the half-life or plasma half-life of the construct. The FuFu deletion mutants that can be used include: Fu1-Fu2 (R22-R31 deletion) Fu1-Fu2 (K25 to R31 deletion) Fu1-Fu2 (R28 to R31 deletion) Fu1-Fu2 (R22 to K27 deletion) and Fu1*Fu2 (S21 to Q38 deletion)

[0079] In some embodiments, the FuFu sequence is linked to a linker (e.g., (G4S)n, where n = 1 to 3), a cleavable linker (e.g., containing valine-citrulline, such as valine-citrulline-PAB), and a cytotoxic moiety (e.g., MMAE). For example, the polypeptide may comprise an Fc domain (e.g., a mutant Fc domain, such as SEQ ID NOs: 13 to 16) and a FuFu region. The FuFu region may be linked to a linker and MMAE (where MMAE has been covalently linked using sortase), such as:

[0080] In some embodiments, the cell targeting moiety comprises a polypeptide containing at least 1, 2, 3, or 4 copies of the Fu1-Fu2 sequence (e.g., two or more copies of SEQ ID NO: 4 or SEQ ID NO: 17). For example, the cell targeting moiety may comprise two copies of the Fu1-Fu2 sequence (SEQ ID NO: 4). The Fu1-Fu2 sequences may be separated by a linker such as G4S (GGGGS, SEQ ID NO: 18), (G4S)2 (SEQ ID NO: 5), (Gly)6, or (EAAAK)3 (SEQ ID NO: 84).

[0081] If the construct comprises an Fc region as described herein (e.g., a mutant Fc region, such as DHS Fc, SEQ ID NO: 13), the construct may dimerize based on the binding of the Fc region in two different molecules. In this way, the total number of, for example, LGR5 / LGR6 binding domains and cytotoxic agents can be effectively doubled in a single dimeric construct. The total number of LGR5 / LGR6 binding domains (e.g., Fu1-Fu2, FuFu(N137A)) in the dimeric construct may be 2, 4, 6, or 8.

[0082] The LGR family of G protein-coupled, 7-transmembrane spanning receptors contains 8 members, all of which have large extracellular domains composed of up to 18 copies of leucine-rich repeat motifs. These 8 receptors fall into 3 groups. The first group consists of LGR1 (which is the FSH receptor), LGR2 (the LH receptor), and LGR3 (the TSH receptor). The second group consists of LGR4, LGR5, and LGR6 (which are receptors for R-spondin (RSPO)), and the third group contains LGR7 and LGR8 (which are receptors for relaxin and insulin-like 3 protein, respectively). LGR5 and LGR6 are well-defined markers of stem cells in the intestine (LGR5) and skin and fallopian tube epithelium (LGR6), as well as in many other tissues and tumor types. LGR5 has been shown to be positively regulated by the Wnt signaling pathway, which controls the proliferation of stem cells that form the epithelium of the colon, small intestine, and stomach. During embryonic development, LGR5 is expressed in a variety of tissues, but in adults, its expression is highly restricted to rare cells in the intestine, mammary gland, ovary, testis, hair follicle, brain, and eye. Using genetic labeling techniques, cells expressing LGR5 were found to function as stem cells and were able to give rise to all other cell types present in the epithelium of the colon and stomach. In contrast to LGR5, LGR6 is not regulated by Wnt signaling. In LGR6-LacZ LacZ knock-in mice, expression was found to be restricted to rare cells in the brain, mammary gland, lung, and hair follicle. Lineage mapping showed that LGR6-positive cells present in the hair follicle bulb were located in a different position from LGR5-positive cells, and that they gave rise to the epidermis and sebaceous glands. Subsequent studies have shown that cells expressing LGR6 are stem cells that produce the new skin required during the wound healing process. There is also evidence that LGR6 is uniquely expressed by tumor stem cells. LGR6 was found to label a subset of cells isolated from human lung adenocarcinoma that were able to form new tumors when injected into mice.

[0083] LGR5 and LGR6 are expressed in many types of tumors, including breast, colon, and endometrial cancers. There is several lines of evidence indicating that LGR6, but not LGR5, uniquely identifies stem cells in the fallopian tube epithelium (FTE) and ovarian cancer. Thus, LGR6 has been shown to be a stem cell in tumors arising from the FTE. Accordingly, some embodiments of the present disclosure relate to the use of LGR6 as a tumor stem cell target, since it is expressed on the cell surface, where it may be accessed by antibodies and other types of tumor-targeting toxins.

[0084] R-spondin (RSPO) is a ligand for LGR5 and LGR6. RSPO is a group of 4 cysteine-rich secreted paralogs (R-spondin 1 to 4). They share 40% to 60% sequence homology and overall similarity in domain structure. All 4 RSPO family members contain an N-terminal secretory signal peptide, 2 tandem furin-like cysteine-rich (Fu-CRD) domains, a thrombospondin type 1 repeat (TSP) domain, and a C-terminal basic amino acid-rich (BR) domain. RSPO1, RSPO2, RSPO3, and RSPO4 can each act as a ligand for both the LGR5 receptor and the LGR6 receptor, and they can bind to the LGR5 receptor and the LGR6 receptor with high affinity. Accordingly, certain embodiments of the present disclosure relate to the use of polypeptides derived from RSPO, which can be used to target therapeutic agents to cells expressing LGR4, LGR5, or LGR6, such as tumor stem cells expressing LGR6. In one specific aspect, the Fu1-Fu2 domain of RSPO1 and / or RSPO2 is conjugated to a cytotoxic agent, such as the toxin monomethyl auristatin E (MMAE), to selectively target tumors expressing high levels of LGR6.

[0085] RSPO may play a role in the development of cancer and the maintenance of stem cells. A small population of stem cells that maintain the epithelium throughout the body proliferate and then differentiate in response to growth factors in their niche, and as they differentiate, their progeny undergo a series of transcriptional states and lose their proliferative potential (Clarke, 2019). Signaling in the WNT pathway controls fate decisions during embryogenesis and in many adult tissues (Raslan and Yoon, 2019). WNT signaling is regulated by a combination of WNT ligands that bind to various types of Frizzled receptors and R-spondin (RSPO) that binds to leucine-rich repeat-containing G protein-coupled receptors (LGR). Cells in the immediate environment of the stem cell niche are the main source of ligands that drive WNT signaling, and the most effective signaling molecules are shown to be transmitted to stem cells over very short distances. In malignant tissues, RSPO1 has a dominant but not exclusive position among the 4 members of the RSPO family, and among the members of the LGR family of receptors, most evidence points to a key role for LGR5 and LGR6 (Yan et al., 2017). II. Cytotoxic Agents

[0086] A variety of cytotoxic moieties can be included in the constructs of the present disclosure. In some embodiments, the cytotoxic moiety is a conjugated drug or polypeptide.

[0087] A variety of conjugated drugs can be used as cytotoxic moieties. Conjugated drugs that can be used include compound classes such as maytansinoids, auristatins, amanitins, calicheamycins, psymberin, duocarmycins, anthracyclins, camptothecin, doxorubicin, taxol, and pyrrolobenzodiazepines Some specific examples of cytotoxic agents include paclitaxel, docetaxel, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, mithramycin, actinomycin, glucocorticoids, puromycin, epirubicin, cyclophosphamide, methotrexate, cytarabine, 5-fluorouracil, platinum, streptozocin, minomycin C, anthracyclins, dactinomycin or actinomycin, bleomycin, mithramycin, anthramycin, duocarmycin, ifosfamide, mitoxantrone, daunomycin, carminomycin, animoterin, melphalan, esperamicin, lexitropsin, auristatins (such as auristatin E, auristatin F, AEB, AEVB, AEFP, MMAE, MMAF), eleuthorobin, netropsin, podophyllotoxin, maytansinoids including maytansine and DM1, deruxtecan, and combretastatin.

[0088] In some embodiments, the conjugated drug is monomethyl auristatin E (MMAE, also known as vedotin). MMAE is a potent antimitotic agent that can inhibit cell division by blocking tubulin polymerization. Auristatin is a synthetic analogue of the antitumor natural product dolastatin, and auristatin has previously been used as a payload for antibody-drug conjugates. The potency of MMAE is 100 to 1000 times stronger than that of doxorubicin (Adriamycin / Rubex).

[0089] In some embodiments, the cytotoxic moiety is a serine protease, such as granzyme B (GrB). GrB can include a variety of mutations, such as those described in U.S. Patent No. 9,096,840 or U.S. Patent Application Nos. 2014 / 0140976 and 2015 / 0010556. For example, in some aspects, the recombinant serine protease is a GrB polypeptide and comprises the sequence Other serine proteases that can be used include cathepsin G (NCBI accession number P08311), chymase (NCBI accession number P23946), myeloblastin (NCBI accession number P24158), kallikrein-14 (NCBI accession number Q9P0G3), complement factor D (NCBI accession number K7ERG9), PRSS3 protein (NCBI accession number A1A508), trypsin-1 (NCBI accession number P07477), serine protease 57 (NCBI accession number Q6UWY2), and PRSSL1 protein (NCPI accession number B7ZMF6), or polypeptides having at least 90% or at least 95% sequence identity.

[0090] In some embodiments, the cytotoxic moiety is a cytotoxic protein. Cytotoxic proteins that can be used include apoptosis factors or apoptosis-related proteins, including AIF, Apaf (e.g., Apaf-1, Apaf-2, Apaf-3), oder APO-2(L), APO-3(L), caspases, Bad, Bak, Bax, Bcl-2, Bcl-xL, Bcl-xs, bik, CAD, calpain, caspases (e.g., caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11), ced-3, ced-9, c-Jun, c-Myc, crm A, cytochrome C, CdR1, DcR1, DD, DED, DISC, DNA-PKCS, DR3, DR4, DR5, FADD / MORT-1, FAK, Fas (Fas-ligand CD95 / fas (receptor)), FLICE / MACH, FLIP, fodrin, fos, G-actin, Gas-2, gelsolin, granzyme A / B, ICAD, ICE, JNK, lamin A / B, MAP, MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD, NF-kB, NuMa, p53, PAK-2, PARP, perforin, PITSLRE, PKCδ, pRb, presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, thymidine kinase from herpes simplex, TRADD, TRAF2, TRAIL-R1, TRAIL-R2, TRAIL-R3, and transglutaminase.

[0091] In other embodiments, the cytotoxic agent can be selected from bispecific antibodies and bioactive compounds (including nucleic acids such as DNA, mRNA, siRNA, and fragments thereof); pharmaceutical compounds (e.g., various therapeutic drugs); and radionuclides and cytotoxins that can be targeted to a desired tissue or cell by a targeting moiety. These agents can act while remaining conjugated to the target protein or a portion thereof, or if the linking group is a group that is cleavable in vivo, they can first dissociate from the target protein.

[0092] Suitable cytotoxic agents for the present disclosure include microtubule inhibitors, topoisomerase I inhibitors (e.g., deruxtecan), intercalating agents, inhibitors of intracellular signaling pathways, kinase inhibitors, transcriptional inhibitors (e.g., siRNA, aRNA, and miRNA), and DNA minor groove binders. Selected cytotoxic agents that can be used in some selected embodiments of the present disclosure include calicheamicin, MMAE, DM1, deruxtecan, SN-38, MMAF, PE38, diphtheria toxin, and yttrium-90. Selected cytotoxic agents that can be used in multiple embodiments are also discussed in Kostova et al. (2021), Chen et al. (2017), and Lambert et al. (2017). A. Maytansine alkaloids

[0093] Maytansine alkaloids (also known as maytansinoid analogs) are semi-synthetic agents derived from the natural product maytansine. Maytansine alkaloids include emtansine, which can disrupt microtubule function. Maytansine can be obtained by fermentation, and the molecule can be synthetically modified to produce maytansine alkaloids DM1, DM3, and DM4, as shown below. Different side chains on the maytansine alkaloids can result in different release and stability characteristics. Maytansine alkaloids include the following compounds: Maytansine alkaloids that can be used in multiple embodiments are also discussed, for example, in Chen et al. (2017). B. Auristatin

[0094] In multiple embodiments of the present disclosure, various auristatins can be used as cytotoxic moieties. Auristatins are generally synthetic compounds that share some structural similarities with the natural compound dolastatin 10. Selected auristatins that can be used in some embodiments of the present disclosure include the following: Selected auristatins that can be used in multiple embodiments are reviewed, for example, in Kostova et al. (2021). In some selected embodiments, the cytotoxic payload is MMAE, MMAF, or PF-06380101. C. Tubulysin

[0095] The cytotoxic moiety can be a tubulysin payload. Tubulysin-based payloads can be derived from naturally occurring tubulysins (e.g., tubulysin A, B, C) and generally act as microtubule destabilizing agents. In some embodiments, the cytotoxic moiety is tubulysin A, tubulysin B, tubulysin C, tubulysin G, or tubulysin I. D. Eribulin

[0096] The cytotoxic moiety can be eribulin. Eribulin is a microtubule-disrupting agent and has the following structure: E. Taxol derivatives

[0097] The cytotoxic moiety can be a taxol derivative. Taxol derivatives inhibit cell growth by stabilizing microtubule filaments. Taxol derivatives include docetaxel and paclitaxel. F. DNA-damaging agents

[0098] In some embodiments, the cytotoxic moiety is calicheamicin. Calicheamicin is an antitumor antibiotic that can cause double-strand DNA breaks. Calicheamicin has the following structure: Additional DNA-damaging agents that can be used include anthramycin-based dimers and esperamicins, anthracyclines, and camptothecins. III. Linkers

[0099] In various embodiments of the present disclosure, a variety of linkers can be used to link the cytotoxic moiety to a cell-targeting moiety (e.g., an R-spondin targeting moiety). Some non-limiting examples of linkers that can be used are provided in Table 1 below. Table 1. Exemplary linkers The selected linkers that can be used in various embodiments of the present disclosure are also discussed in Kostova et al. (2021), Chen et al. (2017), and Lambert et al. (2017). In some embodiments, the linker is MHH, DSDM, sulfo-SPDB, MC-VC-PABC, SMCC, Mal-PEG-NHS, GGFG, or GBC.

[0100] In some embodiments, a peptide linker is included in the constructs of the present disclosure, near or adjacent to the sortase recognition sequence as described herein. The linker (e.g., a G4S linker) can promote or facilitate the conformational freedom of the sortase domain to recognize the sortase recognition motif. In some embodiments, the linker is a (GGS), (GGGS), or (G4S) linker. The linker can be repeated, for example, 1, 2, 3, 4, 5, 6, 7, or 8 times (e.g., (G X S)n, where x = 1 to 4, and n = 1 to 9), more preferably repeated 1 or 2 times (e.g., (GX S)n, where x = 1 to 4, and n = 1 to 3). IV. Mutant Fc Region

[0101] In some embodiments, the constructs of the present disclosure may comprise a wild-type or mutant Fc region. For example, the construct may comprise a cell targeting moiety (e.g., an R-spondin targeting moiety), a cytotoxic moiety (e.g., MMAE or MMAF), and a mutant Fc region (e.g., DHS Fc). The Fc region can cause dimerization of the construct. In some selected embodiments, it has been observed that including the DHS Fc region in the construct can improve the stability, potency, and / or half-life of the construct.

[0102] As used herein, the "DHS Fc" region refers to a polypeptide comprising a variant human IgG Fc domain that can bind human FcRn at acidic pH, wherein the Fc domain has the following substitutions: (i) aspartic acid at position 309 (L / V309D); (ii) histidine at position 311 (Q311H); and (iii) substitution of serine or tyrosine at position 434 (N434Y or N434S); wherein the amino acid position numbering is according to the Kabat system. The DHS Fc region may also optionally comprise the substitution mutation V264E (e.g., mutations L / V309D, Q311H, N434S / Y; and optionally V264E), wherein the DHS Fc region can bind human FcRn at acidic pH. Without wishing to be bound by any theory, including the DHS Fc region in the constructs of the present disclosure may increase the half-life due to binding to human FcRn. The DHS mutants are disclosed in Lee et al. (2019) and U.S. Patent 11059892, which are incorporated herein by reference in their entirety and may be included in the constructs of the present disclosure.

[0103] In some embodiments, mutant or variant human Fc domains are provided that exhibit, compared to the corresponding wild-type Fc domain, (i) enhanced binding to FcRn at pH 5.8, and (ii) reduced binding or no detectable binding at pH 7.4. The mutant or variant Fc domain can be a mutant or variant IgG domain. The mutant or variant Fc domain can be included in a polypeptide (such as an antibody). In some embodiments, the mutant or variant Fc domain can be included in a therapeutic antibody (such as an agonist or antagonist antibody). In some embodiments, there are compositions that include a polypeptide having a mutant or variant Fc domain (an "antibody Fc domain") derived from a human IgG1-4 antibody. The mutant Fc domain can be a variant of the wild-type human IgG1 Fc domain (SEQ ID NO:9), wherein the mutant or variant Fc domain is capable of binding to FcRn with increased affinity at acidic pH, but not at neutral pH. In some embodiments, the engineered Fc domain can exhibit an increased affinity for FcRn, for example, about 5-fold higher than that of the glycosylated wild-type Fc domain. In other embodiments, mutant human Fc domains of all other wild-type IgG subclasses (human IgG2, IgG3, and IgG4) are provided that are capable of binding to FcRn with increased affinity at acidic pH and not at neutral pH. The mutant or variant Fc domain can include mutations (L / V309D, Q311H, N434S / Y) and optionally (V264E) relative to wild-type human IgG1 Fc (SEQ ID NO:1), human IgG2 Fc (SEQ ID NO:10), human IgG3 Fc (SEQ ID NO:11), or human IgG4 Fc (SEQ ID NO:12) to enhance binding of the mutant or variant Fc to FcRn at acidic pH (e.g., pH 5.8) and not at physiological pH (pH 7.4). For example, mutations (L / V309D, Q311H, N434S) were made in human IgG1, IgG2, IgG3, and IgG4, resulting in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively.

[0104] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of greater than about 200 amino acids to the full-length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein", "polypeptide", and "peptide" may be used interchangeably herein.

[0105] As used herein, "amino acid residue" refers to any amino acid, amino acid derivative, or amino acid mimetic known to one of ordinary skill in the art. In certain embodiments, the residues of a protein molecule are contiguous, without any non-amino acid residues interrupting the sequence of amino acid residues. In other embodiments, the sequence may contain one or more non-amino acid moieties. In some specific embodiments, the residue sequence of a protein molecule may be interrupted by one or more non-amino acid moieties.

[0106] As used herein, "unique Fc domain" can be defined as a domain that differs from another Fc by as few as one amino acid. Methods for preparing libraries of unique antibody Fc domains or nucleic acids encoding antibodies are well known in the art. For example, in some cases, the Fc domain can be amplified by error-prone PCR. Additionally, in certain cases, multiple antibody Fc domains may contain a stretch (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of amino acids that have been randomized. In certain cases, specific mutations can be engineered into the Fc domain. For example, in some aspects, residues that are normally glycosylated in the antibody Fc domain can be mutated. Additionally, in certain aspects, residues that are normally glycosylated (or adjacent residues) can be used as insertion sites for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0107] A polypeptide can comprise a mutant or variant antibody Fc domain that is capable of binding an FcR polypeptide. In some aspects, the Fc domain can be further defined as having a specific affinity for an FcR polypeptide under physiological conditions. For example, under physiological conditions, the Fc domain can have an equilibrium dissociation constant of about 10-6 M to about 10-9 M. Additionally, in some aspects, an aglycosylated Fc domain can be defined as comprising one or more amino acid substitutions or insertions relative to a wild-type sequence (e.g., a human wild-type sequence). The Fc domain can be glycosylated or aglycosylated.

[0108] Methods for preparing such polypeptides include those discussed in PCT Publication WO 2008 / 137475, which is incorporated herein by reference. Alternatively, such polypeptides can be prepared directly by genetic engineering techniques, such as by introducing selected amino acid substitutions or insertions into a known Fc background, where, as described above, the insertion or substitution provides improved FcR binding ability for the aglycosylated Fc region. In some embodiments, the Fc domain is engineered to bind one or more specific Fc receptors. As a supplement or alternative, the Fc domain can be engineered so that it does not specifically bind one or more specific Fc receptors.

[0109] In some embodiments, the Fc domain comprises specific binding affinities for FcRs such as human FcγRIA, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, FcγRIIIb, FcαRI or for C1q. In some embodiments, the antibody or polypeptide comprising the Fc domain is glycosylated and exhibits FcR binding similar to, substantially the same as, or the same as that of the wild-type antibody, e.g., compared to the corresponding IgG2, IgG3 or IgG4 antibody. In some embodiments, the antibody is glycosylated. In some embodiments, the antibody or polypeptide comprising the Fc domain is non-glycosylated. The binding affinity of the antibody Fc or other binding proteins can be determined, for example, by the Scatchard analysis of Munson and Pollard (1980). Alternatively, the binding affinity can be determined by surface plasmon resonance or any other well-known method for determining the kinetic and equilibrium constants of protein:protein interactions. Isolated IgG variants are provided in Table 1 below. In various embodiments, mutations can be introduced into the IgG1 Fc domain (e.g., to generate SEQ ID NO:13) as desired, or corresponding mutations can be made in the IgG2 Fc domain (e.g., SEQ ID NO:10), IgG3 Fc domain (e.g., SEQ ID NO:11) or IgG4 Fc domain (e.g., SEQ ID NO:12). Table 1: IgG Variants (Sequence numbers are based on Kabat, and mutations are indicated below) EDHS (V264E, L309D, Q311H, N434S; SEQ ID NO: 38). EDHY (V264E.L309D, Q311H, N434Y; SEQ ID NO: 39), DHS (L309D.Q311H, N434S; SEQ ID NO: 13). DHY (L309D, Q311H, N434Y; SEQ ID NO: 40), IgG2-DHS (V309D, Q311H, N434S; SEQ ID NO: 14), IgG3-DHS (L309D,Q311H, N434S; SEQ ID NO:15), IgG4-DHS (L309D, Q311H, N434S; SEQ ID NO:16)

[0110] As used herein, "position" refers to a position in a protein sequence. Positions can be numbered sequentially or according to a defined format, such as the EU index used for antibody numbering.

[0111] For all positions discussed in the present invention, numbering is according to the EU index. The "EU index" or "EU index in Kabat" or "EU numbering scheme" refers to EU antibody numbering (Edelman et al., 1969; Kabat et al., 1991; both incorporated herein by reference in their entirety).

[0112] In certain embodiments, the size of at least one Fc polypeptide protein molecule can include, but is not limited to, at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 225 or more amino acid residues, and any range derivable therefrom. The compound can comprise from SEQ ID NO: 13 to 16 (human IgG1 to 4 Fc polypeptides) or the above number of consecutive amino acids from SEQ ID NO: 13 to 16, and these can be further determined to have percent sequence identity or homology to the wild-type human IgG Fc domain (e.g., percent sequence identity to any one of SEQ ID NO: 1 to 4). V. Sortase recognition sequence

[0113] In some embodiments, a sortase recognition sequence is included in the constructs of the present disclosure. For example, a sortase recognition sequence can be included to link a cytotoxic moiety to a polypeptide comprising a cell targeting moiety (e.g., a polypeptide targeting R-spondin, such as FuFu). The sortase-catalyzed transacylation reaction can allow for the preparation of head-to-tail protein-protein fusions with high specificity and near quantitative yields (e.g., Popp et al. (2011), Guimaraes et al. (2011), Popp et al. (2007)).

[0114] Sortases, sortase-mediated transacylation reactions, and their use in protein engineered transacylation (sometimes also referred to as transpeptidation) are well known to those skilled in the art (see, for example, International Patent Application PCT / US2010 / 000274 and International Patent Application PCT / US2011 / 033303). The transpeptidation reaction catalyzed by sortase can be used to link polypeptides comprising a transamidase recognition motif to those bearing one or more N-terminal glycine residues. In some embodiments, the sortase recognition motif is an LPXT motif or an LPXT(G)n motif. As is known in the art, replacing the C-terminal residue of the recognition sequence with a moiety that exhibits poor nucleophilicity once released from the sortase can result in more efficient ligation.

[0115] The sortase-mediated transacylation reaction is catalyzed by the transamidase activity of sortase. A transamidase is an enzyme that can form a peptide bond (i.e., an amide bond) between an acyl donor compound and a nucleophilic acyl acceptor containing an NH2-CH2- moiety. In some embodiments, the sortase is sortase A (SrtA) or sortase E. However, any sortase or transamidase that catalyzes the transacylation reaction can be used in some embodiments of the present disclosure.

[0116] In some embodiments, the sortase recognition sequence is LPXT (SEQ ID NO: 41), where X is a standard or non-standard amino acid. In some embodiments, X is selected from D, E, A, N, Q, K, or R. For example, in some embodiments, the recognition sequence is LPET (SEQ ID NO: 42). In some embodiments, the recognition sequence is selected from LPXT (SEQ ID NO: 41), SPXT (SEQ ID NO: 42), LAXT (SEQ ID NO: 43), LSXT (SEQ ID NO: 44), NPXT (SEQ ID NO: 45), VPXT (SEQ ID NO: 46), IPXT (SEQ ID NO: 47), and YPXR (SEQ ID NO: 48). In some embodiments, X is selected to match a naturally occurring transamidase recognition sequence. In some embodiments, the sortase recognition sequences described in PCT International Patent Application WO 2013003555, U.S. Patent 7,238,489, and U.S. Patent Application 2014 / 0030697 can be used. The sortase recognition sequence can be, for example, LPKTG (SEQ ID NO: 49), LPATG (SEQ ID NO: 50), LPNTG (SEQ ID NO: 51), LPETG (SEQ ID NO: 52), LPXAG (SEQ ID NO: 53), LPNAG (SEQ ID NO: 54), LPXTA (SEQ ID NO: 55), LPNTA (SEQ ID NO: 56), LGXTG (SEQ ID NO: 57), LGATG (SEQ ID NO: 58), IPXTG (SEQ ID NO: 59), IPNTG (SEQ ID NO: 60), IPETG (SEQ ID NO: 61), LPKTGG (SEQ ID NO: 62), LPATGG (SEQ ID NO: 63), LPNTGG (SEQ ID NO: 64), LPETGG (SEQ ID NO: 65), LPXAGG (SEQ ID NO: 66), LPNAGG (SEQ ID NO: 67), LPXTAG (SEQ ID NO: 68), LPNTAG (SEQ ID NO: 69), LGXTGG (SEQ ID NO: 70), LGATGG (SEQ ID NO: 71), IPXTGG (SEQ ID NO: 72), IPNTGG (SEQ ID NO: 73), and IPETGG (SEQ ID NO: 74).

[0117] Sortase E can be used to covalently link a cytotoxic moiety to a polypeptide comprising an LGR binding domain. Sortase E can recognize the sequence LAHTGG (SEQ ID NO:106). For this reason, sortase A and sortase E can be used to covalently link a cytotoxic moiety to both the C-terminus and the N-terminus of a polypeptide, respectively. The cytotoxic moieties can have the same structure (e.g., both are MMAE), or different cytotoxic moieties can be covalently bound to the polypeptide. Using both sortase A and sortase E can confer advantages for production. For example, in the case of FcF2-LPETGG-His, the sortase reaction can place the MMAE warhead at both ends of the molecule; however, the challenge is that the sortase A reaction is reversible, and thus, during the addition of a second MMAE to the N-terminus, it can remove the MMAE that has been loaded on the C-terminus. The recently isolated sortase E can be used to link a substrate containing an N-terminal GG motif to the sequence LAHTGG (SEQ ID NO:106) on the C-terminus of other proteins or peptides. Due to the high specificity of sortase A and sortase E for different recognition sequences (LPETGGG vs. LAHTGG), they can be used simultaneously or separately to covalently link a cytotoxic moiety (e.g., MMAE, etc.) at both ends of a polypeptide (containing an LGR binding portion) that contains diglycine at the N-terminus and an LPXT (SEQ ID NO:41) (preferably LPETGG, SEQ ID NO:65) sequence at the C-terminus.

[0118] In some embodiments, the coding sequence recognized by the sortase is operably linked to the coding sequence of a serine protease via a linker. Any suitable linker known to those of skill in the art can be used. In one specific embodiment, the linker is a (GGS), (GGGS; SEQ ID NO:75), or (G4S) linker. In some embodiments, the (G4S) linker can contribute to the conformational freedom of the sortase domain to recognize the sortase recognition motif. VI. Partial reduction of disulfide bonds

[0119] If desired, partial reduction of disulfide bonds can be used to link the cytotoxic moiety to the polypeptide containing the LGR binding domain. The polypeptides provided herein can contain disulfide bonds that can be targeted in the Fc hinge region (e.g., 3 disulfide bonds in the immunoglobulin Fc domain) and / or the LGR binding domain (e.g., 8 in the Fu1-Fu2 domain). Covalent attachment of cytotoxins can be achieved by partial reduction of the Fc domain disulfide bonds or disulfide bonds in the LGR binding domain, followed by reaction with a linker containing a thiol-reactive maleimide group that was previously conjugated to the cytotoxin. These loading methods can be used to conjugate 2, 3, 4, 5, 6, 7, 8 or more cytotoxins per molecule, but should be individualized as overloading can distort the protein structure and reduce plasma half-life. For example, a polypeptide (e.g., FcF2-His) can be exposed to TCEP at a concentration of 0.0005 to 5000 μM and subsequently (e.g., after about 25 minutes) a maleimide-containing cytotoxin (e.g., maleimide-val / cit-PAB-MMAE) can be added to the reaction (e.g., at a ratio of about 1:1 to 1:9, 1:2, 1:8 (protein:cytotoxic agent)). The reaction can then be allowed to continue for a sufficient duration to achieve binding of the cytotoxic agent. In some preferred embodiments, the cytotoxic agent contains a cleavable linker (e.g., containing a valine (Val)-citrulline (Cit) bond). VII. Radiotherapy and imaging agents

[0120] In some embodiments, the therapeutic compounds provided herein can comprise a radiotherapy or imaging agent. For example, the therapeutic compound can be covalently bonded or conjugated to a radioisotope (such as iodine-131, strontium-89, samarium-153 or radium-223). The polypeptides described herein (e.g., SEQ ID NO: 1 to 4, 19 or 20) can be covalently bonded or conjugated to an imaging agent or contrast agent. The imaging agent can be, for example, an iodinated contrast agent, an ionic iodinated contrast agent, an MRI contrast agent (e.g., gadolinium), a diagnostic dye, a non-iodinated contrast agent, a non-ionic iodinated contrast agent or an ultrasound contrast agent. Additional radiotherapy and imaging agents that can be covalently bonded or conjugated to the compounds or polypeptides described herein include, for example, lutetium-177. VIII. Modified proteins and polypeptides

[0121] Some embodiments relate to modified proteins and polypeptides, particularly modified proteins or polypeptides that exhibit at least one functional activity comparable to the unmodified form, but the modified protein or polypeptide has additional advantages relative to the unmodified form, such as inhibiting B cell activation, being easier or cheaper to produce, causing fewer side effects, and / or having better or longer potency or bioavailability. Thus, when the present application refers to the function or activity of a "modified protein" or "modified polypeptide", one of ordinary skill in the art will understand that this includes, for example, such proteins or polypeptides that 1) perform at least one same activity or have at least one same specificity compared to the unmodified protein or polypeptide, but may have a different level of another activity or specificity; and 2) have additional advantages relative to the unmodified protein or polypeptide. Determination of activity can be achieved using assays familiar to those skilled in the art, particularly assays regarding protein activity, and for comparison purposes, can include, for example, using native and / or recombinant forms of the modified or unmodified protein or polypeptide. Embodiments regarding "modified proteins" are particularly contemplated to be practiced relative to "modified polypeptides", and vice versa. In addition to the modified proteins and polypeptides discussed herein, embodiments can relate to domains, polypeptides, and proteins described in PCT Publication WO 2008 / 137475 (which is hereby expressly incorporated by reference).

[0122] Modified proteins can have deletions and / or substitutions of amino acids; thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these modified proteins can further contain inserted or added amino acids, such as fusion proteins or proteins with linkers. This can include insertion of a targeting peptide or polypeptide or just a single residue. Terminal additions referred to as fusion proteins are discussed below.

[0123] A "modified deletion protein" lacks one or more residues of the native protein, but has the specificity and / or activity of the native protein. A "modified deletion protein" can also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is a protein from which amino acid residues have been deleted from at least one antigenic region (i.e., a region of the protein that has been determined to be antigenic in a particular organism, such as the type of organism in which the modified protein may be administered).

[0124] Substitution or replacement variants typically involve the exchange of one amino acid for another at one or more sites within a protein and can be designed to modulate one or more properties of the polypeptide, particularly its effector function and / or bioavailability. The substitution can be or can not be conservative, i.e., one amino acid is replaced by an amino acid of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0125] It should also be understood that amino acid and nucleic acid sequences can contain additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, but are still substantially as described in one of the sequences disclosed herein, provided that the sequence meets the above criteria, including the maintenance of biological protein activity involved in protein expression. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which can, for example, contain a variety of non-coding sequences flanking the 5' or 3' portions of the coding region, or can contain a variety of internal sequences known to exist within the gene, i.e., introns.

[0126] The modified polypeptide can be characterized as having a certain percentage identity with the unmodified polypeptide or with any polypeptide sequence described herein (e.g., SEQ ID NO: 1 to 4, 19 or 20). The percentage identity between the modified polypeptide and the unmodified polypeptide can be at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identity, or any range derivable therefrom. It is contemplated that the percentage identities discussed above can relate to a specific region of the polypeptide compared to the unmodified region of the polypeptide.

[0127] When making such changes, the hydrophilicity index of the amino acids can be considered. The importance of the hydrophilicity index of amino acids in conferring the biological function of interactions of proteins is generally understood in the art. It is recognized that the relative hydrophilic properties of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).

[0128] It should also be understood in the art that amino acid-like substitutions can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101 states that the maximum local average hydrophilicity of a protein is determined by the hydrophilicity of its neighboring amino acids, which is related to the biological properties of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It should be understood that an amino acid can be replaced with another amino acid having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, substitutions of amino acids with hydrophilicity values within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0129] As outlined above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account multiple of the foregoing characteristics are well known to those skilled in the art and include: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0130] In some aspects, it has been observed herein that glycosylation can significantly affect the yield and pharmacology of the polypeptides provided herein. For example, glycosylation alterations caused by mutations or substitutions at asparagine can lead to changes in protein folding, stability, pharmacokinetics, and other characteristics of its pharmacology. As will be understood by those skilled in the art, different amounts of glycosylation can be achieved by including substitution mutations in the polypeptide (e.g., alanine substitution for asparagine) or by using different types of cells (e.g., yeast, insect, human, or bacterial cells) to produce the polypeptides provided herein. IX. Pharmaceutical Formulations

[0131] The pharmaceutical compositions of the embodiments of the present invention comprise an effective amount of one or more compounds of the present invention and at least one additional pharmaceutical agent dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered appropriately to an animal (such as a human). In accordance with the present disclosure, those skilled in the art will be aware of the preparation of pharmaceutical compositions comprising at least one chimeric polypeptide or additional active ingredient, as exemplified by Remington: the Science and Practice of Pharmacy (23rd edition, Elsevier, 2020). Additionally, for administration to an animal (such as a human), it is to be understood that the formulations should conform to the sterility, pyrogenicity, general safety, and purity standards required by the FDA Office of Biological Standards.

[0132] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gelling agents, binders, excipients, disintegrants, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington’s Pharmaceutical Sciences, 18th edition Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is contemplated.

[0133] Cell-targeted cytotoxic agents can comprise different types of carriers, depending on whether they are administered in solid, liquid, or aerosol form, and whether they need to be sterile for such administration routes (such as injection). The treatments of the embodiments of the present invention can be administered by the following: intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrathoracic, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, topical, intratumoral, intramuscular, intraperitoneal, subcutaneous, subconjunctival, intravesicularlly, transmucosal, pericardial, intraumbilical, intraocular, oral, topical, local, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, directly locally perfusing and bathing target cells, through a catheter, by lavage, in a cream, in a lipid composition (e.g., liposome), or by other methods known to those of ordinary skill in the art or any combination of the foregoing (see, e.g., Remington’s Pharmaceutical Sciences, 18th Edition Mack Printing Company, 1990, which is incorporated herein by reference).

[0134] The actual dose of the compositions of the embodiments of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, the severity of the condition, the type of disease being treated, previous or concurrent treatment interventions, the idiopathy of the patient, and the administration route. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dose for the individual subject.

[0135] In certain embodiments, the pharmaceutical composition can comprise, for example, at least about 0.1% of an active compound, such as a therapeutic compound provided herein (e.g., FcF2-MMAE). For example, the active compound can be from about 2% to about 75%, or about 25% to about 60% by unit weight, and any range derivable therefrom. As shown in the following examples, for the selected therapeutic compound (e.g., FcF2-MMAE), subnanomolar potency was observed in vitro, and in vivo activity was detected at a dose of only 0.125 nmol / g (10.6 mg / kg). In some embodiments, a dose of about 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 55 mg / kg, or any range derivable therefrom, can be administered to a mammalian subject (e.g., a human). For example, a dose of about 0.5 to 20 mg / kg, or any range derivable therefrom, can be administered to a human subject.

[0136] The therapeutic compound can be administered to the same subject once or repeatedly. In some embodiments, the therapeutic compound is repeatedly administered to the same subject (e.g., a human patient), wherein the administration intervals are at least 1, 2, 3, 4, 5, 6, 7 days or longer, or 1, 2, 3, or 4 weeks. As shown in the following examples, improved potency was observed when the therapeutic compound (FcF2-MMAE) was injected every 7 days instead of every 4 days. The therapeutic compound can be repeatedly administered to the same subject weekly or over a period of months or longer, or as long as the subject has a disease (e.g., cancer). In some embodiments, the therapeutic compound is administered once every 4 days (q4d), once every 7 days (q7d), once every 21 days (q21d), or once every 28 days (q28d).

[0137] In any case, the composition can contain a variety of antioxidants to delay the oxidation of one or more components. Additionally, prevention of microbial action can be achieved through preservatives (e.g., a variety of antibacterial and antifungal agents), which include but are not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0138] In some embodiments where the composition is provided in liquid form, the carrier can be a solvent or a dispersion medium, including but not limited to water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes), and combinations thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin; by dispersing in a carrier (e.g., a liquid polyol or lipid) to maintain the desired particle size; by using surfactants, such as hydroxypropylcellulose; or a combination of such methods. In many cases, it will be preferred to include isotonic agents, such as sugars, sodium chloride, or combinations thereof.

[0139] A sterile injectable solution is prepared by incorporating the active compound in the required amount into a suitable solvent that optionally has a variety of the other ingredients listed above, and then filtering to sterilize. Generally, a dispersion is prepared by incorporating the various sterilized active ingredients into a sterile carrier that contains a base dispersion medium and / or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions, or emulsions, the preferred method of preparation is vacuum drying or freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered liquid medium. If necessary, the liquid medium should be appropriately buffered and made isotonic with a sufficient dilution of saline or glucose prior to injection. Formulations of highly concentrated compositions for direct injection are also contemplated, where DMSO is envisioned as a solvent to achieve extremely rapid penetration, thereby delivering a high concentration of the active agent to a small area.

[0140] The composition must be stable under manufacturing and storage conditions and protect against the contaminating effects of microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be kept at a minimum at a safe level, such as less than 0.5 ng / mg protein.

[0141] In some specific embodiments, extended absorption of an injectable composition can be achieved by using agents with delayed absorption (such as aluminum monostearate, gelatin, or combinations thereof) in the composition. X. Cancer

[0142] Multiple cancer cells can be treated by the methods and compositions provided herein. In some embodiments, the cancer cells express LGR4, LGR5, and / or LGR6. The cancer cells can be cancer stem cells (CSCs), cancer cells with enhanced stemness, or cancers that express LGR4, LGR5, or LGR6, regardless of functional classification.

[0143] In some embodiments, the cancer includes CSCs. A subset of cells in solid tumors exhibits many characteristics of stem cells, such as the ability to form spheroids or initiate new tumors. For stem cells present in organized epithelia, many CSCs require RSPO for robust growth in culture (Sato et al., 2009; Barker et al., 2010). Elimination of a small fraction of cells (CSCs) in a tumor can reduce or prevent further expansion of the tumor by reducing the supply of more differentiated cells that make up the majority of the population.

[0144] In some embodiments, the cancer is ovarian cancer. High-grade serous ovarian cancer can be caused by ovarian surface epithelium or fallopian tube epithelium, but the latter is dominant (Zhang et al., 2019). Lineage-tracing studies in mice have shown that LGR5 marks the stem cell population in the ovary, and LGR6 marks the stem cells in the fallopian tube epithelium of mice and humans (de Lau et al., 2014; Kessler et al., 2015; Zhang et al., 2019). Data from TCGA show that high-grade serous ovarian cancer can express high levels of LGR5 and LGR6 mRNA. Additionally, ovarian cancer has the highest median expression of RSPO1 mRNA compared to all other tumor types in the database, except for mesothelioma (Schindler et al., 2017). Without being bound by any theory, this data supports the view that ovarian cancer may rely on RSPO1 to support its CSC population. As shown in the following examples, the data provided herein support the view that the compounds provided herein can use the receptor-binding domain of RSPO1 armed with a cytotoxin to selectively bind to cancer cells (e.g., ovarian cancer cells expressing LGR5 / LGR6) and deliver the cytotoxin to the cancer cells.

[0145] Cancer cells treatable with a cell targeting construct according to some embodiments include, but are not limited to, cells from: bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. Additionally, the cancer can specifically be the following histological types, although it is not limited to these: neoplasm; carcinoma; undifferentiated carcinoma; giant cell and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; hepatobiliary carcinoma, combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial adenocarcinoma of colonic polyps; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenocortical carcinoma; endometrioid carcinoma; carcinoma of skin appendages; apocrine adenocarcinoma; sebaceous gland carcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant arrhenoblastoma; sertoli cell carcinoma; malignant leydig cell tumor; malignant lipoma; malignant paraganglioma; malignant mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma;Melanoma in giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Müllerian mixed tumor; Wilms tumor; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymoma; Malignant Brenner tumor; Malignant phyllodes tumor; Synovial sarcoma; Malignant mesothelioma; Dysgerminoma; Embryonal carcinoma; Malignant teratoma; Malignant struma ovarii; Choriocarcinoma; Malignant mesonephroma; Angiosarcoma; Malignant hemangioendothelioma; Kaposi's sarcoma; Malignant hemangiopericytoma; Lymphangiosarcoma; Osteosarcoma; Juxtacortical osteosarcoma; Chondrosarcoma; Malignant chondroblastoma; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Malignant odontogenic tumor; Ameloblastic odontosarcoma; Malignant ameloblastoma; Ameloblastic fibrosarcoma; Malignant pinealoma; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrous astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroblastoma; Primitive neuroectoderm; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant schwannoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's paragranuloma; Malignant lymphoma, small lymphocyte; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular; Mycosis fungoides; Other specified non-Hodgkin lymphoma; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia; Lymphocytic leukemia; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloid leukemia; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia; Mast cell leukemia; Megakaryoblast leukemia; Myeloid sarcoma; And hairy cell leukemia.; VI. Combination Therapy

[0146] To enhance the effectiveness of the therapeutic compounds of the present disclosure, it may be desirable to combine these compositions with other agents effective in treating the target disease. In some embodiments, a therapeutic compound (e.g., a cell-targeted cytotoxic agent comprising auristatin) is administered to a mammalian subject in combination with a second anti-cancer agent or treatment to treat cancer in the subject. In some embodiments, the targeted cytotoxic agents of the present disclosure may be administered to the subject in combination with an immunotherapeutic agent, an anti-bacterial agent (e.g., an antibiotic), or an anti-viral agent to treat a bacterial or viral infection in the subject, respectively.

[0147] As a non-limiting example, the treatment of cancer can be carried out with the therapeutic compounds of the present disclosure in combination with other anti-cancer agents. An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to the tumor or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions will be provided in a combinatorial amount effective to kill or inhibit cell proliferation. The process may involve simultaneous contact of the cells with the agent or agents and the factors. This can be achieved by contacting the cells with a single composition or pharmaceutical formulation containing both agents or by contacting the cells simultaneously with two different compositions or formulations, one of which contains the anti-cancer peptide or nanoparticle complex and the other contains the second agent. In some embodiments, the anti-cancer peptide is one agent and the cell-targeted cytotoxic agent (e.g., a cell-targeted cytotoxic agent comprising auristatin) is the other agent.

[0148] Treatment with the therapeutic compounds of the present disclosure can be separated by minutes to weeks before or after treatment with other agents. In embodiments where another agent and the therapeutic compound are administered separately to the cells, it will generally be ensured that a meaningful period of time has not elapsed between each delivery time such that the agent and the therapeutic compound are still able to have a beneficial combinatorial effect on the cells. In such cases, it is expected that the cells can be contacted with the two forms within about 12 to 24 hours of each other, and more preferably within about 6 to 12 hours of each other. In some cases, it may be desirable to significantly extend the treatment period, with intervals of days (e.g., 2, 3, 4, 5, 6, or 7 days) to weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks) between each administration. A wide variety of dosing regimens can be used.

[0149] A variety of combinations can be used, where treatment with the therapeutic compound is designated as "A" (e.g., administration of a cell-targeted cytotoxic agent comprising auristatin), and the second agent (e.g., radiotherapy, chemotherapy, or an anti-inflammatory agent) is designated as "B":

[0150] In certain embodiments, taking into account the toxicity of the vehicle (if any), administration of the treatment of the embodiments of the present invention to a patient will follow the general protocol for administration of chemotherapeutic agents. It is expected that the treatment cycles can be repeated as needed. It is also expected that a variety of standard treatments as well as surgical interventions can be combined with the treatment of the hyperproliferative cells described. A. Chemotherapy

[0151] Cancer treatment also includes various combination therapies. In some aspects, the therapeutic compounds of the present disclosure (e.g., cell-targeted cytotoxic agents comprising auristatin) are administered (or formulated) in combination with chemotherapeutic agents. For example, in some aspects, the chemotherapeutic agent is a protein kinase inhibitor, such as an EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, P13K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor, or BRAF inhibitor. Some non-limiting examples of protein kinase inhibitors include Afatinib, Axitinib, Bevacizumab, Bosutinib, Cetuximab, Crizotinib, Dasatinib, Erlotinib, Fostamatinib, Gefitinib, Imatinib, Lapatinib, Lenvatinib, Mubritinib, Nilotinib, Panitumumab, Pazopanib, Pegaptanib, Ranibizumab, Ruxolitinib, Saracatinib, Sorafenib, Sunitinib, Trastuzumab, Vandetanib, AP23451, Vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, Miltefosine, Perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, Alvocidib, Genistein, Selumetinib, AZD-6244, Vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or a mixture thereof.

[0152] Other combinatorial chemotherapy includes, for example: alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, prosulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;Nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ωI1); dynemicin, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycins, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrolino doxorubicin, and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, trimetrexate;Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal agents such as mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansine alkaloids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2”-trichloroethylamine; trichothecene (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoid such as paclitaxel and docetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexe such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum class; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoid such as retinoic acid; capecitabine;Carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In certain embodiments, the compositions provided herein can be used in combination with gefitinib. In other embodiments, the embodiments of the present invention can be practiced in combination with Gleevac (e.g., from about 400 mg / day to about 800 mg / day of Gleevac can be administered to a patient). In certain embodiments, one or more chemotherapeutic agents can be used in combination with the compositions provided herein.; B. Radiation Therapy

[0153] Radiation therapy has been widely used for treatment and includes the commonly known gamma rays, X-rays and / or direct delivery of radioactive isotopes to tumor cells. Other forms of radiation therapy are also contemplated, such as microwave and UV irradiation. These factors can affect extensive damage to DNA, precursors of DNA, DNA replication and repair, and chromosome assembly and maintenance. The dose range of X-rays is from a daily dose of 50 to 200 roentgens for an extended period of time (3 to 4 weeks) to a single dose of 2000 to 6000 roentgens. The dose range of radioactive isotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and the uptake of neoplastic cells.

[0154] As used herein, the terms “contact” and “exposure” when applied to cells describe the process of delivering a therapeutic composition and a chemotherapeutic or radiotherapeutic agent to a target cell or placing them in direct adjacency to the target cell. To effect cell killing or stasis, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent cell division. C. Immunotherapy

[0155] Generally, immunotherapy relies on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector can be, for example, an antibody specific for some marker on the surface of the tumor cell. For example, the immunotherapy can be an antibody, such as an anti-PD-L1 antibody or an anti-CTLA4 antibody. In some embodiments, the immunotherapy is an antibody drug conjugate (ADC).

[0156] Individual antibodies can act as effectors of therapy, or they can recruit other cells to actually effect cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used only as targeting agents. Alternatively, the effector can be a lymphocyte carrying surface molecules that interact directly or indirectly with tumor cell targets. A variety of effector cells include cytotoxic T cells and NK cells.

[0157] Accordingly, immunotherapy can be combined with serine protease therapy of embodiments of the present invention as part of a combination therapy. General methods of combination therapy are discussed below. Generally, tumor cells must have some marker that is suitable for targeting (i.e., not present on most other cells). There are many tumor markers, and in the case of embodiments of the present invention, any of these tumor markers can be suitable for targeting. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155. D. Gene Therapy

[0158] In another embodiment, the secondary therapy is gene therapy, in which the therapeutic polynucleotide is administered before, after, or concurrently with the therapeutic composition. Viral vectors for the expression of gene products are well known in the art and include eukaryotic expression systems such as adenovirus, adeno-associated virus, retrovirus, herpes virus, lentivirus, poxvirus (including vaccinia virus), and papillomavirus (including SV40). Alternatively, administration of the expression construct can be accomplished with lipid-based vectors (such as liposomes or DOTAP:cholesterol vesicles). All of these methods are well known in the art (see, e.g., Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).

[0159] Delivery of a vector encoding one of the following gene products will have a combined anti-proliferative effect on the target tissue. A variety of proteins are encompassed by embodiments of the present invention and are well known in the art. E. Surgery

[0160] Approximately 60% of people with cancer will undergo some type of surgery, which includes prophylactic, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in combination with other treatments (e.g., the treatments provided herein, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies).

[0161] Curative surgery includes resections in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, treatments by surgery include laser surgery, cryosurgery, electro-surgery, and microscopically controlled surgery (Mohs’ surgery). Embodiments of the present invention are also expected to be used in combination with the removal of superficial cancer, primary cancer, or an accompanying amount of normal tissue.

[0162] After removing some or all of the cancer cells, tissues, or tumors, a cavity may be formed in the body. Treatment can be accomplished by perfusion, direct injection, or local application of additional anti-cancer treatments in the area. Such treatments can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also have multiple doses. IV. Examples

[0163] The following examples are included to illustrate some preferred embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to function well in the practice of the present invention and can thus be considered to constitute a preferred mode for its practice. However, based on the present disclosure, those skilled in the art should understand that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention and still obtain the same or similar results. Example 1 Compounds for targeting LGR5-expressing stem cells in ovarian cancer Design of FcF2-His

[0164] Figure 1The upper part shows a schematic diagram of FcF2-His. This protein contains a variant human IgG1 Fc domain linked to the Fu1-Fu2 domain of human RSPO1 via two Gly4Ser spacers (Lee et al., 2019). Two additional Gly4Ser spacers separate the Fu1-Fu2 domain from the LPETGG sortase recognition sequence followed by an 8×His tag that facilitates purification. Each Fc-(Fu1-Fu2)-LPETGG-His (abbreviated as FcF2-His) sequence contains 391 amino acids. The Fc domain causes dimerization via three intermolecular disulfide bonds, one of which links the Fc to the light chain in the antibody and the other two link the Fc domains together in its hinge region. The total calculated MW of the resulting protein is 85,284 Da. Glycosylation is present on the Fc domain and on residue N137 (RSPO1 numbering) in the Fu1-Fu2 domain. Sortase-Mediated Conjugation of Monomethyl Auristatin E (MMAE)

[0165] Figure 1 The lower part shows a schematic diagram of the conversion of FcF2-His to FcF2-MMAE. Sortase cleaves the LPETGG sequence between threonine and glycine and forms a transient thioester bond with a cysteine in the active site of the enzyme, which is subsequently attacked by the N-terminal glycine of the protease-sensitive GGG-vc-PAB-MMAE linker. The His tag is lost in this reaction. This results in the precise covalent loading of one molecule of MMAE on each arm of FcF2-His and produces a homogeneous population of conjugated FcF2-MMAE molecules. Generation, Purification, and Characterization of FcF2-MMAE

[0166] FcF2-His was produced by transient transfection of the vector pcDNA3.1-FcF2-8×His into HEK293E cells and purified from the cell supernatant by capture on Ni-NTA resin and subsequent ion exchange chromatography. FcF2-His was then reacted with sortase-His and GGG-vc-PAB-MMAE at 37 °C for 4 h to produce FcF2-MMAE. In 14 batches, the average yield of FcF2-His was 23.4 ± 2.3 mg / L; the sortase efficiency was 77 ± 8.6%; and the yield of the final product was 18.8 ± 3.5 mg / L (mean ± SEM).

[0167] Figure 2 shows the results of the characterization of FcF2-His and FcF2-MMAE by reverse-phase HPLC, size-exclusion chromatography, and reducing and non-reducing SDS-PAGE. In the reverse-phase HPLC analysis using a C4 column, both forms of the molecule ran as a single, well-defined peak (Figures 2A & D). Analysis on an HPLC 300 size-exclusion column indicated that both FcF2-His and FcF2-MMAE were present in solution as dimers and dimers of molecules that were already dimers (hereinafter referred to as tetramers) (Figures 2B & E). A small amount of higher MW material (7%) eluted just before the major tetramer peak of FcF2-His; FcF2-MMAE contained a third, lower MW peak. Non-reducing SDS-PAGE analysis of FcF2-His (Figure 2C) showed that in the presence of SDS detergent in the loading buffer, most of the tetramers ran at an MW (85.3 kD) consistent with the size of the FcF2-His dimer. Under reducing conditions, most of the protein ran as a doublet at ~42 to 46 kD. To better understand this doublet, FcF2-His was treated with PNGase to remove N-linked glycans. As shown in Figure 10A, this resulted in the collapse of the doublet into a single band, consistent with the conclusion that the doublet was due to differential glycosylation. When the single glycosylation site at N137 in the Fu2 domain was mutated to alanine, the doublet disappeared. Extensive comparison of wild-type and N137A forms of R1FF-MMAE showed no change in cytotoxicity or plasma pharmacokinetics, indicating that glycosylation at this site had little functional consequence with respect to these parameters.

[0168] Non-reducing SDS-PAGE analysis of FcF2-MMAE (Figure 2E) showed that in the presence of SDS detergent in the loading buffer, most of the FcF2-MMAE ran at an MW of 72 to 78 kD and, when run under reducing conditions, it ran at 42 to 48 kD in a manner similar to the behavior of FcF2-His under non-reducing and reducing conditions. Western blot analysis of FcF2-MMAE using anti-RSPO1 and anti-MMAE antibodies indicated that the major band stained for protein on the SDS-PAGE gel contained all components of the molecule (Fc, Fu1-Fu2, and MMAE) (Figure 10B), and this was also confirmed by all major peaks visible in the C4 and SEC300 profiles. In Vitro Cytotoxicity and Selectivity of FcF2-MMAE

[0169] HEK293 cells and the human ovarian cancer cell line OVCAR8 were molecularly engineered to stably express elevated levels of LGR5. The parental forms of both lines expressed variable but not well-defined levels of all three LGR receptors. Flow cytometry analysis using an antibody against LGR5 demonstrated that the level of LGR5 in HEK293 / LGR5 cells was 8.7-fold higher than in HEK293 / EV controls; in the case of OVCAR8 cells, the difference was ~25-fold (Figures 11A and 11B, respectively). A cytotoxicity assay was performed using a 120-hour exposure to the drug and CCK8 reagent to assess viability. Figures 3A&B show greater cytotoxicity that is LGR5 receptor-dependent in the isogenic HEK293 pair; for the two tested batches, the sensitivity of HEK293 / LGR5 cells was 21- and 46-fold that of HEK293 / EV cells. Large differential effects were also observed when testing with the OVCAR8 / EV and OVCAR8 / LGR5 isogenic pairs; the two most recent batches showed 77- and 87-fold differential inhibition of cell growth (Figures 3C to D). In the most recent batches meeting all release criteria, for OVCAR8 / EV cells, the mean IC 50 was 4.5 ± 1.1 nM (SEM), and for OVACAR8 / LGR5 cells, the mean IC 50 was 0.059 ± 0.015 nM; the mean ratio of IC 50 values was 76 ± 5 (N = 3).

[0170] The question of whether differential inhibition of growth of LGR5-deficient and LGR5-rich isogenic pairs might be due to differences in sensitivity to free MMAE rather than Fu1-Fu2-directed targeting was addressed by determining the IC 50 value of free MMAE using the same assay. The resistance of HEK293 / LGR5 cells to free MMAE was 1.6-fold that of HEK293 / EV cells to free MMAE (19.2 ± 0.4 vs 30.5 ± 2.5, p = 0.03 N = 3). The resistance of OVCAR8 / LGR5 cells to free MMAE was 1.5-fold that of OVCAR8 / EV cells to free MMAE (IC 50 values were 129 vs 85 nM, respectively). Thus, both LG5R-rich cell types were actually slightly resistant to free MMAE, providing confidence that the selectivity exhibited by FcF2-MMAE is attributable to targeting of the Fu1-Fu2 domain.

[0171] Further evidence was provided by observing that treatment of the OVCAR8 cell population with FcF2-MMAE cleared LGR-positive cells. A mixed OVCAR8 cell population expressing low and higher levels of LGR5 cells was exposed to 10 nM FcF2-MMAE for 96 hours. Before treatment, flow cytometry analysis showed a bimodal distribution of LGR5-expressing cells (Figure 11C). FcF2-MMAE treatment resulted in loss of most cells expressing high levels of LGR5, but had a significantly lower effect on a subset of cells with low levels of LGR5 expression. In addition to IC 50 value differences, this data also provides evidence of differential killing as a function of LGR5 expression level. Cytotoxicity against Human Wild-Type Ovarian Cancer Cell Lines

[0172] To help select a suitable xenograft model for potency testing, the cytotoxicity of FcF2-MMAE against a panel of 8 human ovarian cancer cell lines was determined. The concentration-survival curves shown in Figure 3E indicate that the IC 50 values range from 3.8 to 29.6 nM; the IC 50 < 10 nM for 7 out of 8 cell lines. Thus, although the level of the sum of all LGR expression in these cell lines is unknown, the data indicate that FcF2-MMAE is highly effective in this panel of ovarian cancer cell lines. Determinants of Cytotoxicity Selectivity

[0173] RSPO1 is a bispecific ligand. The Fu1 domain of RPSO1 binds to the ubiquitin ligase receptors ZNRF3 and RNF43, and the Fu2 domain binds to LGR4, LGR5, or LGR6. The relative contribution of each of these types of receptors to the selectivity of FcF2-MMAE for OVCAR8 / EV and OVCAR8 / LGR5 cells was explored by introducing mutations in one or the other Fu domain, which were previously shown to disrupt binding to their cognate receptors (Peng et al., 2013; Xie et al., 2013; Zebisch et al., 2013; Zhang et al., 2020; Cui et al., 2021). The cytotoxicity of the mutant forms was tested in in vitro OVCAR8 / EV and OVCAR8 / LGR5 models. Figure 4 The growth inhibition curves shown in indicate that both domains are important for the selectivity of FcF2-MMAE. The Q71R mutation in Fu1 shifted the IC 50The ratio decreased from 19.5 to 5.1 (p = 0.05), the F106R - F110R mutation in Fu2 decreased the ratio to 2.1 (p = 0.03), and when both mutations were present, the selectivity was abolished. Thus, in this model system, the ability to bind to both types of receptors is important for the successful internalization of FcF2 - MMAE and the release of free MMAE. Pharmacokinetics of FcF2-MMAE in Mice

[0174] BALB / c mice were given a bolus injection of 0.1 nmol / g (9 μg / g) of FcF2 - MMAE and plasma samples were obtained from 3 mice at each sampling time point. The concentration of FcF2 - MMAE was measured using ELISA, which utilized monoclonal capture and polyclonal detection antibodies (LLQ 1.5 pmol / ml) of different species prepared by immunization with RSPO1. Figure 5A shows the complex plasma decay curve; analysis performed with WinNonLin curve fitting software yielded estimates of a distribution half - life of 4.47 hours and a terminal half - life of 29.7 hours. The initial half - life was lower than expected. One mechanism by which FcF2 - MMAE can be removed from the plasma compartment is by binding to red blood cells, white blood cells, or platelets. However, when plasma spiked with FcF2 - MMAE was added to the components formed by sedimentation to reconstitute their respective normal volumes, the drug was not significantly removed from the plasma fraction within 27 hours at 40 °C (Figure 5B). This suggests that the rapid initial half - life is largely due to distribution into tissues. Efficacy of FcF2-MMAE in a Human Ovarian Cancer Xenograft Model

[0175] Isogenic pairs of xenografts generated from OVCAR8 / EV and OVCAR8 / LGR5 cells were used to explore both the in vivo efficacy and selectivity of FcF2-MMAE. In the experiment shown in Figure 6A, once the tumors became palpable, mice were treated via the IP route with 0.5 nmol / g (42 mg / kg) every 4 days for a total of 4 doses. FcF2-MMAE had a much greater effect on OVCAR8 / LGR5 than on OVCAR8 / EV tumors. By day 20, the difference in growth rates was apparent and persisted until day 60, at which time the FcF2-MMAE-treated OVCAR8 / LGR5 tumors were on average only 35% the size of the vehicle-treated tumors. FcF2-MMAE was also effective in slowing the growth rate of OVCAR8 / EV cells, consistent with evidence that they express some combination of LGR receptors, but the curves of the drug-treated and vehicle-treated tumors did not separate until after 30 days, and at 60 days, the FcF2-treated OVCAR8 / EV tumors were on average 63% the size of the control tumors. Notably, for both OVCAR8 / EV and OVCAR8 / LGR5 tumors, the reduction in tumor growth rate was maintained for >1.5 months after the last dose of FcF2-MMAE, an effect consistent with the targeting of cancer stem cells in the tumors, which are difficult to recover growth rate. The lack of dose-limiting toxicity prompted a second efficacy study of the same design but using a dose of 1.5 nmol / g and a q7dx4 schedule, demonstrating that the efficacy of FcF2-MMAE was equally different in both types of xenografts (Figure 12A).

[0176] Figure 6B shows the change in average mouse weight during and after four FcF2-MMAE injections at a dose of 0.5 nmol / g and Figure 12B shows the same data for a dose of 1.5 nmol / g administered according to the q7dx4 schedule. The first injection resulted in a transient decrease in average body weight but rapid recovery, and in both studies, the total mouse body weight increased during the treatment period. In neither the control nor the treatment groups did animals treated with 0.5 nmol / g die due to tumor burden prior to sacrifice. No observable adverse events occurred after the first dose of FcF2-MMAE, including diarrhea, changes in activity level, posture, or grooming, or reduced food consumption.

[0177] The OVCAR8 / LGR5 model was used to explore the potency and toxicity of FcF2-MMAE as a function of the more clinically relevant dosing schedule of every 7 days. The growth curves shown in Figure 7A demonstrate an increase in potency as the dose increases from 0.125 nmol / g to 1.0 nmol / g. Even the dose of 0.125 nmol / g (10.6 mg / kg) resulted in a significant decrease in growth rate (final tumor volume was 66% of untreated control). As shown in Figure 7B, this dose did not result in weight loss. Potency and maximum weight loss increased as the dose reached 0.75 nmol / g; at 1.0 nmol / g, no further increase in either parameter was observed. This experiment suggests a therapeutic window within an 8-fold dose range, but this estimate needs to be refined with FcF2-MMAE produced under GMP conditions. Efficacy of FcF2-MMAE in a Wild-Type Human Ovarian Cancer Xenograft Model

[0178] The potency of FcF2-MMAE was explored in a total of 3 ovarian cancer xenograft models established from cell lines that had not undergone any genetic modification to increase LGR5. When tumors became palpable, in the KF-28 and CAOV3 models, a single IP dose of 1.0 nmol / g resulted in a long-term delay in tumor growth without dose-limiting weight loss (Figure 8A / C). According to the q7dx4 schedule, at a dose of 0.5 nmol / g, FcF2-MMAE was active against IGROV8 and CAOV3 xenografts without significant toxicity in the form of weight loss (Figure 8B / D). These data provide evidence for potency in 3 different human ovarian cancer models without molecular engineering to enhance LGR5 expression. The expression levels of LGR4, LGR5, and LGR6 in these models cannot yet be accurately defined, so sensitivity cannot be correlated with the expression of any one of them. However, the data are consistent with the concept that stem cell-like cells in each of these tumors collectively express sufficient amounts of these receptors to respond to FcF2-MMAE.

[0179] Selectively targeting stem cells in tumors can reduce or limit tumor expansion and the metastatic capacity of cancer. The rationale for using the receptor-binding Fu1-Fu2 domain of RSPO1 to achieve this goal is based on its high affinity (∼3 nM) for LGR5 and LGR6 (Carmon et al., 2011), and evidence that the expression of these receptors marks stem cells in tumors, as they do in normal epithelium. This approach is of particular interest in the case of ovarian cancer, as LGR5 and LGR6 mark stem cells in the ovarian surface and fallopian tube epithelium, from which ovarian cancer arises (Zhang et al., 2019), and these tumors exhibit abnormally high levels of LGR5 and LGR6 expression after transformation (Schindler et al., 2017; Lee et al., 2020). The fact that the Fu1-Fu2 domain binds both LGR and ZNRF3 or RNF43 in a bispecific manner favors specificity, and because it is part of a normal human protein, it reduces the risk of immunogenicity.

[0180] FcF2-MMAE is different from the R1FF-MMAE molecule, which consists only of the Fu1-Fu2 domain of RSPO1 linked to MMAE via a cleavable linker (Yu et al., 2021). Although R1FF-MMAE exhibits LGR5-dependent cytotoxicity and in vivo activity, it does not have optimal pharmaceutical properties. The modifications made to generate FcF2-MMAE involved: a) improving folding by exploiting the chaperone function of Fc to increase protein yield from transient transfection cultures; b) dimerizing it so that it carries two MMAE molecules instead of one; c) increasing the plasma half-life by including a mutant form of Fc with improved FcRn-binding characteristics (Lee et al., 2019); and d) increasing the affinity for binding to LGR and ZNRF3 / RNF43 by including two rather than just one copy of the Fu1-Fu2 domain. Achievement of these goals was demonstrated by a significantly higher yield of the FcF2-His precursor, higher potency and selectivity when tested in syngeneic OVCAR8 / EV and OVCAR8 / LGR5 cells, a 6-fold increase in the terminal plasma half-life, and improved potency in xenograft models. Importantly, dimerization of the two Fu1-Fu2 domains caused by the presence of the Fc domain does not impair the efficiency of the sortase reaction, which retains high efficiency.

[0181] FcF2-MMAE causes LGR5-dependent killing in vitro and differentially depletes cells with the highest levels of this receptor. IC 50The average difference of 76-fold in value was sufficient to generate significantly greater in vivo potency against LGR5-rich OVCAR8 / LGR5 cells. The results of cytotoxicity assays indicated that conjugation to both LGR and ubiquitin ligase receptor was important for optimal selectivity. Given its terminal half-life of 27.4 hours, it was interesting that injection every 7 days was more potent than injection every 4 days. Consistent with its in vitro sub-nanomolar potency, in vivo activity was detected at a dose as low as 0.125 nmol / g (10.6 mg / kg). Potency increased as the dose was raised to levels approaching the maximum tolerated dose, yielding an estimated 8-fold therapeutic window, which was significantly higher than that of many chemotherapeutic agents used to treat ovarian cancer. Most importantly, in a panel of ovarian cell lines, FcF2-MMAE showed cytotoxicity at <10 nM in all but one member, and had in vivo activity in two additional ovarian xenograft models established from cells expressing only endogenous, unmanipulated levels of LGR. Although the sum of expression of each individual member of the LGR4-6 family could not be precisely determined due to the different affinities of available antibodies and the different affinities of each LGR for the dimeric form of the RSPO1 Fu1-Fu2 domain, this provided substantial assurance that the expression was high enough to allow FcF2-MMAE to be effective. LGR5 and LGR6 are expressed in the stem cells of many other types of cancer, supporting the use of FcF2-MMAE to treat a wide variety of cancers expressing LGR5 and / or LGR6.

[0182] Cancers, including ovarian cancer and other cancers, can be tested for LGR5 / LGR6 expression. Expression may be higher in some cancers, such as malignant cells with intact stem cell capacity. Cancers with attenuated stem cell capacity but that continue to express sufficient LGR5 or LGR6 may be sensitive to and benefit from treatment with FcF2-MMAE. Not all cells expressing LGR5 can act as stem cells (Azkanaz et al., 2022). LGR5 expression is not restricted to epithelial cells; LGR5 is expressed in some mesenchymal cells, but it is currently unclear whether these cells have stem cell characteristics (Kim et al., 2022). These results support the use of FcF2-MMAE to disrupt stem cells in tumors, and similar selective killing is expected to be observed in malignant cells.

[0183] Ablation of LGR5-expressing cells in the mouse intestine does not disrupt its epithelial integrity, and recent studies have shown that this is due to the plasticity of transiently amplifying cells to regenerate cells with intact stem cell capacity (Tian et al., 2011; Azkanaz et al., 2022). However, the extent to which ovarian cancer or other types of cancer retain such plasticity remains to be determined. Studies using organoid-derived colon cancer xenografts have shown that ablation of LGR5 can produce a relatively durable response, and ADCs targeting LGR5 have produced good responses in colon cancer xenograft models (Junttila et al., 2015; Gong et al., 2016). Long-lasting growth inhibition was observed in the KF-28 and CAOV3 models using a single dose of FcF2-MMAE.

[0184] The activity of FcF2-MMAE was also tested in vivo using a xenograft mouse model of human colorectal cancer. FcF2-MMAE was administered to mice bearing human colorectal xenografts (LoVo) at a dose of 1 nmol / g every 7 days, with a total of 4 administrations per mouse, and no clinical toxicity was observed at this dosing. Nevertheless, FcF2-MMAE led to a decrease in the average weight of colorectal tumors without altering the body weight of the mice. The results are shown in Figure 21 in.

[0185] At doses that produce anti-tumor activity, FcF2-MMAE produced unexpectedly low toxicity in mice. In addition to possible differences in LGR5 and LGR6 expression between tumor stem cells and normal stem cells, the differential effect on tumor tissue compared to normal tissue may be caused by multiple factors. Without wishing to be bound by any theory, normal epithelium may be more tolerant of the loss of LGR5-positive cells, as shown in the intestine (Tian et al., 2011; Junttila et al., 2015; Gong et al., 2016). In normal tissues, stem cells exist in highly structured and protected niches, surrounded by cells that provide support in the form of WNT, RSPO1, and cytokines. Due to microanatomical and physiological differences, FcF2-MMAE may be much more accessible to tumor stem cells than to stem cells in normal epithelial niches. When LGR and ZNRF3 / RNF43 are in normal polarized niches, the ability of plasma RSPO1 to access stem cells may be limited in terms of the degree to which they are expressed on the luminal surface rather than the basal surface of stem cells (de Vreede et al., 2022). However, after transformation, polarization is lost, and once the drug reaches the stem cell environment, FcF2-MMAE can better access receptors widely distributed on cancer stem cells. FcF2-MMAE contains only the Fu1-Fu2 domain of RSPO1 and lacks the long C-terminal TSP-BR domain, which has been shown to mediate binding to proteoglycans that facilitate accumulation in normal tissue niches (Lebensohn and Rohatgi, 2018). The missing TSP-BR domain has also been reported to limit the ability of the remaining Fu1-Fu2 portion of the molecule to activate WNT signaling through non-LGR-dependent pathways, which is a potential cause of toxicity, but it does not impair receptor binding (Dubey et al., 2020).

[0186] At therapeutically effective doses, FcF2-MMAE produces very few clinically observable adverse events in mice. It is currently unclear whether the Fu1-Fu2 domain can drive some degree of undesired proliferation in both normal and tumor tissues. Systemic administration of high doses of full-length RSPO1 results in a rapid but transient upregulation of WNT signaling in the small intestine, as detected by increased AXIN2 and Ki-67 expression. A response is detectable at 3 hours, peaks at 24 hours, and largely subsides by 48 hours (Kim et al., 2005). These results were not observed to be associated with any adverse clinical consequences. Repeated high doses of endogenous RSPO1 daily can produce a proliferative response in the stem cells of the jejunum (Zhou et al., 2013; Sun et al., 2021), liver (Sun et al., 2021), and skin (Weber et al., 2020), but it is also well tolerated. RSPO1 can promote recovery from enteritis induced by both radiation and chemicals (Zhao et al., 2007; Zhao et al., 2009; Zhou et al., 2013). These results support the view that even if FcF2-MMAE causes an increase in WNT signaling in vivo, when the drug is administered on a weekly schedule, the response may be only transient rather than sustained.

[0187] In the past, it has been difficult to develop high-affinity antibodies that are selective for LGR5 and LGR6. However, some researchers have explored the use of antibody-drug conjugates (ADCs) targeting LGR5 to deplete stem cells in gastrointestinal tumors (Junttila et al., 2015; Gong et al., 2016; Azhdarinia et al., 2018). Although good responses have been observed, the magnitude of LGR5-mediated selectivity is limited.

[0188] This data supports the view that RSPO targeting can have several important advantages over ADCs. ADC development has not progressed, and anti-LGR5 ADCs have not entered clinical trials. Multiple advantages of RSPO targeting may exist, including the following. First, FcF2-MMAE uses a natural ligand that binds with nanomolar affinity and can be rapidly internalized by endocytosis, enabling intracellular delivery of its payload. Second, the cytotoxin-armed Fu1-Fu2 has the potential to target all three LGR family members (LGR4, LGR5, and LGR6) and two ubiquitin ligase receptors, ZNRF3 and RNF43, simultaneously, while an ADC can only target a single LGR at a time. Thus, the cytotoxin-armed Fu1-Fu2 domain has the potential to kill cells that have low expression of one type of LGR or ubiquitin ligase receptor but significant expression of another type of LGR or ubiquitin ligase receptor. Third, the precision of the sortase reaction conjugate MMAE results in a more homogeneous molecular population. Fourth, the bispecific binding of the Fu1-Fu2 domain is beneficial for selectivity as well as increased internalization rate and extent, in terms of the degree to which ZNRF3 / RNF43 is also expressed on CSCs. Fifth, there are some tumors that overexpress ZNRF3 and RNF43 independently of LGR. Since the Fu1 domain binds to these two receptors, it can also target this type of tumor. Sixth, the Fu1-Fu2 domain can engage LGR and ZNRF3 or RNF43 simultaneously. In essence, this is equivalent to a bispecific ADC; this type of ADC is currently of great interest because of their enhanced avidity, specificity, and ability to aggregate receptors and mediate enhanced internalization (Shim, 2020). These results support both Fu1-Fu2 as targeting ligands to deliver cytotoxic payloads to LGR5 / LGR6-expressing cancer cells and also support the use of FcF2-MMAE to treat cancers that may include CSCs. Example 2 Materials and Methods

[0189] The following materials and methods were used in the experiments provided in Example 1.

[0190] Reagents and cell lines: Antibodies were from the following sources: anti-RSPO1, clone OTI11A9, OriGene, Inc; anti-MMAE, clone B11F11, Levena Biopharma; anti-hLGR5 / GPR49 antibody catalog number MAB8078, R&D Systems; PE-conjugated anti-mouse IgG antibody catalog number F0102B, R&D Systems. Ni-NTA resin was purchased from Qiagen, and SP-agarose gel and DEAE resin were from GE healthcare Life Sciences. Propidium iodide was purchased from ThermoFisher (catalog number P3566). All ovarian cancer cell lines were obtained from ATCC or laboratories in the United States; all cell lines were STR-verified at ATCC. The sortase plasmid vector pet30b-7M SrtA was purchased from Addgene. As previously described by Yu et al. 2021, sortase-6×His containing mutations P94R, E105K, E108Q, D160N, D165A, K190E, and K196T was produced in Escherichia coli (E. coli) strain Rosetta and purified using Ni-NTA resin chromatography. (Gly)3-vc-PAB-MMAE was synthesized by Levena Biopharma. The plasma level of FcF2-MMAE was determined using an ELISA kit (DY4645-05) from R&D Systems.

[0191] Synthesis and purification of FcF2-His: FcF2-His was produced by transiently transfecting the pcDNA3.1 vector containing the insert encoding the Fu1-Fu2 domain of RSPO1 into HEK293E cells. The cells were grown in 300 ml of HEK293E medium in a 1 L flask rotating at 130 rpm on a platform. The medium consisted of: 150 mL of Gibco FreeStyle 293 medium (catalog number 12338-026, Thermofisher), 150 mL of HyClone SFM4HEK293 medium (catalog number 82003-356), 6 mL of fetal bovine serum (catalog number 26140-079, ThermoFisher), 333 uL of G418 sulfate (catalog number G8168, Sigma), and 333 uL of anti-coagulant (catalog number 0010057AE, ThermoFisher). After 5 days of culture, the cell supernatant was harvested, centrifuged to precipitate debris and then loaded onto a column containing Ni-NTA resin (catalog number 30250, Qiagen), which was equilibrated with a buffer containing 150 mM NaCl and 20 mM Tris (pH 7.6). After washing, the FcF2-His protein was eluted with a buffer containing 300 mM imidazole, 150 mM NaCl, and 20 mM Tris (pH 7.6). The eluate was diluted 1:3 with 20 mM Tris (pH 7.6) and then loaded onto a DEAE column. The concentration of the flow through of the DEAE column was quantified by reverse phase HPLC analysis using a C4 column.

[0192] Conjugation of FcF2-His with MMAE using sortase: The sortase reaction was carried out at 37 °C for 4 hours with FcF2-His immobilized on SP-agarose gel resin (catalog number 17072901, Cytiva); the reaction mixture contained (Gly)3-vc-PAB-MMAE and FcF2-His at a molar ratio of 20:1, and sortase-6×His and FcF2-His at a molar ratio of 1:4. After washing the SP-agarose gel to remove sortase-6×His and unreacted (Gly)3-vc-PAB-MMAE, the purified FcF2-MMAE was eluted with phosphate buffer containing 1 M NaCl. The FcF2-MAME was diluted to a NaCl concentration of 200 mM, then sterilized with a 0.22 um filter and stored frozen at -80 °C.

[0193] Flow cytometry analysis: After staining with anti-hLGR5 / GPR49 antibody at a final concentration of 12.5 μg / mL in the dark at 25 °C for 30 minutes, the expression of LGR5 in live cells was determined by flow cytometry. Excess anti-hLGR5 / GPR49 antibody was removed by washing with two rounds of PBS, and the cells were incubated with a 1:20 diluted PE-conjugated anti-mouse IgG antibody in the dark at 25 °C for 30 minutes. Excess anti-mouse IgG antibody was removed by washing with two rounds of PBS and the cells were resuspended in 300 μL of PBS containing 0.5 μg / mL propidium iodide. The prepared cell suspension was analyzed on a BD FACSAria II flow cytometer.

[0194] Growth rate inhibition assay: The CCK8 reagent (Dojindo, Inc) was used to determine the effect of FcF2-MMAE on the in vitro cell growth rate. For each drug concentration, cells were seeded in triplicate wells at a density sufficient to yield an OD450 > 1.5 after subtracting the OD determined at the start of drug exposure in the control wells. Survival was calculated as the percentage decrease in the difference between T = 0 and the end of the assay (Hafner et al., 2016). For each concentration of the test drug, all data points represent the mean ± SEM of triplicate cultures.

[0195] Pharmacokinetic study: BALB / c mice were injected IV with FcF2-MMAE at a dose of 0.1 nmol / g and blood was collected from the cheek vein or tail vein at regular intervals into EDTA-coated tubes. Plasma FcF2-MMAE concentrations were determined by ELISA using capture and detection antibodies specific for human RSPO1 that recognize the Fu1 and Fu2 subdomains. The Phoenix WinNonlin version 8.1 (Certara Inc., Princeton, NJ, USA) was used to evaluate the pharmacokinetic parameters.

[0196] Potency study: BALB / c nu / nu mice were obtained from the UCSD breeding colony and inoculated with tumor cells SC, which were harvested from culture and mixed with Matrigel at 2:1 vol / vol before injection of 150 μl of the mixture. The number of cells inoculated varied with tumor type: OVCAR8 / EV and OVCAR8 / LGR5, 2.5×10 6 cells / site; KF-28, 2.5×10 6 cells / site; CAOV3, 5×10 6 cells / site. The formula V=(w 2×L) / 2, and the tumor growth rate was determined from measurements of the cross diameter taken once or twice weekly. Control mice received vehicle (phosphate buffered saline containing 0.02% Tween-20) alone according to the same schedule. Example 3 Containing multiple FuFu regions in a therapeutic compound

[0197] Based on the hypothesis that RSPO1 can be used to selectively target cytotoxic drugs to CSCs, a compound containing two binding domains and armed with the cytotoxin monomethyl auristatin (R1FF-MMAE) was generated. Since the affinity of the ligand can be affected by the number of binding sites, tests were conducted to see if the potency of R1FF-MMAE could be further enhanced by increasing the number of binding domains. A vector capable of expressing a protein containing four modified binding domains was constructed, and the FcST4-His protein was successfully produced in transiently transfected HEK293E cells and purified using nickel and ion exchange resins. After connecting MMAE to the polypeptide using a sortase reaction, the drug (FcST4-MMAE) was characterized using gel electrophoresis, ion exchange, and HPLC analysis on size exclusion columns, Western blotting, and cytotoxicity testing. The results showed that FcST4-MMAE could be purified to high purity and that the compound was significantly stable at 4 °C and in the presence of a low pH of 3.0. Cytotoxicity testing revealed that an increase in the binding domains did not enhance potency, which may be due to an increase in ligand size or a change in the structure mediated by domain-domain interactions, which may interfere with access to the LGR5 / LGR6 receptor.

[0198] The vector construct of the FcST4 molecule was designed to contain an IgG leader sequence at the N-terminus, followed by a mutant Fc domain tandemly linked to two modified receptor-binding domains (ST), with a linker sequence between the two ST domains. A second spacer sequence was inserted exactly upstream of the LPETGG sortase recognition (donor) motif and an 8×His tag was located at the C-terminus. Figure 17 A schematic diagram showing the steps for creating the FcST4 vector using the overlap PCR technique, and the amino acid sequence of FcST4. As Figure 17As shown, the construct contains two LGR binding domains separated by a G4S linker. The nucleotide sequence of FcST4 (SEQ ID NO:103) is provided. Two DNA fragments were generated from the existing FcST2 vector using restriction enzyme double digestion such that both fragments had a common overlap in the linker sequence which would allow the two fragments to anneal. Appropriate primers were first designed to amplify the two fragments separately and then the two fragments were annealed at 70 °C to produce the final product. The final product was ligated into pcDNA3.1. Sanger sequencing using maxiprep was used to confirm that the sequence matched the planned sequence 100% and then the ligated vector was stored at -80 °C for transfection.

[0199] Full-length protein was produced in HEK293e cells. The FcST4.pcDNA3.1 vector was transiently transfected into HEK293e cells. Prior to transfection, HEK293e cells were allowed to grow in Freestyle293 medium for 5 days. On the day of transfection, the cell medium was changed and a mixture containing the FcST4.pcDNA31 vector and PEI 25K (1 mg / mL) was added dropwise to a 150 mL transfection flask and incubated at 130 rpm in 5% CO2 for 4 hours. Four hours after transfection, 150 mL of HySFM293 medium and 30 mL of Freestyle 293 medium were added to the flask to bring the final volume to 300 mL. The cells were allowed to grow. Approximately 24 hours after transfection, valproic acid and a 1:1000 v / v anti-coagulant were added to the flask containing the transfected HEK293E cells. Cells were harvested on day 5. The average cell viability of the 7 batches of FcST4 produced was 79% on the day of harvest.

[0200] After harvest, the cell supernatant was processed through an initial step of purification. The first purification step was based on the ability of the 8×His tag to bind to Ni resin. Particulate matter was removed by high-speed centrifugation of the cell supernatant and then it was loaded onto a gravity flow column packed with pre-loaded Ni-NTA resin. FcST4-His was eluted using 300 mM imidazole and then the protein was passed through a DEAE column and the flow-through was characterized using HPLC reverse phase C4 column and non-reducing SDS / PAGE gel analysis.

[0201] The results of the initial Ni purification step were examined in more detail by collecting and analyzing fractions from successive washing and elution steps with 300 mM imidazole and successive flow-through and washing steps of the DEAE column. The ratio of band sizes did not change with successive elution, indicating that selective purification of the 120 kD FcST4-His dimer could not be achieved with this strategy. The DEAE step successfully removed some of the HMW forms; on average, 28% of the FcST4-His was lost in this step. However, this loss was mainly the result of the successful retention of the HMW forms on the DEAE resin. The use of successive Ni-NTA and DEAE steps produced a reasonably pure form of FcST4, which was then used as the input for a sortase reaction to conjugate MMAE to the molecule. The first step of Ni-NTA purification and the second step of DEAE purification produced a reasonably pure FcST4-His protein, as demonstrated by the presence of a single peak in the HPLC-C4 profile. This protein was successfully captured from the cell supernatant by Protein A resin and was successfully eluted with PBS containing 1 M NaCl, as demonstrated by a band of the correct size on an SDS / PAGE gel.

[0202] After Ni-NTA and DEAE purification, the sortase reaction was used to link FcST4-His to the cytotoxin monomethyl auristatin (MMAE). The sortase cleaves between the threonine and glycine in the LPETGG tag, removing the GG-8×His tag. The sortase reaction was carried out with the precursor FcST4-His loaded on the cation exchange resin SP-Sepharose gel. This allowed the excess G3-val-cit-PAB-MMAE and the sortase to be washed away before eluting the SP-Sepharose gel with 1 M NaCl. The sortase reaction efficiency for all batches of FcST4-His was 100 ± 37% (SD). However, after the sortase reaction, the average yield percentage of the final product was 90 ± 22% (SD).

[0203] To evaluate the stability of the FcST4-MMAE molecule, the samples were subjected to multiple freeze-thaw cycles. Even after 4 cycles, the SEC130 profile of the FcST4-MMAE molecule did not change significantly.

[0204] After generating 7 batches of FcST4-MMAE, batches 2 to 6 were used for cytotoxicity analysis. HEK293e suspension cells were grown and treated with the drug at a series of concentrations starting from 0 mM to 400 mM in a 96-well plate. On day 6, CCK8 reagent was added and the OD450 of the experimental plate was read. Consistently, for FcST4-MMAE batches 2 to 6, although a difference in targeted killing was observed between HEK293e empty vector cells and cells overexpressing the LGR5 receptor, the difference was not statistically significant. The cytotoxicity assay of FcST4-MMAE was also performed using the OVCAR8 cell line. Drug concentrations starting from 400 nM to 0 mM were tested. Similar to the results in HEK293e cells, although a difference in targeted killing was observed between OVCAR8 cells expressing only the empty vector and cells engineered to overexpress the LGR5 receptor, the difference was not statistically significant. The IC50 values ranged from 1.9 to 3.8 nM. The potency of FcST2-MMAE against 8 human ovarian cancer cell lines was tested, and the results are as Figure 18 shown. FcST2-MMAE was significantly effective in killing cells with IC 50 values ranging from 3.8 to 29 nM, and the IC 50 of all cell lines except one was less than 20 nM. Example 4 FcF2-MMAE without linker

[0205] The FcF2-MMAE compound was prepared as described in the above example, and the modification was that the polypeptide did not contain a G4S linker. A schematic diagram of the linker-free FcF2 polypeptide portion (FcF2Δlinker-His) of the compound is shown in Figure 19 shown.

[0206] The molecular weight of the resulting monomer decreased from 42,642 to 41,363, a decrease of 1,279, and the size decreased by ~3%. ExpiCHO cells were used to produce the FcF2Δlinker-His polypeptide. Ni-NTA and DEAE purification steps were performed. Based on SDS page experiments and HPLC reversed-phase C4 spectra, the production and purification of the FcF2Δlinker-His polypeptide were consistent with the yield and purity of the FcF2-His polypeptide. Sortase described in the above example was used to covalently link MMAE to FcF2Δlinker.

[0207] Cytotoxicity experiments were performed on OVCAR8 cancer cells (OVCAR8 / EV) and OVCAR8 cancer cells overexpressing the LGR5 receptor (OVCAR8 / LGR5) using FcF2-MMAE and FcF2Δ linker-MMAE. Both FcF2-His and FcF2Δ linker-His from which MMAE was formed were produced in ExpiCHO. MMAE was conjugated using the above sortase reaction. As Figure 20 shown, both FcF2-MMAE and FcF2Δ linker-MMAE led to killing of cancer cells, and increased cancer cell death was observed in cancer cells overexpressing the LGR5 receptor (OVCAR8 / LGR5). Overall, no significant differences in IC 50 values or degree of selectivity were observed. FcF2Δ linker-MMAE was shown to be less potent against both LGR5-deficient and LGR5-rich cells. These results suggest that therapeutic cancer killing can be observed in the FcF2-MMAE construct in which the polypeptide portion of the therapeutic molecule does not contain a linker. Example 5 In vivo potency of FcF2-MMAE

[0208] The potency of FcF2-MMAE was tested in an additional human tumor xenograft model in which tumors grew subcutaneously from a previously established tumor cell line. The LoVo cell line is a colorectal line containing a KRAS G13D mutation and having a high level of WNT signaling. LoVo cells were SC inoculated in nu / nu mice; there were a total of 20 tumors per group. Administration of FcF2-MMAE was initiated on the first day any tumor became detectable. The control group received only saline; the experimental group received FcF2-MMAE injected IP q7dx4 at a dose of 1 nmol / g body weight and was then discontinued from further administration. As Figure 22 shown, FcF2-MMAE delayed the onset of tumor growth and slowed the growth rate after tumor establishment relative to tumors in the untreated control group. There was no significant weight loss in either the control or experimental group mice. These results suggest that FcF2-MMAE is active in this model. The results are shown in Figure 21 and Figure 22 .

[0209] The efficacy of FcF2-MMAE was further tested using tumors grown from the AGS human gastric cancer cell line. This cell line was chosen for testing for two reasons: it has a high level of WNT pathway signaling and it represents a tumor type with unmet medical need. AGS cells were inoculated subcutaneously (SC) in nu / nu mice; there were a total of 20 tumors per group. Administration of FcF2-MMAE was initiated on the first day any tumor became detectable. The control group received only saline; the experimental group received FcF2-MMAE injected IP q7dx4 at a dose of 1 nmol / g body weight and was then discontinued from further administration. As Figure 23 shown, in the experimental group, FcF2-MMAE significantly inhibited the outgrowth of tumors. Of the 20 inoculations in the control group, tumors developed in 11; of the 20 inoculations in the experimental group, tumors did not develop in any (p < 0.0037 chi-square). By 160 days after inoculation, no tumors were present in the experimental group. There was no significant weight loss in either the control or experimental group mice. These results indicate that FcF2-MMAE was curative in these mice that received it. The results are shown in Figure 23 ...

[0210] The efficacy of FcF2-MMAE was also tested using tumors grown from the SKNAS neuroblastoma cell line. This cell line was chosen for testing for two reasons: it has a high level of WNT pathway signaling and it is a very aggressive tumor. SKNAS cells were inoculated SC in nu / nu mice; there were a total of 20 tumors per group. Administration of FcF2-MMAE was initiated on the first day any tumor became detectable (day 7 after inoculation). The control group received only saline; the experimental group received FcF2-MMAE injected IP q7dx4 at a dose of 1 nmol / g body weight and was then discontinued from further administration. As Figure 24 shown, FcF2-MMAE increased the time to first detection and increased the time until explosive tumor growth began from 15 days to 38 days. There was no significant weight loss in either the control or experimental group mice. These results indicate that FcF2-MMAE was highly active in this model. The results are shown in Figure 24 ... Example 6 Conjugation of FcF2-His with Cytotoxins

[0211] As shown in this example, FcF2-His can be conjugated to a variety of cytotoxins. Although monomethyl auristatin (MMAE) is a very potent cytotoxin, even more potent cytotoxins can be used in some antibody-drug conjugates. To test and demonstrate the versatility of FcF2 as a targeting moiety, sortase A was used to conjugate two additional classes of warheads to FcF2-His. The same reaction conditions of sortase A used for conjugating (Gly)3-val / cit-PAB-MMAE were used for conjugating PNU159682 and deruxtecan.

[0212] Figure 25A shows the SDS-PAGE analysis of FcF2-His conjugated to PNU159682, an anthracycline derivative. As detected by SDS-PAGE, conjugation to this molecule does not alter the structural characteristics of FcF2-His. FcF2-PNU159682 was tested against OVCAR8 / EV cells lacking LGR5 and OVCAR8 / LGR5 cells expressing approximately 10-fold higher levels of the LGR5 receptor. The IC 50 for OVCAR8 / LGR5 cells was 300 pM, while the IC 50 for OVCAR8 / EV cells was >3,000 pM, thus resulting in an IC 50 ratio of ~10 (Figure 25B). Figure 25C shows the structure of PNU-159682.

[0213] As the cytotoxin contained in the antibody-drug conjugate Fam-trastuzumab deruxtecan-nxki, deruxtecan has shown very high potency in breast cancer patients. To further demonstrate the feasibility of conjugating FcF2-His to other types of warheads, sortase A reaction was used to conjugate deruxtecan to FcF2-His. The first step was to construct a linker for deruxtecan, which would serve as the substrate for the sortase A reaction. To do this, conditions were developed for the peptide GGGC to react with commercially available maleimide-GGFG-deruxtecan to produce the GGGC-MA-GGFG-deruxtecan linker. This required optimizing the conditions such that the linker remained soluble in aqueous buffer. Figure 26A shows a schematic of the reaction, and Figure 26B shows the reverse-phase HPLC analysis of the GGGC-MA-GGFG-deruxtecan linker documenting its purity.

[0214] The sortase reaction was used to conjugate the GGGC-MA-GGFG-drutac linker to FcF2-LEPTGG-His. Figure 27A shows the reverse-phase HPLC (C4 column) analysis of FcF2-drutac, documenting its purity and the absence of unreacted substrates. The standard CCK8 cytotoxicity assay and 120-hour exposure were used to determine the ability of FcF2-drutac to inhibit the growth of OVCAR8 / EV cells lacking LGR5 versus OVCAR8 / LGR5 cells enriched in LGR5. As shown in Figure 27B, the assay demonstrated that FcF2-drutac was very potent against OVCAR8 / LGR5 and OVCAR8 / EV cells, with IC 50 values of 0.7532 and 9.045 nM. Most importantly, FcF2-drutac retained a 12-fold selectivity advantage against LGR5-enriched cells.

[0215] Collectively, these results indicate that FcF2-His can be successfully conjugated to a wide variety of different types of cytotoxic agents without significant structural damage or loss of selectivity for the LGR5 stem cell receptor. Example 7 Mutant forms of FcF2-MMAE with extended plasma half-life

[0216] All protein therapeutics are gradually degraded by plasma proteases and cellular clearance mechanisms during their circulation in the bloodstream. Western blot analysis of FcF2-MMAE incubated in human plasma at 37 °C identified low molecular weight fragments, indicating that the first part of the Fub1 domain of FcF2-MMAE was cleaved. This is the region that early studies indicated was cleaved by enterokinase. Vectors expressing mutant forms of FcF2-LPETGG-His containing 4 short deletions in this region were prepared, and their proteins were produced and tested for their degradation rates in human plasma. This effort highlighted sub-regions of particular interest. A series of additional FcF2-LPETGG-His vectors containing single alanine substitution mutants were constructed, and their protein products were produced and tested. This screen identified mutations R28A and R30A as particularly interesting, but several other single point mutations also slowed plasma degradation.

[0217] Generation of the FcF2(R28A) mutant using the nucleotide sequence:

[0218] The FcF2(R28A) mutant as an amino acid sequence:

[0219] The Fu1-Fu2(R28A) mutant has the amino acid sequence:

[0220] Generate the FcF2(R30A) mutant using the nucleotide sequence:

[0221] The FcF2(R30A) mutant has the amino acid sequence:

[0222] The Fu1-Fu2(R30A) mutant has the nucleotide sequence:

[0223] Generate variants of the FcF2 construct using the Fu1-Fu2 region containing additional deletion mutations as follows. The following additional deletion mutants of the Fu1-Fu2 region were generated: R22 to R31 (RSPO1 nucleotides 64 to 93), K25 to R31 (RSPO1 nucleotides 73 to 93), R28 to R31 (RSPO1 nucleotides 82 to 93), and R22 to K27 (RSPO1 nucleotides 64 to 81). These deletion mutants can be used to bind to LGR in a manner similar to other Fu1-Fu2 regions. The deletion mutants have the following sequences: Fu1-Fu2 (R22 to R31 deletion) Fu1-Fu2 (K25 to R31 deletion) Fu1-Fu2 (R28 to R31 deletion) Fu1-Fu2 (R22 to K27 deletion) and Fu1-Fu2 (S21 to Q38 deletion) These deletion mutants can lead to an increase in plasma half-life.

[0224] Test FcF2-MMAE using OVCAR8 / EV cells versus OVCAR8 / LGR5 cells (WT), potencies and selectivities of FcF2-R28A-MMAE and FcF2-R30A-MMAE. Figure 28 Data showing concentration-survival curves were presented, and it was observed that neither the R28A nor the R30A mutation altered the selective potency against LGR5-rich cells.

[0225] Pharmacokinetic studies were then conducted in BALB / c mice. Mice were given intravenous (IV) injections of FcF2-MMAE (WT), FcF2-R28A-MMAE, or FcF2-R30A-MMAE, and the plasma concentration of each was determined in timed samples by ELISA using capture and detection antibodies against different parts of the molecule. Figure 29 It was shown that the R28A, but not the R30A, mutation prolonged the initial half-life of the molecule and increased the AUC 0-120 by 3.4-fold. Of particular importance, during the early phase of the plasma decay curve, when drug penetration into tumors is likely to occur at the highest rate, the concentration of FcF2-R28A-MMAE was two (>2) orders of magnitude higher than the concentration of FcF2-MAME. These results suggest that the R28A mutation can improve the half-life of FcF2 constructs containing cytotoxic constructs.

[0226] The plasma half-life of the FcF2-MMAE construct was compared with the plasma half-life of the R1FF-MMAE (“RSPO1-MMAE”) construct described by Yu et al. (2021) using BALB / C female mice. It was observed that the in vivo distribution half-life of FcF2-MMAE was approximately 15-fold longer than the in vivo distribution half-life of R1FF-MMAE. The results are shown in Figure 34 below. Example 8 Improving MMAE loaded onto FcF2-His using sortase E

[0227] Some studies using antibody-drug conjugates support the view that, in some cases, increasing the number of cytotoxins per antibody molecule (DAR) can improve potency and efficacy. In the case of FcF2-LPETGG-His, the sortase reaction can place the MMAE warhead at both ends of the molecule. The major challenge is that the sortase A reaction is reversible, and thus during the process of adding a second MMAE to the N-terminus, it may remove the MMAE already loaded on the C-terminus. The inventors then worked on developing another method to link additional cytotoxic moieties to FcF2.

[0228] Figure 30A schematic diagram of the method is shown. The recently isolated sortase E ligates a substrate containing an N-terminal GG motif to the sequence LAHTGG (SEQ ID NO:106) on the C-terminus of other proteins or peptides. Since sortase A and sortase E have a high specificity for different recognition sequences (LPETGGG versus LAHTGG), it is possible to operate together to load MMAE onto both ends of the FcF2 molecular variant, which contains diglycine at the N-terminus and the LPETGG sequence at the C-terminus.

[0229] Construct a vector expressing GG-FcF2-LEPTGG-His, produce and purify the vector in HEK293 cells; tests show that when using sortase A to load MMAE, adding two glycines at the N-terminus does not change the potency or selectivity.

[0230] Construct a gene containing the sortase E sequence, clone the gene into a bacterial expression vector, synthesize the protein in the Rosetta strain of Escherichia coli, and purify it using Ni-NTA chromatography. To facilitate the testing of sortase E, a form of the FcF2-LPETGG-His vector was constructed in which the LPETGG sequence was replaced with the sortase E recognition sequence LAHTGG (SEQ ID NO:106). This protein was produced and purified from HEK293 cells. As a test substrate for the sortase E reaction, the inventors prepared a linker consisting of the peptide GGGC conjugated to the maleimide-containing fluorescent molecule Dye650. Figure 31 It is shown that sortase E is able to load GGGC-MA-Dye650 onto FcF2-LAHTGG-His, thus determining that both sortase A and sortase E can be used to load substrates with N-terminal glycine onto FcF2 containing the appropriate sortase recognition sequence. Repeating these experiments will further support and, if necessary, can be used for statistical analysis of these results. Example 9 Improve MMAE loaded onto FcF2-His using partial reduction of disulfide bonds

[0231] The FcF2-LEPTGG-His molecule contains 3 disulfide bonds in the immunoglobulin Fc domain and 8 disulfide bonds in the Fu1-Fu2 domain. Antibody-drug conjugates are typically loaded with cytotoxins by partially reducing their Fc domain disulfide bonds and then reacting them with a linker containing a thiol-reactive maleimide group pre-conjugated to the cytotoxin. This allows 2 to 8 or more cytotoxins to be loaded per molecule, but it must be individualized because overloading will distort the protein structure and reduce the plasma half-life. To determine whether this same method could be used to load MMAE onto FcF2-His without using a sortase reaction, a series of experiments were conducted to evaluate the loading as a function of TCEP concentration.

[0232] Aliquots of FcF2-His were exposed to TCEP at concentrations ranging from 0.0005 to 5000 μM, and after 25 minutes, maleimide-val / cit-PAB-MMAE (MA-MMAE) was added to the reaction at a ratio of 1:2 or 1:8 (protein:MMAE). The reaction was then allowed to proceed overnight. Figure 32 Western blot analysis of these samples probed with anti-RSPO1 and anti-MMAE is shown. In the absence of MA-MMAE, no MMAE signal was detected; however, an increase in TCEP concentration led to a gradually large amount of MMAE being loaded onto FcF2-His. A higher protein:MA-MMAE ratio led to higher loading. TCEP at a concentration of 5000 μM reduced all disulfide bonds, so only FcF2-His monomers were visible, but a very large amount of MMAE was loaded when all disulfide bonds were reduced.

[0233] This data indicates that the method of partial disulfide bond reduction can be used to load MMAE onto FcF2-His. Additional studies can be conducted to measure the number of MMAE molecules loaded, and how potency, selectivity, and plasma half-life vary with loading. Increasing the loading of the cytotoxic moiety can lead to increased potency or enhanced killing of the construct due to the presence of additional molecules of the cytotoxic moiety (e.g., MMAE). As shown above, covalent bonding of additional cytotoxic moieties to the construct (e.g., at the N- and C-termini of FcF2) can be covalently linked by a variety of techniques, including partial reduction of disulfide bonds and covalent linkage using sortases (e.g., sortase A and sortase E). ***

[0234] According to the present disclosure, all of the methods disclosed and claimed herein can be made and implemented without undue experimentation. Although the compositions and methods of the present invention have been described in terms of some preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods described herein, as well as in the steps or the order of the steps of the methods, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein, while the same or similar results would be achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. 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Claims

1. A compound comprising one or more cytotoxic agents conjugated to a polypeptide comprising one or more LGR-binding domains, wherein (i) the polypeptide further comprises an Fc region, and / or (ii) the polypeptide comprises at least two copies of the LGR-binding domain; and wherein each LGR-binding domain comprises a polypeptide having at least 90%, more preferably at least 95% sequence identity to at least one of SEQ ID NO:4, 78 to 83, 85 to 89, 90 to 96, 98 or 102.

2. The compound of claim 1, wherein each LGR-binding domain independently comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:

87.

3. The compound of claim 1, wherein the LGR-binding domain is from human R-spondin-1 (hR-spondin-1), human R-spondin-2 (hR-spondin-2), human R-spondin-3 (hR-spondin-3), or human R-spondin-4 (hR-spondin-4).

4. The compound of claim 1, wherein the LGR-binding domain independently comprises an amino acid sequence selected from FuFu (SEQ ID NO:4) or FuFu N137A (SEQ ID NO:17).

5. The compound of claim 1, wherein the LGR-binding domain comprises a substitution mutation at position R28 or R30, numbering according to Kabat.

6. The compound of claim 5, wherein the substitution mutation is a substitution of arginine with alanine.

7. The compound of claim 6, wherein the substitution mutation is R28A.

8. The compound of claim 7, wherein the LGR-binding domain comprises the Fu1-Fu2(R30A) mutant (SEQ ID NO:91) or the Fu1-Fu2(R30A) mutant (SEQ ID NO:100).

9. The compound of claim 7, wherein the LGR-binding domain comprises the Fu1-Fu2(R30A) mutant (SEQ ID NO:100).

10. The compound of claim 1, wherein the LGR-binding domain comprises Fu1-Fu2(R22 to R31 deletion) (SEQ ID NO:92), Fu1-Fu2(K25 to R31 deletion) (SEQ ID NO:93), Fu1-Fu2(R28 to R31 deletion) (SEQ ID NO:94), or Fu1-Fu2(R22 to K27 deletion) (SEQ ID NO:95).

11. The compound of claim 1, wherein the polypeptide comprises FcST4 (SEQ ID NO:105).

12. The compound according to claim 1, wherein the Fc region is located at the N-terminus relative to the LGR binding domain, and / or wherein the polypeptide comprises, in the N-to-C direction: the Fc region and the LGR binding domain.

13. The compound according to any one of claims 1 to 12, wherein the Fc region is an IgG Fc domain.

14. The compound according to claim 13, wherein the polypeptide comprises SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:

12.

15. The compound according to claim 1, wherein the polypeptide comprises SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, or SEQ ID NO:88; and wherein the polypeptide does not comprise SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, or SEQ ID NO:

89.

16. The compound according to claim 1, wherein the polypeptide comprises SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, or SEQ ID NO:89; and wherein the polypeptide does not comprise SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, or SEQ ID NO:

88.

17. The compound according to claim 12, wherein the human IgG Fc domain is capable of binding to human FcRn at acidic pH, and wherein the Fc domain has the following substitution mutations: (i) an aspartic acid substitution mutation at position 309 (L / V309D); (ii) a histidine substitution mutation at position 311 (Q311H); and (iii) a serine substitution mutation at position 434 (N434S) or a tyrosine substitution mutation at position 434 (N434Y); wherein the amino acid position numbering is according to the Kabat system; and wherein the Fc domain binds to FcRn with a higher affinity than the wild type at acidic pH.

18. The compound according to claim 17, wherein the substitution mutation at position 434 is serine (N434S).

19. The compound according to claim 17, wherein the substitution mutation at position 434 is tyrosine (N434Y).

20. The compound according to any one of claims 17 to 19, wherein the polypeptide comprises SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:

16.

21. The compound according to claim 20, wherein the polypeptide comprises SEQ ID NO:

13.

22. The compound according to any one of claims 17 to 21, wherein the Fc domain is glycosylated.

23. The compound according to claim 22, wherein the Fc domain has substantially the same binding affinity for FcγR as the wild type Fc domain.

24. The compound according to claim 22, wherein the Fc domain has the same or substantially the same binding affinity for one, two, or all of FcγRI, FcγRII, and FcγRIII as compared to the wild type.

25. The compound according to claim 17, wherein the Fc domain does not bind detectably or selectively to FcRn at neutral pH, and / or does not exhibit or exhibits substantially no binding to FcRn at neutral pH.

26. The compound according to claim 25, wherein the Fc domain exhibits: (i) enhanced binding to FcRn at pH 5.8, and (ii) reduced binding to FcRn or undetectable binding to FcRn at pH 7.4 as compared to the wild type.

27. The compound according to any one of claims 17 to 21, wherein the Fc domain is aglycosylated.

28. The compound according to claim 27, wherein the Fc domain has a substitution mutation of glutamic acid at position 264 (V264E).

29. The compound according to any one of claims 17 to 28, wherein the IgG is IgG1, IgG2, IgG3, or IgG4.

30. The compound according to any one of claims 17 to 28, wherein the IgG is IgG1.

31. The compound according to any one of claims 17 to 28, wherein the Fc domain comprises the following substitution mutations: (i) IgG1-Fc EDHS (V264E; L309D; Q311H; N434S), (ii) IgG1-Fc EDHY (V264E; L309D; Q311H; N434Y), (iii) IgG1-Fc DHS (L309D; Q311H; N434S), (iv) IgG1-Fc DHY (L309D; Q311H; N434Y), (v) IgG2-DHS (V309D; Q311H; N434S), (vi) IgG3-DHS (L309D; Q311H; N434S), or (vii) IgG4-DHS (L309D; Q311H; N434S).

32. The compound according to claim 31, wherein the Fc domain is IgG1-Fc DHS (L309D; Q311H; N434S).

33. The compound according to any one of claims 12 to 32, wherein the compound dimerizes through a disulfide bond formed in the Fc domain.

34. The compound according to any one of claims 12 to 33, wherein the Fc domain is separated from the LGR binding domain by a linker.

35. The compound according to claim 34, wherein the linker comprises G4S (SEQ ID NO:18) or (G4S)2 (SEQ ID NO:5).

36. The compound according to any one of claims 12 to 33, wherein the Fc domain is not separated from the LGR binding domain by a linker, or wherein the polypeptide does not comprise a linker.

37. The compound according to any one of claims 34 to 36, wherein the polypeptide comprises, from the N-terminus to the C-terminus: the Fc domain and the LGR binding domain; or wherein the Fc domain is closer to the N-terminus of the polypeptide than the LGR binding domain.

38. The compound according to any one of claims 1 to 37, wherein the compound comprises two copies of FuFu (SEQ ID NO:4) or FuFu N137A (SEQ ID NO:17).

39. The compound according to claim 38, wherein the two copies of FuFu (SEQ ID NO:4) or FuFu N137A (SEQ ID NO:17) are separated by a linker, preferably a G4S linker (SEQ ID NO:18) or a (G4S)2 linker (SEQ ID NO:5).

40. The compound according to claim 39, wherein the compound comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

19.

41. The compound according to any one of claims 1 to 40, wherein the compound comprises SEQ ID NO:

1.

42. The compound according to claim 1, wherein the compound comprises SEQ ID NO:

20.

43. The compound according to any one of claims 1 to 42, wherein the polypeptide comprises a leader sequence.

44. The compound according to claim 43, wherein the leader sequence is an endogenous leader sequence, an IgG leader sequence, or an IgK leader sequence.

45. The compound according to claim 44, wherein the IgG leader sequence is an IgGk leader sequence (SEQ ID NO:8).

46. The compound according to any one of claims 1 to 42, wherein the polypeptide does not comprise a leader sequence.

47. The compound according to any one of claims 1 to 46, wherein the first cytotoxic agent is a conjugated drug.

48. The compound according to claim 47, wherein the drug is maytansine alkaloid, auristatin, amanitin, calicheamicin, psymberin, duocarmycin, anthracycline, camptothecin, doxorubicin, paclitaxel, tubulin, eribulin, or pyrrolobenzodiazepine 49. The compound according to claim 48, wherein the drug is an auristatin.

50. The compound according to claim 49, wherein the auristatin is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or PF-06380101.

51. The compound according to claim 49, wherein the auristatin is monomethyl auristatin E (MMAE).

52. The compound according to claim 47, wherein the drug is a camptothecin analogue.

53. The compound according to claim 52, wherein the camptothecin analogue is topotecan, irinotecan, belotecan, or derotecan.

54. The compound according to claim 47, wherein the drug is an anthracycline analogue.

55. The compound according to claim 54, wherein the anthracycline analogue is PNU-159682 (CAS No.: 202350-68-3).

56. The compound according to any one of claims 47 to 51, wherein the conjugated drug is linked to the polypeptide through a linker.

57. The compound according to claim 56, wherein the linker is a protease-cleavable linker.

58. The compound according to claim 57, wherein the protease-cleavable linker is citrulline-valine.

59. The compound according to any one of claims 1 to 58, wherein the compound comprises at least one spacer located between the cytotoxic agent and the LGR-binding domain.

60. The compound according to claim 59, wherein the compound comprises two linkers.

61. The compound according to any one of claims 59 to 60, wherein the linker comprises G4S (SEQ ID NO:18) or (G4S)2 (SEQ ID NO:5).

62. The compound according to any one of claims 1 to 41, wherein the cytotoxic moiety is a cytotoxic protein.

63. The compound according to claim 62, wherein the cytotoxic protein is a serine protease.

64. The compound according to claim 63, wherein the serine protease is granzyme B (GrB).

65. The compound according to claim 1, wherein the compound comprises SEQ ID NO:1 covalently linked to monomethyl auristatin E (MMAE).

66. The compound according to claim 1, wherein the compound comprises SEQ ID NO:2 covalently linked to monomethyl auristatin E (MMAE).

67. The compound according to any one of claims 12 to 66, wherein the compound dimerizes through a disulfide bond in the Fc domain.

68. The compound according to any one of claims 1 to 67, wherein the cytotoxic agent has been covalently bound to the polypeptide through a sortase linker.

69. The compound according to claim 68, wherein the sortase is a sortase A linker or a sortase E linker.

70. The compound according to any one of claims 1 to 67, wherein the compound comprises a sortase linker between the LGR-binding domain and the cytotoxic agent.

71. The compound according to claim 70, wherein the cytotoxic agent has been covalently bound to the polypeptide through a sortase.

72. The compound according to claim 71, wherein the sortase is sortase A or sortase E. The compound according to claim 70, wherein the sortase linker comprises the sequence LPXT(G) n or LAHTGG (SEQ ID NO: 106), wherein n = 1 to 10.

74. The compound according to claim 73, wherein the sortase linker is LPETGG (SEQ ID NO:6).

75. The compound according to any one of claims 1 to 74, wherein the compound further comprises a second cytotoxic agent.

76. The compound according to claim 75, wherein the first cytotoxic agent and the second cytotoxic agent are each independently selected from the cytotoxic agents described in claims 47 to 64.

77. A compound according to any one of claims 75 to 76, wherein the first cytotoxic agent and the second cytotoxic agent have been covalently bound to the polypeptide via a sortase linker.

78. The compound according to claim 77, wherein the first cytotoxic agent is covalently linked to a first sortase linker on the N-terminal side of the polypeptide, and wherein the second cytotoxic agent is covalently linked to a second sortase linker on the N-terminal side of the polypeptide.

79. The compound according to claim 78, wherein the first sortase linker comprises the sequence LPXT(G)n, where n = 1 to 10.

80. The compound according to claim 79, wherein the first sortase linker is LPETGG (SEQ ID NO:6).

81. The compound according to any one of claims 78 to 80, wherein the second sortase linker is LAHTGG (SEQ ID NO:106).

82. The compound according to any one of claims 78 to 80, wherein the first cytotoxic moiety has been covalently bound to the first sortase linker using sortase A, and wherein the second cytotoxic moiety has been covalently bound to the second sortase linker using sortase E.

83. The compound according to claim 82, wherein the first cytotoxic moiety and the second cytotoxic moiety are each independently a conjugated drug according to any one of claims 48 to 55 or a cytotoxic protein according to any one of claims 62 to 64.

84. The compound according to claim 83, wherein the first cytotoxic moiety and the second cytotoxic moiety are different conjugated drugs.

85. The compound according to claim 83, wherein the first cytotoxic moiety and the second cytotoxic moiety have the same structure.

86. The compound according to claim 85, wherein both the first cytotoxic moiety and the second cytotoxic moiety are monomethyl auristatin E (MMAE).

87. A compound according to any one of claims 75 to 76, wherein the first cytotoxic agent or the second cytotoxic agent is linked to the polypeptide via a disulfide bond, preferably wherein the disulfide bond is present in the Fc region or the LGR binding domain.

88. The compound according to claim 87, wherein the disulfide bond is included in a maleimide group.

89. The compound according to claim 88, wherein the maleimide group is covalently bound to a cleavable linker.

90. The compound according to claim 89, wherein the cleavable linker comprises a valine (Val)-citrulline (Cit) bond.

91. The compound according to claim 87, wherein the first cytotoxic agent is covalently bound to the polypeptide to a sortase linker, the sortase linker comprising the sequence LPXT(G) n or LAHTGG (SEQ ID NO: 106), where n = 1 to 10; and wherein the second cytotoxic agent has been linked to the polypeptide via a disulfide bond.

92. The compound according to any one of claims 87 to 91, wherein the disulfide bond is present in the Fc region.

93. The compound according to any one of claims 87 to 91, wherein the disulfide bond is present in the LGR binding domain.

94. The compound according to any one of claims 1 to 74, wherein the polypeptide comprises SEQ ID NO:

76.

95. The compound according to claim 94, wherein the polypeptide comprises SEQ ID NO:

77.

96. The compound according to claim 95, wherein the polypeptide is covalently linked to -PABA-MMAE.

97. The compound according to any one of claims 1 to 96, wherein the compound is comprised in a pharmaceutical composition.

98. A pharmaceutical composition comprising the compound according to any one of claims 1 to 97.

99. The pharmaceutical composition according to claim 98, wherein the pharmaceutical composition is formulated for intravenous, intraperitoneal, subcutaneous, intratumoral, intrathecal, inhalation, intraarterial, or intrapleural administration.

100. A nucleic acid encoding the polypeptide according to any one of claims 1 to 96.

101. A host cell comprising the nucleic acid according to claim 100.

102. The host cell according to claim 101, wherein the cell is a bacterial cell.

103. The host cell according to claim 101, wherein the cell is a eukaryotic cell.

104. The host cell according to claim 101, wherein the cell is a eukaryotic cell, a human cell, an insect cell, or a yeast cell.

105. The host cell according to claim 104, wherein the human cell is a HEK293 cell, a Chinese hamster ovary (CHO) cell, or a variant thereof.

106. A method of producing a therapeutic compound that binds to an LGR receptor, wherein the method comprises: (a) expressing in a cell a polypeptide encoded by the nucleic acid according to claim 100, wherein the polypeptide comprises a sortase linker at the terminus of the polypeptide; (b) obtaining the polypeptide; and (c) contacting a first cytotoxic agent and the polypeptide with a first transpeptidase, thereby covalently conjugating the first cytotoxic compound to the polypeptide.

107. The method according to claim 106, wherein the cell is a bacterial cell.

108. The method according to claim 106, wherein the cell is a mammalian cell or an insect cell.

109. The method according to claim 108, wherein the mammalian cell is a HEK293 cell, a Chinese hamster ovary (CHO) cell, or a variant thereof.

110. The method according to any one of claims 106 to 109, wherein the first transpeptidase is sortase A or sortase E.

111. The method according to any one of claims 106 to 110, wherein prior to step (c), the cytotoxic moiety comprises a C-terminal sortase donor sequence and the polypeptide comprises an N-terminal sortase acceptor sequence.

112. The method according to claim 111, wherein the C-terminal sortase donor sequence is LPXT(G) n , where n = 1 to 10.

113. The method according to claim 112, wherein the C-terminal sortase donor sequence is LPETGG (SEQ ID NO:6). The method according to claim 113, wherein the sortase linker further comprises –(His) n –, where n = 1 to 10.

115. The method according to claim 113, wherein the N-terminal sortase acceptor sequence comprises 1 to 10 glycine residues.

116. The method according to claim 115, wherein the N-terminal sortase acceptor sequence is GGG.

117. The method according to any one of claims 106 to 116, wherein the cytotoxic agent is a conjugated drug.

118. The method according to claim 116, wherein the drug is maytansine alkaloid, auristatin, amanitin, calicheamicin, psymberin, duocarmycin, anthracycline, camptothecin, doxorubicin, paclitaxel, tubulin lysine, eribulin, or pyrrolobenzodiazepine 119. The method according to claim 118, wherein the drug is auristatin.

120. The method according to claim 119, wherein the auristatin is monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), or PF-06380101.

121. The method according to claim 119, wherein the auristatin is monomethyl auristatin E (MMAE).

122. The method according to claim 116, wherein the drug is a camptothecin analogue.

123. The method according to claim 122, wherein the camptothecin analogue is topotecan, irinotecan, belotecan, or derotecan.

124. The method according to claim 116, wherein the drug is an anthracycline analogue.

125. The method according to claim 124, wherein the anthracycline analogue is PNU-159682 (CAS No.: 202350-68-3).

126. The method according to any one of claims 106 to 125, wherein the LGR receptor is LGR4, LGR5, or LGR6.

127. The method according to any one of claims 106 to 116, wherein before step (c), the cytotoxic moiety comprises an N-terminal sortase donor sequence, and the polypeptide comprises a C-terminal sortase acceptor sequence.

128. The method according to claim 111, wherein the C-terminal sortase donor sequence is LAHTGG (SEQ ID NO: 106).

129. The method according to claim 113, wherein the N-terminal sortase acceptor sequence comprises 1 to 10 glycine residues.

130. The method according to claim 115, wherein the N-terminal sortase acceptor sequence is GG or GGG.

131. The method according to any one of claims 106 to 116, wherein the cytotoxic agent is a conjugated drug.

132. The method according to claim 131, wherein the conjugated drug is the conjugated drug according to any one of claims 47 to 55 or 62 to 64.

133. The method according to claim 132, wherein the conjugated drug is monomethyl auristatin E (MMAE), PNU-159682, topotecan, irinotecan, belotecan, or derotecan.

134. The method according to any one of claims 106 to 133, wherein the method further comprises (d) covalently binding a second cytotoxic compound to the polypeptide by any one of the following: (i) contacting the second cytotoxic agent and the polypeptide with a second transpeptidase, or (ii) covalently binding the second cytotoxic agent to the polypeptide by forming a disulfide bond through a partial disulfide bond reaction.

135. The method according to claim 134, wherein the partial disulfide bond reaction bonds the second cytotoxic agent to the Fc region or the LGR binding domain.

136. The method according to claim 135, wherein the second cytotoxic compound comprises a linker, and wherein the partial disulfide bond reaction binds the linker to the polypeptide.

137. The method according to claim 136, wherein the linker comprises a thiol-reactive maleimide group.

138. The method according to claim 137, wherein the linker further comprises a cleavable bond.

139. The method according to claim 138, wherein the cleavable bond comprises a valine (Val)-citrulline (Cit) bond.

140. The method according to claim 134, wherein the second transpeptidase is sortase A or sortase E.

141. The method according to claim 140, wherein the first transpeptidase is sortase A and the second transpeptidase is sortase E.

142. The method according to any one of claims 134 to 141, wherein the second cytotoxic agent is the cytotoxic agent according to any one of claims 47 to 55 or 62 to 64, preferably MMAE.

143. A method for producing a polypeptide, comprising: (a) expressing the nucleic acid according to claim 100 in a cell under conditions for producing the encoded polypeptide; and (b) purifying the polypeptide from the cell.

144. A method for treating a subject suffering from a cell proliferative disease, comprising administering to the subject an effective amount of the compound according to any one of claims 1 to 97 and / or the pharmaceutical composition according to claim 98 or 99.

145. The method according to claim 144, wherein the cell proliferative disease is an autoimmune disease.

146. The method according to claim 144, wherein the cell proliferative disease is cancer or a pre-cancerous condition.

147. The method according to claim 146, wherein the cancer or the pre-cancerous condition is characterized by the presence of cancer stem cells.

148. The method according to claim 147, wherein the cancer stem cells present LGR on their surface.

149. The method according to claim 148, wherein the LGR is selected from LGR4, LGR5 and LGR6, preferably LGR5.

150. The method according to claim 146, wherein the cancer is ovarian cancer, myeloma, lymphoma, lung cancer, breast cancer, brain cancer, prostate cancer, spleen cancer, pancreatic cancer, cervical cancer, uterine cancer, head and neck cancer, esophageal cancer, liver cancer, skin cancer, kidney cancer, leukemia, bone cancer, testicular cancer, colon cancer, basal cell carcinoma, hepatocellular carcinoma, hepatobiliary cancer, colorectal cancer, or bladder cancer.

151. The method according to claim 146, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, colon cancer, gastric cancer, cholangiocarcinoma, lung cancer, liver cancer, skin cancer, neuroblastoma, or leukemia.

152. The method according to claim 151, wherein the cancer is ovarian cancer or acute lymphoblastic leukemia.

153. The method according to any one of claims 146 to 151, wherein the cancer is metastatic cancer.

154. The method according to any one of claims 146 to 153, further comprising administering to the subject at least a second anti-cancer treatment.

155. The method according to claim 154, wherein the second anti-cancer treatment is surgical treatment, chemotherapy, radiotherapy, gene therapy, or immunotherapy.

156. A method for killing / treating cancer stem cells, the method comprising contacting the cancer stem cells with the compound according to any one of claims 1 to 97 or the pharmaceutical composition according to claim 98 or 99.

157. A method for inhibiting the proliferation of cancer stem cells, the method comprising contacting the cancer stem cells with the compound according to any one of claims 1 to 97 or the pharmaceutical composition according to claim 98 or 99.

158. A method for treating cancer, the method comprising contacting the cancer stem cells with the compound according to any one of claims 1 to 97 or the pharmaceutical composition according to claim 98 or 99.

159. A method for reducing the spread of cancer cells and / or cancer stem cells, the method comprising contacting the cancer stem cells with the compound according to any one of claims 1 to 97 or the pharmaceutical composition according to claim 98 or 99.

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