Cell targeting constructs and their uses

Abstract

Provided herein is a cytotoxic compound that targets cells. Such compounds can be used to selectively bind to and kill cancer cells, such as cancer stem cells that express LGR4, LGR5, or LGR6. In some embodiments, the cytotoxic compound comprises an LGR-binding polypeptide, a cytotoxic agent (e.g., MMAE), and an Fc region (e.g., a mutant Fc domain). Also provided is a method for using the compound to treat cell proliferation diseases, such as cancer. TIFF2026503181000087.tif105146

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 381,050, filed October 26, 2022, U.S. Provisional Patent Application No. 63 / 386,777, filed December 9, 2022, and U.S. Provisional Patent Application No. 63 / 478,428, filed January 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 this invention.

[0003] This application contains an ST.26 XML Sequence Listing, which has been submitted electronically and is incorporated herein by reference in its entirety. The Sequence Listing was created on October 24, 2023, is named CLFR.P0508WO_ST26, and is 100 KB.

[0004] 1. Field The present disclosure relates generally to the fields of molecular biology and medicine. More particularly, the present disclosure relates to cell-targeted cytotoxic constructs. [Background technology]

[0005] 2. Description of Related Technology Immunotoxins and immunoconjugates have shown clinical promise for the treatment of diseases such as cancer, but significant clinical limitations remain. The successful development of targeted therapeutic agents, for example for cancer applications, relies on the identification of target cell-specific ligands and antigens, the creation of molecules capable of specifically targeting these components, and finally, the use of highly toxic molecules to kill the target cells. Immunoconjugates composed of antibodies and small toxic drugs or radioisotopes have been successfully tested in vitro and in animal models, and have demonstrated activity in clinical settings. In addition to using small molecules as toxin components, several highly cytotoxic protein components, such as diphtheria toxin, ricin A chain, Pseudomonas exotoxin, and gelonin (rGel), have been used in targeted therapy. However, issues such as capillary leak syndrome, immunogenicity, and unintended toxicity (to non-target cells) continue to limit successful implementation of these therapies, especially for long-term or chronic applications.

[0006] Treating cancers involving cancer stem cells (CSCs) poses a particularly challenging clinical problem. CSCs are typically a small subpopulation of cells within tumors that possess the ability to self-renew, differentiate, and form tumors when transplanted into an animal host. Numerous cell surface markers, such as CD44, CD24, and CD133, are often used to identify and enrich CSCs from tumors (Yu et al., 2012). CSCs are often resistant to conventional chemotherapy and radiation treatments, and CSCs can contribute to the origin of cancer metastasis. CSCs remain a significant clinical problem for cancer treatment, and the development of approaches to kill CSCs is a major goal in the field of oncology. Highly specific and highly active toxin molecules, and cell-targeting moieties containing such molecules, that exhibit improved therapeutic efficacy and pharmacological properties remain in demand. In particular, there is a real need for improved treatments for cancers whose growth is dependent on CSCs. Summary of the Invention

[0007] overviewThe present disclosure overcomes limitations in the prior art by providing improved constructs that can be used to deliver toxins to cells (e.g., cancerous 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., a Fu1-Fu2 domain) and a cytotoxic payload (e.g., MMAE, deruxtecan, PNU159682). A variety of cytotoxic payloads can be used (e.g., Figures 25-27, 29), and multiple cytotoxic payloads can be included in the construct (e.g., Figures 30-32). The constructs can further contain a mutant Fc domain (e.g., a "DHS" mutant), and the Fc can dimerize the construct (see, e.g., the schematic diagram in Figure 1). Because the constructs provided herein can target or selectively bind to LGRs (e.g., LGR4, LGR5, LGR6), they may be particularly useful for killing cancer stem cells and / or treating cancers that involve cancer stem cells (e.g., Figures 3A-E, 4). The LGR targeting moiety may also selectively bind to ZNRF3 and / or RNF43. As shown in the Examples below, the constructs can exhibit improved potency and half-life, and mutant forms of the LGR targeting moiety (e.g., R28A) and Fc hinge region (e.g., DHS Fc) are provided that have been observed to increase the plasma half-life of the construct in vivo without altering its potency (e.g., Figures 28-29). To covalently link one or more additional cytotoxic moieties to the construct, sortase A, sortase E, and / or partial reduction of one or more disulfides in the LGR binding moiety or Fc hinge region can be used to covalently link the additional cytotoxic moieties to the construct.The anti-cancer effects of the constructs provided herein were observed in vivo using multiple cancer animal models, including in vivo xenograft animal models of colorectal cancer, gastric cancer, neuroblastoma, and ovarian cancer (see, e.g., Figures 6A-B, 7A-B, 8A-D, 9, 12A-B, 18A-B, and Figures 21-24). Additional copies of the LGR targeting moiety can be used to increase binding to cancer cells expressing LGR, such as cancer stem cells (Figure 17). Linkers can be included in or omitted from the constructs provided herein (e.g., Figures 19-20). Provided herein are anti-cancer compounds that are chemically distinct from the constructs provided in Yu et al. (2021) and may further offer advantages in potency, efficacy, and / or in vivo pharmacokinetic half-life; for example, compared to R1FF-MMAE (Yu et al., 2021), the FcF2 compounds provided herein surprisingly exhibited approximately 15-fold longer distribution phase half-life than R1FF-MMAE when measured in vivo using plasma pharmacokinetic analysis in mice (e.g., Figure 34). Also provided are methods for treating diseases (e.g., cancer) using the constructs. The therapeutic compounds provided herein may be particularly useful for treating cancers whose growth depends on CSCs.

[0008] In some aspects, provided is a compound that can selectively target the cancer that expresses R-spondin receptor LGR4, LGR5 and / or LGR6.These receptors are expressed in various cells, including epithelial stem cells, normal tissues and tumors, and play an important role in embryogenesis, tissue homeostasis and regeneration.LGR5 and LGR6 are expressed at increased levels in various different types of cancer, CSC, and stem cells in the ovarian surface and fallopian tube epithelium where ovarian cancer develops.High-grade serous ovarian cancer is one of the tumors that express abnormally high levels of LGR5 mRNA and LGR6 mRNA.R-spondin is the natural ligand for LGR5 and LGR6, and binds to them with nanomolar affinity. As shown in the Examples below, to target cancer stem cells (e.g., in ovarian cancer and other tumors), the sortase reaction was used to site-specifically conjugate the potent cytotoxin monomethyl auristatin E (MMAE) via a protease-cleavable linker to two furin-like domains of RSPO1 (Fu1-Fu2), which mediate RSPO1 binding to LGR5 and LGR6 and their co-receptors ZNRF3 and RNF43, and an immunoglobulin Fc domain was included at the N-terminus and acted to dimerize the receptor-binding domains, such that each molecule carried two MMAEs. The resulting molecule, FcF2-MMAE, demonstrated: (1) selective, LGR5-dependent, low-nanomolar cytotoxicity against ovarian cancer cells in vitro; (2) selectivity that was dependent on binding to both the LGR receptor and the ubiquitin ligase co-receptor; (3) favorable stability and plasma pharmacokinetic properties upon IV administration, including an elimination half-life of 29.7 hours; (4) selective in vivo inhibition of LGR5-rich tumors as opposed to LGR5-poor isogenic tumors; and (5) therapeutic efficacy in two different aggressive wild-type human ovarian cancer xenograft models.FcF2-MMAE may offer advantages over the previously generated R1FF-MMAE (Yu et al., 2021), including: (a) increased protein yield from transiently transfected cultures by leveraging the chaperone function of Fc to improve folding; (b) dimerizing the resulting molecule so that it carries two molecules of MMAE instead of one; (c) increased plasma half-life by including a mutant form of Fc with improved FcRn binding characteristics; and (d) increased avidity for binding to LGR and ZNRF3 / RNF43 by including two copies of the Fu1-Fu2 (FuFu) domain instead of just one. Achievement of these goals was evidenced by significantly greater yields of the FcF2-His precursor, greater potency and selectivity when tested in isogenic OVCAR8 / EV and OVCAR8 / LGR5 cells, a six-fold increase in terminal plasma half-life, and improved efficacy in xenograft models. 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, remaining high. These results support the idea 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 that express stem cell markers. While not wishing to be bound by any theory, the results are consistent with the idea that the Fu1-Fu2 portion of the compound can simultaneously engage both (i) LGRs (e.g., LGR4, LGR5, or LGR6) and (ii) ZNRF3 or RNF43. Exemplary amino acid sequences, and nucleotide sequences encoding the polypeptides, included in the therapeutic compounds of the present disclosure are also shown, for example, in Figures 13-16.In some embodiments, provided herein are compounds comprising a polypeptide comprising (from N- to C-terminus) an Fc domain (e.g., a mutated 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 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 each LGR-binding domain comprises a polypeptide having at least 95% sequence identity to at least one of SEQ ID NOs: 4, 78-83, 85-89, 90-96, 98, or 102. The LGR-binding domains can each comprise an amino acid sequence independently 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 comprise an amino acid sequence independently selected from FuFu (SEQ ID NO: 4) or FuFu N137A (SEQ ID NO: 17). The LGR binding domain may comprise a substitution mutation at position R28 or at R30, numbered according to Kabat. The substitution mutation may be arginine to 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 the Fu1-Fu2(R30A) mutant (SEQ ID NO:100). The LGR-binding domain may comprise Fu1-Fu2(R22-R31 deletion) (SEQ ID NO:92), Fu1-Fu2(K25-R31 deletion) (SEQ ID NO:93), Fu1-Fu2(R28-R31 deletion) (SEQ ID NO:94), or Fu1-Fu2(R22-K27 deletion) (SEQ ID NO:95). The polypeptide may comprise FcST4 (SEQ ID NO:105).In some embodiments, the Fc region is N-terminal to the LGR-binding domain, or the polypeptide comprises an Fc region and an LGR-binding domain in an N-to-C orientation. In some embodiments, the Fc region is an IgG Fc domain. The polypeptide can 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 the polypeptide does not comprise SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, and 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 the polypeptide does not comprise SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, and SEQ ID NO:88. In some embodiments, the human IgG Fc domain is capable of binding to human FcRn at acidic pH, and the Fc domain has the following substitution mutations: (i) aspartic acid at position 309 (L / V309D); (ii) histidine at position 311 (Q311H); and (iii) serine (N434S) or tyrosine (N434Y) at position 434; the numbering of amino acid positions is according to the Kabat system; and the Fc domain binds to FcRn at acidic pH with higher affinity than 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 can have the same or essentially the same binding affinity for FcγR compared to a wild-type Fc domain. The Fc domain can have the same or essentially the same binding affinity for one, two, or all of FcγRI, FcγRII, and FcγRIII compared to a wild-type Fc domain.In some embodiments, the Fc domain does not detectably or selectively bind to FcRn at neutral pH and / or exhibits no or essentially no binding to FcRn at neutral pH. In some embodiments, the Fc domain exhibits (i) enhanced binding at pH 5.8 and (ii) reduced or no detectable binding to FcRn at pH 7.4 compared to wild-type. The Fc domain may be aglycosylated. In some embodiments, the Fc domain has a substitution mutation at position 264 that is glutamic acid (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) 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). The Fc domain may comprise or consist of IgG1-Fc DHS (L309D; Q311H; N434S). The compound may dimerize via a disulfide bond 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 the polypeptide does not comprise a linker. The polypeptide may comprise the Fc domain and the LGR-binding domain from the N-terminal to the C-terminal end; or 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) can be separated via 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 can comprise SEQ ID NO:20. In some embodiments, the polypeptide comprises a leader sequence. The leader sequence can 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 can be a conjugated drug. In some embodiments, the drug is a maytansinoid, auristatin, amanitin, calicheamicin, psymberin, duocarmycin, anthracycline, camptothecin, doxorubicin, taxol, tubulysin, eribruin, 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 analog (e.g., topotecan, irinotecan, belotecan, or deruxtecan). The drug can be an anthracycline analog (e.g., PNU-159682; CAS Number: 202350-68-3). The conjugated drug can be linked to the polypeptide via a linker. The linker may be a protease-cleavable linker, such as, for example, citrulline-valine. In some embodiments, the compound includes at least one spacer or linker (e.g., 1, 2, or 3 spacers or linkers) positioned between the cytotoxic agent and the LGR binding domain. The compound may include two linkers or spacers.The linker or spacer may comprise G4S (SEQ ID NO:18) or (G4S)2 (SEQ ID NO:5). The cytotoxic moiety may be a cytotoxic protein, such as, for example, a serine protease. The serine protease may 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 may dimerize via a disulfide bond in the Fc domain. In some embodiments, the cytotoxic agent is covalently linked to the polypeptide via a sortase linker. The sortase may 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 is covalently attached to the polypeptide via a sortase, such as by having the sortase catalyze the covalent attachment of the cytotoxic agent to the polypeptide. The sortase can be a sortase A linker or a sortase E. The sortase linker has the sequence LPXT(G). n(where n=1-10, or any range derivable therein (e.g., n=4-9)), 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 attached to the polypeptide via a sortase linker. In some embodiments, the first cytotoxic agent is covalently linked to a first sortase linker at the N-terminus of the polypeptide, and the second cytotoxic agent is covalently linked to a second sortase linker at the N-terminus of the polypeptide. The first sortase linker comprises the sequence LPXT(G)n, where n=1-10. The first sortase linker can be LPETGG (SEQ ID NO:6). The second sortase linker can be LAHTGG (SEQ ID NO:106). In some embodiments, the first cytotoxic moiety is covalently attached to the first sortase linker using sortase A, and the second cytotoxic moiety is covalently attached 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 can be different conjugated drugs. The first cytotoxic moiety and the second cytotoxic moiety can have the same structure. In some preferred embodiments, the first cytotoxic moiety and the second cytotoxic moiety are both 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 in the Fc region or the LGR-binding domain.The disulfide bond may be contained in a maleimide group. The maleimide group may be covalently linked to a cleavable linker. The cleavable linker may comprise a valine (Val)-citrulline (Cit) bond. In some embodiments, the first cytotoxic agent has the sequence LPXT(G). n or LAHTGG (SEQ ID NO:106), where n=1-10, and the second cytotoxic agent is linked to the polypeptide via a disulfide bond. The disulfide bond may be in the Fc region. The disulfide bond may be 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, inhalation, intraarterial, or intrapleural administration.

[0010] Another aspect of the present disclosure relates to a pharmaceutical composition comprising the compound described hereinabove. The pharmaceutical composition may be formulated for intravenous, intraperitoneal, subcutaneous, intratumoral, intrathecal, inhalation, intraarterial, or intrapleural administration.

[0011] Yet another aspect of the present disclosure pertains to nucleic acids encoding the polypeptides described above or herein.

[0012] Another aspect of the present disclosure relates to a host cell comprising the nucleic acid 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] Yet another aspect of the present disclosure relates to a method for producing a therapeutic compound that binds to an LGR receptor, the method comprising: (a) expressing in a cell a polypeptide encoded by a nucleic acid described above or herein, wherein the polypeptide comprises a sortase linker at the terminus of the polypeptide; (b) obtaining the polypeptide; and (c) contacting the cytotoxic agent and the polypeptide with a first transpeptidase, thereby covalently linking 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 an 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 is LPXT(G) n (where n=1-10). The C-terminal sortase donor sequence can be LPETGG (SEQ ID NO:6). The sortase linker can be -(His) n- (where n=1-10, preferably n=4-9). The N-terminal sortase acceptor sequence may comprise 1-10 glycine residues. In some embodiments, the N-terminal sortase acceptor sequence is GGG. The cytotoxic agent may be a conjugated drug, such as a maytansinoid, auristatin, amanitin, calicheamicin, psymberin, duocarmycin, anthracycline, camptothecin, doxorubicin, taxol, tubulysin, eribruin, or pyrrolobenzodiazepine. In some embodiments, the drug is an auristatin. The auristatin may 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 analog (e.g., topotecan, irinotecan, belotecan, or deruxtecan). The drug can be an anthracycline analog (e.g., PNU-159682; CAS Number: 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 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, for example, as described above or herein. The conjugate drug can be monomethyl auristatin E (MMAE), PNU-159682, topotecan, irinotecan, belotecan, or deruxtecan.The method may further include (d) covalently linking 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 linking the second cytotoxic agent to the polypeptide via a partial disulfide reaction to form a disulfide bond. The second cytotoxic agent may be linked to the Fc region or the LGR-binding domain via a partial disulfide reaction. The second cytotoxic compound may include a linker, and the linker may be linked to the polypeptide via a partial disulfide reaction. The linker may include a thiol-reactive maleimide group. The linker may further include a cleavable bond. The cleavable bond may include a valine (Val)-citrulline (Cit) bond. The second transpeptidase may preferably be sortase A or sortase E. In some preferred embodiments, the first transpeptidase is sortase A and the second transpeptidase is sortase E. The second cytotoxic agent can be a cytotoxic agent 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 a nucleic acid as described above or herein in a cell under conditions to produce the encoded polypeptide; and (b) purifying the polypeptide from the cell.

[0015] Yet another aspect of the present disclosure relates to a method for treating a subject with a cell proliferative disorder, comprising 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 disorder may be an autoimmune disease. In some embodiments, the cell proliferative disorder is cancer or a precancerous condition. The cancer or precancerous condition may be characterized by the presence of cancer stem cells. The cancer stem cells may display an LGR on their surface. The LGR may be selected from LGR4, LGR5, and LGR6, preferably LGR5. The cancer may 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, bile duct cancer, 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 at least a second anti-cancer therapy to the subject. The second anti-cancer therapy can be surgery, chemotherapy, radiation therapy, gene therapy, or immunotherapy.

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

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

[0019] Yet another aspect of the present disclosure relates to a method for reducing the spread of cancer cells and / or cancer stem cells, 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" refers to a property of a polypeptide, such as an Fc domain (e.g., a mutant IgG Fc domain or a variant IgG Fc domain), that has the ability to bind to FcRn at acidic pH (e.g., pH 5.8), and preferably, the polypeptide or Fc domain has the ability to exhibit increased binding of FcRn at acidic pH compared to a wild-type Fc domain (e.g., a wild-type Fc IgG domain). In some embodiments, an Fc domain or polypeptide that pH-selectively binds to FcRn also exhibits either reduced binding of FcRn at physiological pH compared to a wild-type (e.g., a wild-type IgG Fc domain), or no detectable binding of FcRn at physiological pH.

[0021] As used herein, "essentially free" with respect to a specified component means that the specified component is not intentionally formulated into the composition at all and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any unintentional contamination of the composition is significantly less than 0.05%, preferably less than 0.01%. Most preferred is a composition in which the amount of the specified component cannot be detected by standard analytical methods.

[0022] As used herein, "essentially the same binding affinity" means that two molecules exhibit statistically indistinguishable reversible binding to a ligand as observed based on at least one binding assay, or that the equilibrium constant for the reversible binding of two molecules to a ligand is greater than or equal to K D Indicates a difference of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in the value. D Values ​​can be determined using standard methodologies known in the art, such as enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance (SPR). on and K. off It can be calculated based on observed binding properties.

[0023] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, K D The affinity of a binding domain for its target can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM); alternatively, the affinity can be 100 nM to 1 nM or 0.1 nM to 10 nM. Furthermore, when there is an affinity between two agents within the affinity ranges discussed above, the agents are considered to bind specifically.

[0024] As used herein, "LGR binding domain" refers to a polypeptide that can selectively bind to an LGR receptor, preferably an LGR4, LGR5, or LGR6 receptor. The polypeptide may contain natural amino acids and / or unnatural 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 LGRs with equal affinity. In some aspects, polypeptides that selectively bind to LGR4, LGR5, and LGR6 are provided herein.

[0025] As used herein, the terms "encode" or "encoding" in reference to nucleic acids are used to enable those skilled in the art to readily understand the invention; however, these terms may be used interchangeably with "comprise" or "comprising," respectively.

[0026] As used herein, "a" or "an" may mean one or more. As used herein in the claims, the words "a" or "an," when used in conjunction with the word "comprising," may mean one or more than one.

[0027] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not mutually exclusive, although the present disclosure supports a definition that refers to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0028] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device being used to measure the variable, the inherent variation of error for the method being employed to determine the value, or the inherent variation among study subjects.

[0029] Other objects, features, and advantages of the present invention will become apparent from the detailed description which follows. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0030] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0031] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0032] [Figure 1] Schematic illustration of FcF2-His and its conversion to FcF2-MMAE using a sortase reaction. [Figure 2]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. [Figure 3] Figure 3A-E: Cytotoxicity of FcF2-MMAE against cell lines. Potency and selective cytotoxicity of two representative batches of FcF2-MMAE in the HEK293 / EV vs. HEK293 / LGR5 models (Figure 3A and Figure 3B), and potency and selective cytotoxicity of two other representative batches in the OVCAR8 / EV vs. OVCAR8 / LGR5 models (Figure 3C and Figure 3D). Figure 3E, Potency of FcF2-MMAE against eight human ovarian cancer cell lines expressing endogenous levels of LGR. Each curve represents growth inhibition during 120 hours of exposure to increasing concentrations of FcF2-MMAE. Viability was determined using CCK8 reagent. [Figure 4] Relative contribution of the Fu1 and Fu2 domains to the selective cytotoxicity of FcF2-MMAE. Effect of disabling binding of Fu1 (FcF2-Q71R-MMAE), Fu2 (FcF2-F106R-F110R-MMAE), or both (FcF-Q71R-F106R-F110R-MMAE) on the selectivity of growth inhibition of OVCAR8 / EV and OVCAR8 / LGR5 cells exposed for 120 hours. Values ​​are the mean ± SEM of three independent experiments, each performed in triplicate. [Figure 5] Figure 5A-B: Plasma pharmacokinetics of FcF2-MMAE. Figure 5A, Plasma concentration of FcF2-MMAE as a function of time after IV injection of 0.1 nmol / g (9 μg / g) into BALB / c mice, as determined by ELISA. Figure 5B, Concentration of FcF2-MMAE in the plasma fraction of mouse whole blood as a function of time during incubation, as determined by ELISA. [Figure 6]Figures 6A-B: Efficacy and toxicity of FcF2-MMAE against human ovarian cancer OVCAR8 / EV (LGR5-low) xenografts and human ovarian cancer OVCAR8 / LGR5 (LGR5-rich) xenografts. Figure 6A, Tumor volume as a function of time. Figure 6B, Mouse weights during and after the course of treatment. Dose schedule: 0.5 nmol / g (42.65 mg / kg) q4d x 4 IP. N=8 / group. Vertical arrows indicate dose days. Vertical bars, ±SEM. [Figure 7] Figures 7A-B: Efficacy and toxicity of FcF2-MMAE as a function of dose administered IP every 7 days for six doses in an OVCAR8 / LGR5 xenograft model. Figure 7A, Tumor growth as a function of dose. Figure 7B, Mouse weight during and after the course of treatment as a function of dose. Vertical arrows indicate the days on which doses were delivered. Vertical bars, ±SEM. [Figure 8] Figures 8A-D: Efficacy of a single dose of FcF2-MMAE 1.0 nmol / g in CAOV3 and KF-28 xenograft models. A. Mean tumor volume (Figure 8A) and animal weight (Figure 8B) for KF-28 xenografts. B. Mean tumor volume (Figure 8C) and animal weight (Figure 8D) for CAOV3. Vertical arrows indicate the day the dose was injected. Vertical bars, ±SEM. [Figure 9] Efficacy of FcF2-MMAE at four doses of 0.5 nmol / g against wild-type OVCAR8 and CAOV3 xenografts. A and C, Tumor growth as a function of time. B and D, Mouse weights during and after the course of treatment. Vertical arrows indicate the days on which doses were injected. Vertical bars, ±SEM. [Figure 10] Figure 10A-B: Demonstration of differential glycosylation forms of FcF2-His. Figure 10A, FcF2-His samples before and after treatment with PNGase 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). [Figure 11]Figures 11A-C: Flow cytometric demonstration of differential expression of LGR5 in isogenic pairs of HEK293 and OVCAR8 cells and FcF2-MMAE-mediated depletion of LGR5-expressing cells. 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 and OVCAR8 / LGR5 cells before and after exposure to 10 nm FcF2-MMAE for 120 hours (gray and black curves, untreated, unstained cells and FcF2-MMAE-treated unstained cells; green and blue curves, untreated and treated OVCAR8 / EV and OVCAR8 / LGR5 cells stained with anti-LGR5). [Figure 12] Figures 12A-B: Efficacy and toxicity of FcF2-MMAE against human ovarian cancer OVCAR8 / EV (LGR5-low) xenografts and human ovarian cancer OVCAR8 / LGR5 (LGR5-rich) xenografts at dose schedule: 1.5 nmol / g (128 mg / kg) q7d x 4 IP. Figure 12A, Tumor volume as a function of time. Figure 12B, Mouse weights during and after the course of treatment. N=8 / group. Vertical arrows indicate dose days. Vertical bars, ±SEM. [Figure 13] The amino acid sequence of FcF2 containing an 8xHis tag. The amino acid sequence contains an IgG kappa leader sequence followed by a DHS variant of IgG1 Fc (Lee et al., 2019) linked to the Fu1-Fu2 domains. The amino acids highlighted in blue in the Fc portion of the construct are used to indicate 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 with the corresponding cysteine ​​in another individual Fc region to form a dimerized molecule. [Figure 14]Nucleotide sequence for generating the FcF2 construct in Figure 13, except that the Fc portion of the construct does not contain the DHS mutation. The nucleotide sequence shown includes codon optimization. Codons highlighted in purple within the Fc portion of the construct indicate regions that will later be modified using GeneArt to contain the DHS mutant version of the Fc region. The color coding of the different regions corresponds to the regions otherwise described in Figure 13. [Figure 15] Sequence containing DHS mutations before codon optimization. Codons highlighted in blue in the Fc region correspond to the DHS mutations in the DHS Fc here. The color coding of the different regions corresponds to the regions described in Figure 13, while the other regions correspond to the regions described in Figure 13. [Figure 16] FcF2-8xHis nucleotide (nt) sequence after codon optimization for expression in human cells. This nucleotide sequence corresponds to the amino acid sequence shown in Figure 13. [Figure 17] Construction of FcST4. The amino acid sequence of FcST4 is shown (top). The vector construct for the FcST4 molecule was designed to contain an IgG leader sequence at the N-terminus, followed by a mutated Fc domain connecting two modified receptor-binding domains (STs), with the STs connected in tandem by a linker sequence between the two ST domains. A second spacer sequence was inserted immediately upstream of the LPETGG sortase recognition (donor) motif, and an 8xHis tag was placed at the C-terminus. [Figure 18] Figures 18A-B: Analysis of the efficacy of FcST2-MMAE against eight human ovarian cancer cell lines. Figure 18A, Viability as a function of FcST2-MMAE concentration for eight human ovarian cancer cell lines. Figure 18B, IC50 values ​​for the eight cell lines tested. [Figure 19] Linkerless FcF2. A linkerless FcF2 construct is shown (bottom). In contrast to the linker-containing FcF2 (top), the linkerless FcF2 does not contain a G4S linker in the polypeptide. [Figure 20] Cytotoxicity of FcF2-MMAE and FcF2Δlinker-MMAE. [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 caused a decrease in mean tumor weight without changing mouse body weight. [Figure 22] Efficacy of FcF2-MMAE in a colon cancer LoVo xenograft model. Dose schedule: FcF2-MMAE IP injection q7d x 4. Each data point is the mean size of all tumors in a group. Vertical bars are SEM. [Figure 23] Efficacy of FcF2-MMAE in a gastric cancer AGS xenograft model. Dose schedule: FcF2-MMAE IP injection q7d x 4. Each data point is the mean size of all tumors in a group. Vertical bars are SEM. [Figure 24] Efficacy of FcF2-MMAE in a neuroblastoma SKNAS xenograft model. Dose schedule: FcF2-MMAE IP injection q7d x 4. Each data point is the mean size of all tumors in a group. Vertical bars are SEM. [Figure 25] Figures 25A-C: Analysis of the structural integrity and selective cytotoxicity of FcF2-PNU159682. Figure 25A, SDS-PAGE analysis of two different batches of FcF2-PNU159682. Figure 25B, Growth inhibition of OVCAR8 / EV cells versus OVCAR8 / LGR5 cells as a function of FcF2-PNU159685 concentration (pM). Data are mean ± SEM of triplicate cultures. Figure 25C, Schematic representation of the structure of PNU159683. [Figure 26] Figures 26A-B: Construction of the GGGC-GGFG-deruxtecan linker. Figure 26A, Schematic of the conjugation of GGGC with maleimide-GGFG-deruxtecan. Figure 26B, Reverse-phase HPLC analysis of the GGGC-MA-GGFG-deruxtecan linker, C4 column. [Figure 27]Figures 27A-B: Production of FcF2-deruxtecan and demonstration of its cytotoxicity and cytotoxic selectivity. Figure 27A, Reverse-phase HPLC (C4 column) analysis of FcF2-deruxtecan demonstrating purity. Figure 27B, Growth inhibition of OVCAR8 / EV cells versus OVCAR8 / LGR5 cells as a function of FcF2-deruxtecan concentration. Data are mean ± SEM of triplicate cultures. [Figure 28] Demonstration that FcF2-MMAE containing mutations R28A and R30A retains potency and cytotoxic selectivity. Growth inhibition of OVCAR8 / EV cells versus OVCAR8 / LGR5 cells as a function of concentration of FcF2-MMAE (WT), FcF2-R28A-MMAE, and FcF2-R30A-MMAE. Data are mean ± SEM of triplicate cultures. [Figure 29] Pharmacokinetics of FcF2-MMAE (WT), FcF2-R28A-MMAE, and FcF2-R30A-MMAE. A, Plasma concentrations of each form as a function of time after IV injection in BALB / c mice. B, Table of areas under the curve from 0 to 120 hours for each form. [Figure 30] Schematic diagram for loading MMAE onto both ends of the FcF2 molecule using both sortase A and sortase E. [Figure 31] Demonstration of loading of GGGC-MA-Dye650 onto FcF2-LAHTGG-His by sortase E. The figure shows SDS-PAGE analysis of sortase E reaction products (lanes 7 and 8) detected using Dye650 fluorescence after elution from SP-Sepharose resin. [Figure 32] Western blot analysis demonstrating MMAE incorporation using partial reduction of disulfide bonds with TCEP. Top panel, blot probed with anti-RSPO1 antibody; bottom panel, blot probed with anti-MMAE. [Figure 33-1]Nucleotide and amino acid sequences of the FcF2(R28A) construct. The Kozak sequence is shown in light blue. The IgGk leader sequence is shown in green highlight. The Fc sequence is shown in gray highlight, with the DHS mutation underlined and highlighted in yellow. The G4SG4S linker is highlighted in red. The LPETG sequence is highlighted in purple. The 8xHis tag is highlighted in light blue. The stop x2 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 with black highlighting. [Figure 33-2] See description of Figure 33-1. [Figure 34] In vivo plasma distribution half-life of R1FF-MMAE ("RSPO1-MMAE") and FcF2-MMAE in female BALB / C mice. The FcF2-MMAE construct exhibited a roughly 15-fold longer half-life than R1FF-MMAE. Distribution half-life (T1 / 2) and elimination half-life (T1 / 2) values ​​are shown. DETAILED DESCRIPTION OF THE INVENTION

[0033] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present disclosure overcomes limitations in the prior art by providing, in some aspects, constructs that selectively target and kill cancer cells, including cancer stem cells. The constructs may include a polypeptide having a region capable of selectively binding to LGR4, LGR5, and / or LGR6 receptors expressed by cancerous cells or CSCs. The constructs may also include a cytotoxic moiety (e.g., MMAE) linked to the polypeptide via a cleavable linker such that the construct can be endocytosed into the cancerous cells and subsequently released into the cells. Thus, delivery of the cytotoxic moiety to cancerous cells, such as cancer stem cells, can result in selective killing of the cancerous cells. The present disclosure is based, in part, on the discovery that including an Fc region (e.g., a mutant Fc region, such as the DHS Fc mutants provided herein) in a polypeptide can result in improvements to the pharmacological profile (e.g., improved half-life of the construct in vivo) and / or improved therapeutic efficacy. The inclusion of an Fc region can also result in a dimerization construct, which 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 the inclusion of two or more LGR binding domains in a polypeptide can result in improved therapeutic properties of constructs for treating cancer, such as cancers involving cancer stem cells. Methods for treating cancer and for selectively targeting cancer stem cells are also provided herein.

[0034] In various aspects, therapeutic constructs are provided. The therapeutic constructs may include (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 constructs may include, for example, 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 may 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 Fc domain or a mutant Fc domain (e.g., DHS mutant Fc; SEQ ID NO:13). The Fc domain can cause dimerization of two copies of the therapeutic construct, which can effectively double the number of cell targeting moieties and cytotoxic moieties in the dimerized construct. As shown in the examples below, the inclusion of a DHS Fc domain resulted in an increase in the half-life of the construct in vivo. In some embodiments, the therapeutic construct can comprise a radioisotope, imaging agent, or radiotherapy. The therapeutic construct can be included in a pharmaceutical composition, such as a formulation for injection (e.g., intravenous or intratumoral injection), inhalation, or inclusion in a liposome or nanoparticle. Methods of using the therapeutic construct are also provided herein. In some embodiments, the therapeutic constructs may be used to treat cancers that express LGR4, LGR5, or LGR6.

[0035] In some aspects, the therapeutic compounds provided herein may comprise the Fu1-Fu2 receptor binding domain of R-spondin to target cancer cells expressing 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 linked to monomethyl auristatin E (MMAE) for ovarian cancer cells rich in stem cell receptor LGR5. A modified IgG1 Fc domain (Lee et al., 2019) containing a half-life extension modification was linked to the N-terminal end of the Fu1-Fu2 domain, which has a sortase recognition sequence at its C-terminal end. During the synthesis of this protein in HEK293E cells, the two chains are linked by an intermolecular disulfide bond between the Fc domains, resulting in a dimeric molecule "FcF2-His" containing 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 generate "FcF2-MMAE" (e.g., Figure 1). This molecule exhibited both low-nM cytotoxicity in a panel of human ovarian cancer cell lines and a favorable stability and pharmacokinetic profile. Furthermore, FcF2-MMAE selectively killed LGR5-rich tumor cells in vitro and differentially inhibited the growth of isogenic LGR5-poor and LGR5-rich tumors in vivo. FcF2-MMAE has shown activity in two different human ovarian cancer xenograft models at a clinically relevant dose schedule and at a dose that produces only transient adverse side effects.These results in ovarian cancer models demonstrate that these compositions and approaches can be used to selectively target and kill cancer stem cells that express LGR5 / LGR6.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.

[0036] I. LGR-binding polypeptides In some aspects, the therapeutic compounds provided herein may comprise an LGR-binding domain polypeptide capable of selectively binding to 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). The therapeutic constructs provided herein can target selected cell types, such as cancer cells or stem cells, by selectively binding to LGR4, LGR5, and / or LGR6. In some embodiments, the LGR-binding domain can selectively bind to LGR5 and LGR6. The LGR-binding domain may be a polypeptide that may contain natural and / or unnatural amino acids.

[0037] In some embodiments, the LGR binding domain comprises or consists of a polypeptide sequence. The polypeptide may comprise a FurinL sequence. The FurinL sequence is each of the two cysteine-rich furin-like domains in the R-spondin polypeptide (De Lau et al., 2012). In some embodiments, the polypeptide contains a first FurinL sequence (also referred to as "Fu1" or "FurinL repeat 1") and a second FurinL sequence (also referred to as "Fu2" or "FurinL repeat 2"). The LGR binding domain can comprise the FurinL sequence from an R-spondin protein, 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 in, for example, De Lau et al. (2012) and Jin-Gen et al. (2015). The first FurinL sequence (Fu1) and the second FurinL sequence (Fu2) can comprise or consist of the FurinL sequence 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 domain (e.g., the Fu1-Fu2 domain) of an R-spondin protein, 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). In some embodiments, the LGR binding domain is the Fu1-Fu2 sequence (SEQ ID NO:4). The LGR binding domain can be included multiple times in the therapeutic compounds or therapeutic 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, such as Fu1-Fu2(N137A) (SEQ ID NO: 17), compared with the LGR binding domain in human R-spondin protein. The LGR binding domain may comprise a polypeptide having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any of the R-spondin or FurinL sequences provided above or herein.

[0038] As used herein, "Fu1-Fu2" and "FuFu" are used interchangeably herein to refer to a polypeptide that contains both the Fu1 FurinL sequence and the Fu2 FurinL sequence from R-spondin.In some embodiments, the Fu1 region and the Fu2 region can be derived from the same R-spondin polypeptide (for example, derived from human RSPO1).Nevertheless, in some embodiments, a polypeptide can comprise the Fu1 region from a first R-spondin protein and the Fu2 region from a second R-spondin protein.For example, the FurinL sequences from different human R-spondin proteins, such as the Fu1 region from RSPO1 and the Fu2 region from RSPO2, can be included in a single polypeptide to selectively bind to LGR receptors such as LGR4, LGR5, and / or LGR6.

[0039] In some embodiments, the LGR binding domain has the following "Fu1-Fu2" sequence (SEQ ID NO:4): TIFF2026503181000002.tif18160; or one or more copies of a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9 of sequence identity.

[0040] Other LGR binding domains may include the Fu1 and / or Fu2 domains from human RSPO2, RSPO3, or RSPO4, or may include polypeptides 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 one or SEQ ID NOs:78-83). Fu1 and Fu2 domains that can be used include the following: TIFF2026503181000003.tif103161TIFF2026503181000004.tif179160

[0041] In some embodiments, instead of a polypeptide containing both the Fu1 and Fu2 regions, either the Fu1 or Fu2 region can be included in the construct 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 (e.g., Fc-Fu1-MMAE or Fc-Fu2-MMAE) that is cytotoxic and selective for LGR4, LGR5, and / or LGR6. A single Fu1 or Fu2 region can be included in the polypeptides described herein in combination with another cell-targeting polypeptide, such as an scFv region. For example, the Fu1 region and an scFv (e.g., an scFv that selectively targets a liver antigen) can be included in a single polypeptide that is covalently linked to a drug (e.g., a cytotoxic moiety such as MMAE) and can selectively direct the drug to the scFv's target tissue (e.g., the liver) while reducing or avoiding intestinal toxicity that would be mediated by the Fu2 domain.

[0042] The LGR binding domain can contain both the Fu1 and Fu2 regions described herein. In some embodiments, the inclusion of both the Fu1 and Fu2 regions can result in a construct with improved cytotoxicity and / or selectivity compared to the inclusion of only a single Fu1 region or a single Fu2 region.

[0043] The above Fu1-Fu2 sequence may include a substitution mutation 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 has the sequence (SEQ ID NO:17): TIFF2026503181000005.tif18160; or a polypeptide having at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9 of sequence identity to FuFu(N137A) (SEQ ID NO:17).

[0044] The Fu1-Fu2 sequence may contain one, two, three, four, five, or more substitution mutations that may provide additional beneficial effects, such as altering the pharmacokinetics or increasing the half-life of the construct after administration to a mammalian subject. For example, the Fu1-Fu2 sequence may contain the (R28A) substitution mutation: TIFF2026503181000006.tif18160. The Fu1-Fu2 sequence may contain the (R30A) substitution mutation: As shown in Example 7 and Figure 28, neither the R28A nor the R30A mutation altered the potency of selectivity for LGR5-enriched cells; however, the R28A mutation extended the initial half-life of the molecule in vivo and increased the AUC 0-120The R28A mutation increased the half-life (e.g., plasma half-life) of Fu1-Fu2 by 3.4-fold, whereas the R30A mutation did not. In some preferred embodiments, the Fu1-Fu2 sequence may contain an R28A mutation, which may result in an increased half-life (e.g., plasma half-life) after administration to a mammalian subject in vivo.

[0045] Deletion mutants of the FuFu sequence can also be used. The following deletion mutants may increase the half-life or plasma half-life of the construct. FuFu deletion mutants that can be used include: Fu1-Fu2 (R22-R31 deletion) TIFF2026503181000008.tif18161, Fu1-Fu2 (K25-R31 deletion) TIFF2026503181000009.tif18160, Fu1-Fu2 (R28-R31 deletion) TIFF2026503181000010.tif18160, Fu1-Fu2 (R22~K27 deletion) TIFF2026503181000011.tif18160, and Fu1-Fu2 (S21~Q38 deletion) TIFF2026503181000012.tif11159.

[0046] In some embodiments, the FuFu sequence is linked to a linker (e.g., (GS)n, where n=1-3), a cleavable linker (e.g., one containing valine-citrulline, such as valine-citrulline-PAB), and a cytotoxic moiety (e.g., MMAE). For example, a polypeptide can include an Fc domain (e.g., a variant Fc domain, such as SEQ ID NOs:13-16) and a FuFu region. The FuFu region can be linked to a linker and MMAE (where the MMAE is covalently linked using a sortase enzyme), e.g., TIFF2026503181000013.tif25168.

[0047] In some embodiments, the cell targeting moiety comprises a polypeptide containing at least one, two, three, or four 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, for example, G4S(GGGGS, SEQ ID NO:18), (G4S)2 (SEQ ID NO:5), (Gly)6, or (EAAAK)3 (SEQ ID NO:84).

[0048] When a construct comprises an Fc region described herein (e.g., a variant Fc region such as the DHS Fc of SEQ ID NO: 13), the construct can dimerize based on the association of the Fc regions in two different molecules. In this way, the total number of LGR5 / LGR6 binding domains and cytotoxic agents can be effectively doubled in a single dimerization construct. The total number of LGR5 / LGR6 binding domains (e.g., Fu1-Fu2, FuFu(N137A)) in a dimerization construct can be 2, 4, 6, or 8.

[0049] The LGR family of G protein-coupled seven-transmembrane spanning receptors contains eight members, all of which have a large extracellular domain consisting of up to 18 copies of leucine-rich repeat motifs. The eight receptors are divided into three groups: the FSH receptor LGR1, the LH receptor LGR2, and the TSH receptor LGR3. The second group consists of receptors for R-spondin (RSPO), LGR4, LGR5, and LGR6. The third group contains receptors for relaxin and insulin-like 3 protein, LGR7 and LGR8, respectively. LGR5 and LGR6 are well-defined markers of stem cells in the intestine (LGR5), 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 multiple tissues, but in adults, its expression is highly restricted to rare cells in the intestine, breast, ovary, testis, hair follicles, brain, and eye. Using gene marking techniques, it has been shown that cells expressing LGR5 function as stem cells capable of giving rise to all other cell types found in the colonic epithelium and gastric epithelium. In contrast to LGR5, LGR6 is not regulated by Wnt signaling. LGR6-LacZ LacZ In knock-in mice, expression was found to be restricted to rare cells in the brain, breast, lung, and hair follicles. Lineage mapping showed that LGR6-positive cells, resident in the bulb of hair follicles, are located differently from LGR5-positive cells and give rise to the epidermis and sebaceous glands. Subsequent studies demonstrated that LGR6-expressing cells are stem cells that generate new skin, which is necessary during the wound healing process. There is also evidence that LGR6 is uniquely expressed by tumor stem cells. LGR6 was found to characterize a subpopulation of cells isolated from a human lung adenocarcinoma that could form new tumors when injected into mice.

[0050] LGR5 and LGR6 are expressed in many types of tumors, including breast, colon, and endometrial cancers. Several lines of evidence suggest that LGR6, but not LGR5, uniquely identifies stem cells in fallopian tube epithelium (FTE) and ovarian cancer. Thus, LGR6 appears to be a stem cell in tumors arising from FTE. Therefore, an embodiment of the present disclosure relates to the use of LGR6 as a target for tumor stem cells, since LGR6 is expressed on the cell surface, potentially making it accessible to antibodies and other types of tumor-targeting toxins.

[0051] R-spondins (RSPOs) are ligands for LGR5 and LGR6. RSPOs are a group of four secreted cysteine-rich paralogs (R-spondins 1-4), which share 40-60% sequence identity and overall similarity in domain organization. All four RSPO family members contain an N-terminal secretory signal peptide, two 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 ligands for both the LGR5 and LGR6 receptors, to which they can bind with high affinity. Therefore, certain aspects of the present disclosure relate to the use of RSPO-derived polypeptides that can be used to target therapeutic agents to cells expressing LGR4, LGR5, or LGR6, such as LGR6-expressing tumor stem cells. In one particular aspect, the Fu1-Fu2 domains of RSPO1 and / or RSPO2 are linked to a cytotoxic agent, such as the toxin monomethylaurostatin E (MMAE), to selectively target tumors that express high levels of LGR6.

[0052] RSPOs may play a role in cancer development and stem cell maintenance. Subpopulations of stem cells that support epithelia throughout the body proliferate and subsequently differentiate in response to growth factors in their niches, and their progeny progress through a series of transcriptional states as they differentiate 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 frizzled receptors and R-spondins (RSPOs) that bind to leucine-rich repeat-containing G protein-coupled receptors (LGRs). Cells in the immediate environment of the stem cell niche are the primary source of ligands that drive WNT signaling, and the most effective signaling molecules appear to be transmitted to stem cells over very short distances. RSPO1 has a predominant, but not exclusive, position among the four members of the RSPO family in malignant tissues, and among the members of the LGR receptor family, most evidence implies a central role for LGR5 and LGR6 ( Yan et al., 2017 ).

[0053] II. Cytotoxic agents 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.

[0054] A variety of conjugated drugs can be used as cytotoxic moieties, including compound classes such as maytansinoids, auristatins, amanitins, calicheamicins, psymberins, duocarmycins, anthracyclines, camptothecins, doxorubicins, taxols, and pyrrolobenzodiazepines. Specific examples of cytotoxic agents include paclitaxel, docetaxel, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, mithramycin, actinomycin, glucorticoids, puromycin, epirubicin, cyclophosphamide, methotrexate, cytarabine, f-fluorouracil, platins, streptozotocin, minomycin C, anthracyclines, dactinomycin or actinomycin, bleomycin, mithramycin, Included are anthramycin, duocarmycins, ifosfamide, mitoxantrone, daunomycin, carminomycin, animoterin, melphalan, esperamicins, lexitropsins, auristatins (e.g., auristatin E, auristatin F), AEB, AEVB, AEFP, MMAE, MMAF), eleuthrobin, netropsin, podophyllotoxins, maytansiods including maytansine and DM1, deruxtecan, and combretestin.

[0055] In some embodiments, the conjugate 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. Auristatins are synthetic analogs of the antitumor natural product dolastatin, and auristatins have previously been used as payloads in antibody-drug conjugates. MMAE is 100-1000 times more potent than doxorubicin (Adriamycin / Rubex).

[0056] In some embodiments, the cytotoxic moiety is a serine protease, such as granzyme B (GrB). GrB can include various 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 has the sequence TIFF2026503181000014.tif54168. Other serine proteases that can be utilized 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 (NCBI accession number B7ZMF6), or polypeptides having at least 90% or at least 95% sequence identity.

[0057] In some embodiments, the cytotoxic moiety is a cytotoxic protein. Cytotoxic proteins that can be used include AIF, Apaf (e.g., Apaf-1, Apaf-2, Apaf-3), order APO-2(L), APO-3(L), ​​apopain, Bad, Bak, Bax, Bcl-2, Bcl-x, and the like. L , Bcl-x S , 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-PK CS, 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- カッパ These include apoptotic factors or apoptosis-related proteins, including B, NuMa, p53, PAK-2, PARP, perforin, PITSLRE, PKC delta, pRb, presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, thymidine kinase from herpes simplex, TRADD, TRAF2, TRAIL-R1, TRAIL-R2, TRAIL-R3, and transglutaminase.

[0058] In a further embodiment, the cytotoxic agent can be selected from bioactive compounds including bispecific antibodies, nucleic acids such as DNA, mRNA, siRNA, and fragments thereof; pharmaceutical compounds such as various therapeutic agents; and radionuclides and cytotoxins, which can be targeted to desired tissues or cells by targeting moieties. These agents can act while they remain conjugated to the targeting protein or its portion, or if the linking group is easily cleavable in vivo, the agent can first be cleaved from the targeting protein.

[0059] Cytotoxic agents suitable for use in the present disclosure include microtubule inhibitors, topoisomerase I inhibitors (e.g., deruxtecan), intercalating agents, inhibitors of intracellular signaling pathways, kinase inhibitors, transcription inhibitors such as siRNA, aRNA, and miRNA, and DNA minor groove binders. Selected cytotoxic agents that can be used in selected embodiments of the present disclosure include calicheamicin, MMAE, DM1, deruxtecan, SN-38, MMAF, PE38, diphtheria toxin, and 90-yttrium. Selected cytotoxic agents that can be used in various embodiments are also discussed in Kostova et al. (2021), Chen et al. (2017), and Lambert et al. (2017).

[0060] A. Maytansinoids Maytansinoids (also called maytansine analogs) are semisynthetic agents derived from the natural product maytansine. Maytansinoids include emtansine, which can disrupt microtubule function. Maytansine can be obtained through fermentation, and the molecule can be synthetically modified to produce maytansinoids DM1, DM3, and DM4, as shown below. Different side chains on maytansinoids can confer different release and stability profiles. Maytansinoids include the following compounds: TIFF2026503181000015.tif56128 Maytansinoids that can be used in various embodiments are further discussed in, for example, Chen et al. (2017).

[0061] B. Auristatins A variety of auristatins can be used as cytotoxic moieties in various embodiments of the present disclosure. Auristatins are typically synthetic compounds that share some structural similarity with the natural compound dolastatin 10. Selected auristatins that can be used in embodiments of the present disclosure include: TIFF2026503181000016.tif96132 Selected auristatins that can be used in various embodiments are discussed, for example, in Kostova et al. (2021). In selected embodiments, the cytotoxic payload is MMAE, MMAF, or PF-06380101.

[0062] C. Tubulysins 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 typically function as microtubule-destabilizing agents. In some embodiments, the cytotoxic moiety is tubulysin A, tubulysin B, tubulysin C, tubulysin G, or tubulysin I. TIFF2026503181000017.tif39169

[0063] D. Eribulin The cytotoxic moiety can be eribulin, which is a tubulin-disrupting agent and has the following structure: TIFF2026503181000018.tif48128

[0064] E. Taxol derivatives The cytotoxic moiety can be a taxol derivative. Taxol derivatives can inhibit cell growth by stabilizing microtubule filaments. Taxol derivatives include docetaxel and paclitaxel.

[0065] F. DNA damaging agents In some embodiments, the cytotoxic moiety is calicheamicin. Calicheamicin is an antitumor antibiotic capable of causing double-stranded breaks in DNA. Calicheamicin has the following structure: TIFF2026503181000019.tif75128 Additional DNA damaging agents that may be used include anthramycin-based dimers and duocarmycins, anthracyclines, and camptothecins.

[0066] III. Linker In various embodiments of the present disclosure, a variety of linkers can be used to connect cytotoxic moieties to cell targeting moieties (e.g., R-spondin targeting moieties).Non-limiting examples of linkers that can be used are provided in Table 1 below.

[0067] Table 1: Examples of linkers TIFF2026503181000020.tif161161 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.

[0068] In some embodiments, a peptide linker is included in the constructs of the present disclosure near or adjacent to the sortase recognition sequence described herein. The linker (e.g., a G4S linker) may facilitate or aid in 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 XS)n, where x=1 to 4 and n=1 to 9), more preferably, can be repeated once or twice (e.g., (G X S)n, where x = 1–4 and n = 1–3).

[0069] IV. Variant Fc Regions In some embodiments, the constructs disclosed herein may contain wild-type Fc regions or mutant Fc regions. For example, the constructs may contain 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 may induce dimerization of the construct. In selected embodiments, it has been observed that the inclusion of a DHS Fc region in the construct can improve the stability, efficacy, and / or half-life of the construct.

[0070] As used herein, the term "DHS Fc" refers to a polypeptide comprising a variant human IgG Fc domain capable of binding to human FcRn at acidic pH, the Fc domain having the following substitutions: (i) aspartic acid at position 309 (L / V309D); (ii) histidine at position 311 (Q311H); and (iii) a substitution of either serine or tyrosine at position 434 (N434Y or N434S); the numbering of amino acid positions is according to the Kabat system. The DHS Fc region may optionally further comprise the substitution mutation V264E (e.g., mutations L / V309D, Q311H, N434S / Y; and optionally V264E), allowing the DHS Fc region to bind to human FcRn at acidic pH. Without wishing to be bound by any theory, inclusion of the DHS Fc region in the constructs of the present disclosure may increase half-life due to binding to human FcRn. DHS variants are disclosed in Lee et al. (2019) and US Patent 11059892, which are incorporated by reference in their entireties, and can be included in the constructs of the present disclosure.

[0071] In some embodiments, mutant or variant human Fc domains are provided that exhibit (i) enhanced binding at pH 5.8 and (ii) reduced or no detectable binding at pH 7.4 for FcRn compared to the corresponding wild-type Fc domain. The mutant or variant Fc domain may be a mutant or variant IgG domain. The mutant or variant Fc domain may be comprised in a polypeptide such as an antibody. In some embodiments, the mutant or variant Fc domain may be comprised in a therapeutic antibody, such as an agonist or antagonist antibody. In some embodiments, there are compositions comprising a polypeptide having a mutant or variant Fc domain ("antibody Fc domain") derived from a human IgG1-4 antibody. The variant Fc domain may be a variant of the wild-type human IgG1 Fc domain (SEQ ID NO:9), where the variant or variant Fc domain is capable of binding with increased affinity to FcRn at acidic pH and not at neutral pH. In some embodiments, the engineered Fc domain may exhibit increased affinity for FcRn, e.g., about 5-fold greater, than a glycosylated wild-type Fc domain. In further embodiments, variant human Fc domains of all other wild-type IgG subclasses (human IgG2, IgG3, and IgG4) are provided, which are capable of binding with increased affinity to FcRn at acidic pH and not at neutral pH. The variant or variant Fc domain may contain mutations (L / V309D, Q311H, N434S / Y), and optionally (V264E), compared 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 increase binding of the variant or variant Fc to FcRn at acidic pH (e.g., pH 5.8), but not at physiological pH (pH 7.4).For example, human IgG1, IgG2, IgG3, and IgG4 were mutated (L / V309D, Q311H, N434S) to generate SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively.

[0072] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids, up to the full-length sequence translated from a gene; a polypeptide of more 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" are used interchangeably herein.

[0073] As used herein, "amino acid residue" refers to any amino acid, amino acid derivative, or amino acid mimetic known to those skilled in the art. In certain embodiments, the residues of a proteinaceous molecule are consecutive, without any non-amino acid residues interrupting the sequence of amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a proteinaceous molecule may be interrupted by one or more non-amino acid moieties.

[0074] As used herein, a "distinct Fc domain" may be defined as a domain that differs from another Fc by at least one amino acid. Methods for generating libraries of nucleic acids encoding distinct antibody Fc domains or antibodies are well known in the art. For example, in some cases, Fc domains may be amplified by error-prone PCR. Furthermore, in certain cases, multiple antibody Fc domains may contain stretches of randomized amino acids (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In certain cases, specific mutations may be engineered into an Fc domain. For example, in some aspects, residues that are normally glycosylated in an antibody Fc domain may be mutated. Furthermore, in certain aspects, a normally glycosylated residue (or adjacent residues) may be used as a site for inserting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0075] The polypeptide may comprise a mutant or variant antibody Fc domain capable of binding to an FcR polypeptide. In some aspects, the Fc domain may be further defined as having a specified affinity for an FcR polypeptide under physiological conditions. Illustratively, the Fc domain may have an equilibrium dissociation constant of about 10 M to about 10 M under physiological conditions. Furthermore, in some aspects, an aglycosylated Fc domain may be defined as comprising one or more amino acid substitutions or insertions compared to a wild-type sequence, such as a human wild-type sequence. The Fc domain may be glycosylated or aglycosylated.

[0076] Means 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 the insertions or substitutions result in improved FcR binding ability relative to aglycosylated Fc regions, as discussed above. In some embodiments, the Fc domain is engineered to bind to one or more designated Fc receptors. Additionally or alternatively, the Fc domain can be engineered so that it does not specifically bind to one or more designated Fc receptors.

[0077] In some embodiments, the Fc domain comprises specific binding affinity for an FcR, such as human FcγRIA, FcγRIIA, FcγRIIB, FcγRIIc, FcγRIIIA, FcγRIIIb, FcαRI, or C1q. In some embodiments, the antibody or polypeptide containing the Fc domain is glycosylated and exhibits FcR binding similar to, essentially the same as, or the same as that of a wild-type antibody (e.g., compared to a corresponding IgG2, IgG3, or IgG4 antibody). In some embodiments, the antibody is glycosylated. In some embodiments, the antibody or polypeptide containing the Fc domain is aglycosylated. The binding affinity of an antibody Fc or other binding protein can be determined, for example, by the Scatchard analysis of Munson and Pollard (1980). Alternatively, binding affinity can be determined by surface plasmon resonance or any other well-known method for determining the kinetics and equilibrium constants of protein:protein interactions. Isolated IgG variants are provided below in Table 1. In various embodiments, if desired, mutations can be introduced into the IgG1 Fc domain (e.g., to give rise to SEQ ID NO:13), 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).

[0078] Table 1. IgG variants (sequence numbering is based on Kabat, mutations are specified below) TIFF2026503181000021.tif47128

[0079] As used herein, "position" means a location in the sequence of a protein. Positions may be numbered consecutively or according to established formats, such as the EU index for antibody numbering.

[0080] For all positions discussed herein, numbering is according to the EU index. "EU index" or "EU index as in Kabat" or "EU numbering scheme" refers to the numbering of the EU antibody (Edelman et al., 1969; Kabat et al., 1991; both of which are incorporated herein by reference in their entirety).

[0081] In certain embodiments, the size of at least one Fc polypeptide proteinaceous 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 therein. The compounds may comprise SEQ ID NOs:13-16 (human IgG1-4 Fc polypeptides) or the above-recited number of consecutive amino acids from SEQ ID NOs:13-16, and may be further defined as having a percent sequence identity or homology to a wild-type human IgG Fc domain (e.g., a percent sequence identity to any one of SEQ ID NOs:1-4).

[0082] V. Sortase Recognition Sequence 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 containing a cell targeting moiety (e.g., an R-spondin targeting polypeptide such as FuFu). Sortase-catalyzed acyl transfer reactions can enable 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)).

[0083] Sortases, sortase-mediated acyl transfer reactions, and their use in acyl transfer (sometimes also referred to as transpeptidation) for protein engineering are well known to those of skill in the art (see, e.g., International Patent Application Nos. PCT / US2010 / 000274 and PCT / US2011 / 033303). Sortase-catalyzed transpeptidation reactions can be used to ligate polypeptides containing a transamidase recognition motif to polypeptides bearing one or more N-terminal glycine residues. In some embodiments, the sortase recognition motif is an LPXT motif or an LPXT(G) motif. n As is known in the art, more efficient ligation can occur by replacing the C-terminal residue of the recognition sequence with a moiety that exhibits poor nucleophilicity upon release from the sortase.

[0084] Sortase-mediated acyl transfer reactions are catalyzed by the transamidase activity of a sortase. A transamidase is an enzyme capable of forming a peptide linkage (i.e., an amide linkage) between an acyl donor compound and a nucleophilic acyl acceptor containing an NH-CH moiety. In some embodiments, the sortase is sortase A (SrtA) or sortase E. However, any sortase or transamidase that catalyzes an acyl transfer reaction can be used in embodiments of the present disclosure.

[0085] 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, 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. Sortase recognition sequences include, for example, It could be TIFF2026503181000022.tif61161.

[0086] Sortase E can be used to covalently attach cytotoxic moieties to polypeptides containing an LGR-binding domain. Sortase E recognizes the sequence LAHTGG (SEQ ID NO:106). For this reason, sortase A and sortase E can be used separately to covalently attach cytotoxic moieties to both the C-terminus and N-terminus of a polypeptide. The cytotoxic moieties can have the same structure (e.g., both MMAE), or different cytotoxic moieties can be covalently attached to a polypeptide. The use of both sortase A and sortase E can provide advantages in production. For example, in the case of FcF2-LPETGG-His, MMAE warheads can be placed at both ends of the molecule by the sortase reaction; however, because the sortase A reaction is reversible, the process of adding a second MMAE to the N-terminus may remove the MMAE already present at the C-terminus. The recently isolated sortase E can be used to ligate substrates containing an N-terminal GG motif to the sequence LAHTGG (SEQ ID NO:106) on the C-terminal end of other proteins or peptides. Because sortase A and sortase E have high specificity for different recognition sequences (LPETGGG vs. LAHTGG), they can be used simultaneously or separately to covalently tether a cytotoxic moiety (e.g., MMAE) to both ends of a polypeptide (containing an LGR-binding moiety) containing a diglycine at the N-terminus and an LPXT (SEQ ID NO:41) (preferably LPETGG, SEQ ID NO:65) sequence at the C-terminus.

[0087] In some embodiments, the sortase recognition coding sequence is operably linked to the serine protease coding sequence via a linker. Any suitable linker known to those of skill in the art can be used. In certain embodiments, the linker is a (GGS), (GGGS; SEQ ID NO:75), or (G4S) linker. In some embodiments, the (G4S) linker can aid in the conformational flexibility of the sortase domain to recognize the sortase recognition motif.

[0088] VI. Partial Reduction of Disulfides If desired, partial reduction of disulfide can be used to link cytotoxic moieties to polypeptides containing LGR binding domains.The polypeptides provided herein can contain targetable disulfides in either the Fc hinge region (for example, the three disulfide bonds in immunoglobulin Fc domains) and / or the LGR binding domain (for example, the eight disulfide bonds in Fu1-Fu2 domains).Partial reduction of the disulfide bonds in the Fc domain or the disulfide bonds in the LGR binding domain can be used to covalently link cytotoxins by subsequently reacting with a linker containing a thiol-reactive maleimide group that has been previously coupled to the cytotoxin.These loading methods can be used to link 2, 3, 4, 5, 6, 7, 8 or more cytotoxins per molecule, but because excessive loading may distort protein structure and reduce plasma half-life, the method should be individualized. For example, a polypeptide (e.g., FcF2-His) can be exposed to TCEP at a concentration of 0.0005-5000 μM, followed (e.g., about 25 minutes later) by the addition of a maleimide-containing cytotoxin (e.g., maleimide-val / cit-PAB-MMAE) to the reaction (e.g., at a ratio of about 1:1 to 1:9, 1:2, or 1:8 (protein:cytotoxic agent)). The reaction can then be allowed to continue for a period of time sufficient to achieve binding of the cytotoxic agent. In some preferred embodiments, the cytotoxic agent comprises a cleavable linker (e.g., comprising a valine (Val)-citrulline (Cit) bond).

[0089] VII. RADIOTHERAPY AND IMAGING AGENTS In some embodiments, the therapeutic compounds provided herein can include radiotherapy or imaging agents. For example, the therapeutic compounds can be covalently bound or conjugated to a radioisotope, such as iodine-131, strontium-89, samarium-153, or radium-223. The polypeptides described herein (e.g., SEQ ID NOs: 1-4, 19, or 20) can be covalently bound or conjugated to an imaging or contrast agent. The imaging agent can be, for example, an iodinated contrast medium, an ionic iodinated contrast medium, an MRI contrast agent (e.g., gadolinium), a diagnostic dye, a non-iodinated contrast medium, a non-ionic iodinated contrast medium, or an ultrasound contrast medium. Additional radiotherapy and imaging agents that can be covalently bound or conjugated to the compounds or polypeptides described herein include, for example, lutetium-177.

[0090] VIII. Modified Proteins and Polypeptides Some embodiments concern modified proteins and polypeptides, particularly those that exhibit at least one functional activity comparable to the unmodified version, but which further possess additional advantages over the unmodified version, such as inhibiting B-cell activation, being easier or cheaper to produce, inducing fewer side effects, and / or having better or longer efficacy or bioavailability. Thus, when the present application refers to the function or activity of a "modified protein" or "modified polypeptide," those skilled in the art will understand that this includes, for example, proteins or polypeptides that (1) exert at least one of the same activities or have at least one of the same specificities as the unmodified protein or polypeptide, but may have a different level of another activity or specificity; and (2) possess additional advantages over the unmodified protein or polypeptide. Determining activity, particularly with respect to protein activity, can be accomplished using assays familiar to those skilled in the art and can include, for example, the use of native and / or recombinant versions of either the modified or unmodified protein or polypeptide for comparison purposes. It is specifically contemplated that embodiments involving "modified proteins" can be implemented with respect to "modified polypeptides," and vice versa. In addition to the modified proteins and polypeptides discussed herein, embodiments may include the domains, polypeptides, and proteins described in PCT Publication WO 2008 / 137475, which is specifically incorporated herein by reference.

[0091] Modified proteins can have amino acid deletions and / or substitutions; 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 those with fusion proteins or proteins with linkers. This can include the insertion of a targeting peptide or targeting polypeptide, or simply the insertion of a single residue. Terminal additions, called fusion proteins, are discussed below.

[0092] A "modified deletion protein" lacks one or more residues of the native protein, but retains the specificity and / or activity of the native protein. A "modified deletion protein" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is one in which amino acid residues have been deleted from at least one antigenic region (i.e., a region of the protein determined to be antigenic in a particular organism, such as the type of organism to which the modified protein may be administered).

[0093] Substitution or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and can be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or bioavailability. Substitutions may or may not be conservative, that is, one amino acid is replaced with one 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.

[0094] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, but still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including maintenance of the biological protein activity involved in protein expression. The addition of terminal sequences applies particularly to nucleic acid sequences that may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or various internal sequences, i.e., introns, that are known to be present within genes.

[0095] A modified polypeptide can be characterized as having a certain percentage of identity to an unmodified polypeptide or to any polypeptide sequence described herein (e.g., SEQ ID NOs: 1-4, 19, or 20). The percentage of identity can be at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity between the modified and unmodified polypeptide, or any range derivable therein. It is contemplated that the percentage of identity discussed above can refer to a particular region of the polypeptide compared to an unmodified region of the polypeptide.

[0096] When making such changes, the hydropathic index of amino acids can be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art. It is recognized that the relative hydropathic characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0097] It is also understood in the art that substitution of like amino acids can be made effectively based on hydrophilicity. U.S. Patent No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as influenced by the hydrophilicity of its neighboring amino acids, correlates with a biological property 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 is understood that one amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0098] As outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into consideration the various aforementioned characteristics are well known to those of skill in the art and include: arginine for lysine; glutamic acid for aspartic acid; serine for threonine; glutamine for asparagine; and valine for leucine and isoleucine.

[0099] It has been observed herein that in some aspects, glycosylation can have a significant effect on the yield and pharmacology of polypeptides provided herein.For example, glycosylation changes, such as those caused by mutation or substitution in asparagine, can cause changes in protein folding, stability, pharmacokinetics and other properties of its pharmacology.As those skilled in the art will understand, different amounts of glycosylation can be achieved by either including substitution mutations (for example, asparagine to alanine) in polypeptide, or by using different types of cells, such as yeast, insect, human or bacterial cells, to produce polypeptides provided herein.

[0100] IX. Pharmaceutical Preparations Pharmaceutical compositions according to embodiments of the present invention comprise an effective amount of one or more compounds of the present invention and at least one additional active ingredient, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, such as a human, as appropriate. The preparation of pharmaceutical compositions containing at least one chimeric polypeptide or additional active ingredient is described in detail in Remington: The Science and Practice of Pharmacy (23 rd It will be known to those of skill in the art in light of the present disclosure, as exemplified by the publication of the American Society of Biological Chemists (ASCI), 2014, pp. 117-119, 2020. Moreover, it will be understood that for animal (e.g., human) administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0101] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizing agents, gels, binders, excipients, disintegrating agents, lubricants, sweetening agents, flavoring agents, dyes, and the like, as well as combinations thereof, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions, except insofar as it is incompatible with the active ingredient.

[0102] The cell-targeted cytotoxic agent may comprise different types of carriers depending on whether it is administered in solid, liquid, or aerosol form, and whether it needs to be sterile for the route of administration, such as injection. Therapies of embodiments of the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, local irrigation to directly bathe target cells, via a catheter, via lavage, in a cream, in a lipid composition (e.g., liposomes), or by other methods known to those skilled in the art or any combination of the foregoing (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[0103] The actual dosage amount of the compositions of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of the condition, the type of disease being treated, previous or ongoing therapeutic interventions, the patient's idiopathic condition, etc., as well as based on the route of administration. In any event, the practitioner responsible for administration will determine the concentration of active ingredient(s) in the composition and appropriate dose for the individual subject.

[0104] In certain embodiments, pharmaceutical compositions may contain at least about 0.1% of an active compound, such as, for example, a therapeutic compound provided herein (e.g., FcF2-MMAE). The active compound may comprise, for example, from about 2% to about 75%, or from about 25% to about 60% of the weight of the unit, and any range derivable therein. As shown in the Examples below, subnanomolar potency was observed in vitro for select therapeutic compounds (e.g., FcF2-MMAE), and in vivo activity was detected at doses of only 0.125 nmol / g (10.6 mg / kg). In some embodiments, a dosage 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 therein, can be administered to a mammalian subject, such as a human. For example, a dosage of about 0.5-20 mg / kg, or any range derivable therein, can be administered to a human subject.

[0105] The therapeutic compound can be administered once or repeatedly to the same subject. In some embodiments, the therapeutic compound is administered repeatedly to the same subject (e.g., a human patient) with at least 1, 2, 3, 4, 5, 6, 7 days or more, or 1, 2, 3, or 4 weeks between administrations. As shown in the following examples, improved efficacy was observed when the therapeutic compound (FcF2-MMAE) was injected every 7 days instead of every 4 days. The therapeutic compound can be administered repeatedly to the same subject every week, or for a period of several months or more, 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).

[0106] In any case, the composition may contain various antioxidants to slow the oxidation of one or more components. Additionally, the prevention of the action of microorganisms can be achieved by preservatives, such as various antibacterial and antifungal agents, including, but not limited to, parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0107] In embodiments in which the composition is provided in liquid form, the carrier can be a solvent or 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. The appropriate fluidity can be maintained, for example, by using a coating such as lecithin; by maintaining the required particle size by dispersion in a carrier, such as a liquid polyol or lipid; by using surfactants, such as hydroxypropylcellulose; or by a combination of such methods. In many cases, it is preferable to include an isotonic agent, such as, for example, sugars, sodium chloride, or a combination thereof.

[0108] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in a suitable solvent with various other ingredients listed above, if necessary, and then filtering and sterilizing. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for preparing sterile injectable solutions, suspensions, or emulsions, the preferred preparation method is vacuum drying or freeze-drying, where a powder of the active ingredient and any desired additional ingredients is obtained from a liquid medium that has previously been sterile-filtered. The liquid medium should be suitably buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose before injection. The preparation of highly concentrated compositions for direct injection is also contemplated, in which case DMSO is used as a solvent, which is expected to cause extremely rapid penetration and deliver a high concentration of the active agent to a small area.

[0109] The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be minimized to safe levels, e.g., less than 0.5 ng / mg protein.

[0110] In certain embodiments, prolonged absorption of injectable compositions can be achieved by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin, or combinations thereof.

[0111] X. Cancer The methods and compositions provided herein can treat a variety of cancer cells.In some embodiments, cancer cells express LGR4, LGR5, and / or LGR6.Cancer cells can be cancer stem cells (CSCs), cancer cells with increased stemness, or cancers that express LGR4, LGR5, or LGR6 regardless of functional classification.

[0112] In some embodiments, cancer contains CSC.Solid tumors have cell subpopulations that exhibit many characteristics of stem cells, such as the ability to form spheroids or initiate new tumors.As opposed to stem cells that reside in organized epithelia, many CSCs require RSPO to actively grow in culture (Sato et al., 2009; Barker et al., 2010).Removing a small percentage of cells that are CSCs in tumors can reduce or stop the further expansion of tumors by reducing the supply of more differentiated cells that make up the majority of the population.

[0113] In some embodiments, the cancer is ovarian cancer. High-grade serous ovarian cancer can arise from either the ovarian surface epithelium or the fallopian tube epithelium, with the latter predominating (Zhang et al., 2019). Lineage tracing studies in mice suggest that LGR5 marks stem cell populations in the ovary, while LGR6 marks stem cells in the fallopian tube epithelium in mice and humans (de Lau et al., 2014; Kessler et al., 2015; Zhang et al., 2019). TCGA data indicate that high-grade serous ovarian cancer may express high levels of LGR5 mRNA and LGR6 mRNA. In addition, with the exception of mesothelioma, ovarian cancer has the highest median expression of RSPO1 mRNA when compared to all other tumor types in the database (Schindler et al., 2017). Without being bound by any theory, this data supports the idea that ovarian cancers may rely on RSPO1 to support their CSC population. As shown in the examples below, data is provided herein that supports the idea that the compounds provided herein can utilize the receptor binding domain of RSPO1 equipped 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.

[0114] Cancer cells that can be treated with cell-targeting constructs according to this embodiment include, but are not limited to, cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, cervix, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer can be specifically of the following histological types, but the cancer is not limited to these: malignant neoplasms; 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; mixed hepatobiliary carcinoma with hepatocellular carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenomatous intrapolypoid adenocarcinoma; familial polyposis coli adenocarcinoma; solid tumors; malignant carcinoid tumors; brachytherapy; chioro-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; Adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; malignant breast Adenoma; Malignant ovarian stromal tumor; Malignant theca cell tumor; Malignant granulosa cell tumor; Malignant androblastoma; Sertoli cell carcinoma; Malignant Leydig cell tumor; Malignant lipid cell tumor; Malignant paraganglioma; Malignant extramammary paraganglioma; Pheochromocytoma; Glomus angiosarcoma; 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; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; malignant mixed tumor; Müllerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; parosteal osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastoma; malignant ameloblastoma;Ameloblastic fibrosarcoma; Malignant pinealoma; Chordoma; Malignant glioma; Ependymoma; Astrocytoma; Protoplasmic astrocytoma; Fibrillary astrocytoma; Astroblastoma; Glioblastoma; Oligodendroglioma; Oligodendroglioma; Anaplastic neuroectodermal; Cerebellar sarcoma; Ganglioneuroblastoma; Neuroblastoma; Retinoblastoma; Olfactory neurogenic tumor; Malignant meningioma; Neurofibrosarcoma; Malignant neurilemmoma; Malignant granular cell tumor; Malignant lymphoma; Hodgkin's disease; Hodgkin's; Paragranuloma; Small lymphocytic malignancy Lymphoma; diffuse large cell lymphoma; follicular lymphoma; mycosis fungoides; other specified non-Hodgkin's 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; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0115] VI. Combination Therapy To increase 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 disease of interest. In some embodiments, a therapeutic compound (e.g., a cell-targeted cytotoxic agent, including an auristatin) is administered to a mammalian subject in combination with a second anti-cancer agent or therapy to treat cancer in the subject. In some embodiments, the targeted cytotoxic agents of the present disclosure can be administered to a subject in combination with an immunotherapeutic agent, an antibacterial agent (e.g., an antibiotic), or an antiviral agent to treat a bacterial or viral infection, respectively, in the subject.

[0116] As a non-limiting example, cancer treatment can be achieved using the therapeutic compounds of the present disclosure in combination with other anti-cancer agents. "Anti-cancer" agents can negatively affect cancer in a subject by, for example, killing cancer cells, inducing apoptosis in 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 tumors or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting cancer progression, or increasing the lifespan of a subject with cancer. More generally, these other compositions are provided in a combined amount effective to kill or inhibit cell proliferation. This process can involve contacting cells with the agents or multiple factors simultaneously. This can be achieved by contacting cells with a single composition or pharmacological formulation containing both agents, or by simultaneously contacting cells with two separate compositions or formulations, one composition containing an anti-cancer peptide or nanoparticle complex and the other composition containing a second agent. In some embodiments, an anti-cancer peptide is one agent and a cell-targeted cytotoxic agent (e.g., a cell-targeted cytotoxic agent, including an auristatin) is the other agent.

[0117] Treatment with a therapeutic compound of the present disclosure may precede or follow treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the therapeutic compound are applied to cells separately, it is generally ensured that no significant period of time passes between the times of delivery so that the agent and the therapeutic compound can still exert their advantageously combined effect on the cells. In such instances, it is contemplated that cells may be contacted with both modalities within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. In some circumstances, it may be desirable to extend the period of treatment significantly, with several days (e.g., 2, 3, 4, 5, 6, or 7 days) to several weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapsed between each administration. A wide range of dosing schedules may be employed.

[0118] Various combinations can be employed, where the therapeutic compound-based therapy is "A" (e.g., administration of cell-targeted cytotoxic agents, including auristatins) and a secondary agent, such as radiation therapy, chemotherapy, or an anti-inflammatory agent, is "B": TIFF2026503181000023.tif18128

[0119] In certain embodiments, administration of the therapies of the present invention to patients will follow typical protocols for the administration of chemotherapeutic agents, taking into account the toxicity, if any, of the vector. It is expected that treatment cycles will be repeated as necessary. It is also contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described hyperproliferative cell therapies.

[0120] A. Chemotherapy Cancer therapy also includes various combination therapies. In some aspects, the therapeutic compounds disclosed herein (e.g., cell-targeted cytotoxic agents, including auristatins) 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 inhibitor of EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor, or BRAF. 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, Vemura phenib, 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.

[0121] Still further combination chemotherapy agents include, for example, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, 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); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictyin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, fenesterine, prednisolone Nitrogen mustards, such as mustard, trofosfamide, and uracil mustard; nitrosuureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 11 and calicheamicin omega 11); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related enediyne antibiotic chromophores, aclacinomycins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potf anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, trimetrexate; fludarabine, 6-mercaptopurine, thiaminopyramine, thiaminopyramine; Purine analogues such as phosphate and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as mitotane and trilostane; folic acid supplements such as florinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziconazole; elformithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan;Lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracrin A) A), Roridin A, and Anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel, gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein tranferase inhibitors, transplatin, and pharmaceutically acceptable salts, acids, or derivatives of any of the above. In certain embodiments, the compositions provided herein may be used in combination with gefitinib. In other embodiments, the embodiments of the present invention may be practiced in combination with Gleevac (e.g., about 400 to about 800 mg / day of Gleevac may be administered to a patient). In certain embodiments, one or more chemotherapeutic agents may be used in combination with the compositions described herein.

[0122] B. Radiation therapy Radiation therapy is widely used in treatment and includes what is commonly known as gamma rays, X-rays, and / or the directed delivery of radioisotopes to tumor cells. Other forms of radiation therapy, such as microwave and UV irradiation, are also contemplated. These factors can affect a wide range of damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray dose ranges range from daily doses of 50 to 200 roentgens over prolonged periods (3 to 4 weeks) to single doses of 2000 to 6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by the neoplastic cells.

[0123] The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic composition and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell. To achieve cell killing or stasis, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent cell division.

[0124] C. Immunotherapy Immunotherapeutics generally rely 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 to some marker on the surface of tumor cells. 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).

[0125] Antibodies can act alone 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 drugs, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and act simply as targeting agents. Alternatively, effectors can be lymphocytes bearing surface molecules that interact either directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0126] Thus, immunotherapy can be used as part of a combined therapy in conjunction with serine protease therapy in accordance with embodiments of the present invention. General approaches to combined therapy are discussed below. Generally, tumor cells must possess some marker that is amenable to targeting, i.e., not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of embodiments of the present invention. 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, erbB, and p155.

[0127] D. Gene Therapy In yet another embodiment, the secondary treatment is gene therapy, in which a therapeutic polynucleotide is administered before, after, or simultaneously with the therapeutic composition. Viral vectors for the expression of gene products are well known in the art, including eukaryotic expression systems such as adenovirus, adeno-associated virus, retrovirus, herpesvirus, lentivirus, poxvirus including vaccinia virus, and papillomavirus including SV40. Alternatively, administration of the expression construct can be achieved using lipid-based vectors such as liposomes or DOTAP:cholesterol vesicles. All of these methods are well known in the art (see, for example, Sambrook et al., 1989; Ausubel et al., 1998; Ausubel, 1996).

[0128] Delivery of a vector encoding one of the following gene products will have a combined anti-hyperproliferative effect on the target tissue: A variety of proteins are encompassed within the embodiments of the present invention and are well known in the art.

[0129] E.Surgery Approximately 60% of people with cancer will undergo some type of surgery, including preventative surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatments provided herein, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0130] Curative surgery includes resection, 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 part of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and miscopy-controlled surgery (Mohs surgery). It is further contemplated that embodiments of the present invention can be used in conjunction with the removal of superficial cancers, pre-cancers, or incidental amounts of normal tissue.

[0131] When all parts of cancerous cells, tissues, or tumors are removed, a cavity may be formed in the body. Treatment can be achieved by perfusion, direct injection, or local application of additional anti-cancer therapy to the area. Such treatment 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 be at different dosages. [Example]

[0132] IV. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art will recognize that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and can thus be considered to constitute preferred modes for its practice. However, those of skill in the art, in light of the present disclosure, will recognize that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0133] Example 1 Compounds for targeting LGR5-expressing stem cells in ovarian cancer Design of FcF2-His A schematic diagram of FcF2-His is presented at the top of Figure 1. The protein contains a variant human IgG1 Fc domain (Lee et al., 2019) connected to the Fu1-Fu2 domain of human RSPO1 via two Gly4Ser spacers. Two additional Gly4Ser spacers separate the Fu1-Fu2 domain from the LPETGG sortase recognition sequence, which is followed by an 8xHis tag to aid in purification. Each Fc-(Fu1-Fu2)-LPETGG-His (abbreviated FcF2-His) sequence contains 391 amino acids. The Fc domain dimerizes through three intermolecular disulfide bonds: one bond connecting the Fc to the light chain in antibodies and two bonds connecting the Fc domains together at their hinge regions. The resulting protein has a calculated overall MW of 85,284 Da. Glycosylation is present on the Fc domain and on residue N137 (RSPO1 numbering) in the Fu1-Fu2 domain.

[0134] Sortase-mediated conjugation of monomethyl auristatin E (MMAE) The bottom part of Figure 1 shows a schematic diagram of the conversion of FcF2-His to FcF2-MMAE. The sortase enzyme cleaves the LPETGG sequence between the threonine and glycine and forms a transient thioester bond with the cysteine ​​in the enzyme's active site that is subsequently attacked by the N-terminal glycine of the protease-sensitive GGG-vc-PAB-MMAE linker. The His tag is lost during the reaction. This results in precise covalent loading of one MMAE molecule on each arm of FcF2-His, generating a homogenous population of conjugated FcF2-MMAE molecules.

[0135] Generation, purification, and characterization of FcF2-MMAE FcF2-His was produced by transient transfection of HEK293E cells with the vector pcDNA3.1-FcF2-8xHis and purified from the cell supernatant by capture on Ni-NTA resin followed by ion exchange chromatography. FcF2-His was then reacted with sortase-His and GGG-vc-PAB-MMAE at 37°C for 4 hours to produce FcF2-MMAE. Across 14 batches, the average yield of FcF2-His was 23.4±2.3 mg / L; sortase efficiency was 77±8.6%; and the final product yield was 18.8±3.5 mg / L (mean±SEM).

[0136] Figure 2 shows the results of characterization of FcF2-His and FcF2-MMAE by reversed-phase HPLC, size-exclusion chromatography, and reducing and non-reducing SDS-PAGE. In reversed-phase HPLC analysis using a C4 column, both forms of the molecule run as a single, clearly defined peak (Figures 2A and 2D). Analysis on an HPLC 300 size-exclusion column indicated that FcF2-His and FcF2-MMAE exist in solution both as dimers and as dimers of already dimeric molecules, hereafter referred to as tetramers (Figures 2B and 2E). A minor high-MW species (7%) eluted immediately before the major tetramer peak of FcF2-His; FcF2-MMAE contained a third, lower-MW peak. Nonreducing SDS-PAGE analysis of FcF2-His (Figure 2C) showed that in the presence of SDS detergent in the loading buffer, the majority of the tetramer migrated at a molecular weight consistent with the size of the FcF2-His dimer (85.3 kD). Under reducing conditions, the majority of the protein migrated as a doublet at approximately 42–46 kD. To better understand this doublet, FcF2-His was treated with PNGase to remove N-linked sugars. As shown in Figure 10A, this resulted in the doublet converging 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 was abolished. Extensive comparison of the wild-type and N137A forms of R1FF-MMAE indicated no change in cytotoxicity or plasma pharmacokinetics, indicating that glycosylation at this site has little functional consequence on these parameters.

[0137] Nonreducing SDS-PAGE analysis of FcF2-MMAE (Figure 2E) showed that the majority of FcF2-MMAE migrated at a MW of 72-78 kD in the presence of SDS detergent in the loading buffer, and under reducing conditions, it migrated at 42-48 kD, similar to the behavior of FcF2-His under nonreducing and reducing conditions. Western blot analysis of FcF2-MMAE using anti-RSPO1 and anti-MMAE antibodies indicated that the major protein-staining band on the SDS-PAGE gel contained all components of the molecule (Fc, Fu1-Fu2, and MMAE) (Figure 10B), which was also confirmed for all major peaks visible in the C4 and SEC300 profiles.

[0138] In vitro cytotoxicity and selectivity of FcF2-MMAE HEK293 cells and the human ovarian cancer cell line OVCAR8 were molecularly engineered to stably express increased levels of LGR5. Both parental forms of these lines express variable, but not clearly defined, levels of all three LGR receptors. Flow cytometry analysis using an antibody against LGR5 demonstrated 8.7-fold higher levels in HEK293 / LGR5 cells than in HEK293 / EV controls; in the case of OVCAR8 cells, the difference was approximately 25-fold (Figures 11A and 11B, respectively). To assess viability, cytotoxicity assays were performed using 120-hour exposure to drug and CCK8 reagent. Figures 3A and 3B demonstrate higher LGR5 receptor-dependent cytotoxicity in isogenic HEK293 pairs; for the two batches tested, HEK293 / LGR5 cells were 21- and 46-fold more sensitive than HEK293 / EV cells. Significant differential effects were also observed when testing using the isogenic pairs OVCAR8 / EV and OVCAR8 / LGR5; the two most recent batches demonstrated differential inhibition of cell growth by 77-fold and 87-fold (Figure 3C-D). Across the most recent batches that met all release criteria, the average IC against OVCAR8 / EV cells was 1.25. 50The mean IC for OVACAR8 / LGR5 cells was 4.5 ± 1.1 nM (SEM). 50 was 0.059 ± 0.015 nM; IC 50 The mean ratio of values ​​was 76±5 (N=3).

[0139] To address the question of whether the differential inhibition of growth in LGR5-poor versus LGR5-rich isogenic pairs could be due to differences in sensitivity to free MMAE rather than Fu1-Fu2-directed targeting, we used the same assay to measure the IC of free MMAE. 50 This was addressed by determining IC values. HEK293 / LGR5 cells were 1.6-fold more resistant to free MMAE than HEK293 / EV cells (19.2 ± 0.4 vs. 30.5 ± 2.5, p = 0.03 N = 3). OVCAR8 / LGR5 cells were 1.5-fold more resistant to free MMAE than OVCAR8 / EV cells (IC 50 (Values ​​were 129 nM vs. 85 nM, respectively.) Thus, both LG5R-rich cell types were indeed slightly resistant to free MMAE, providing evidence that the selectivity exhibited by FcF2-MMAE is due to targeting via the Fu1-Fu2 domain.

[0140] Further evidence was provided by the observation that treating a population of OVCAR8 cells with FcF2-MMAE resulted in the depletion of LGR-positive cells from the population. A mixed population of OVCAR8 cells expressing low and higher levels of LGR5 was exposed to 10 nM FcF2-MMAE for 96 hours. Prior to treatment, flow cytometry analysis demonstrated a bimodal distribution of LGR5-expressing cells (Figure 11C). FcF2-MMAE treatment caused the loss of a large proportion of cells expressing high levels of LGR5, but produced a significantly lesser effect on the subset of cells expressing low levels of LGR5. This data supports the IC 50 The difference in values ​​provides evidence of differential killing as a function of LGR5 expression levels.

[0141] Cytotoxicity against human wild-type ovarian cancer cell lines To aid in the selection of an appropriate xenograft model for efficacy testing, the cytotoxicity of FcF2-MMAE was assessed on a panel of eight human ovarian cancer cell lines. The concentration-survival curves shown in Figure 3E demonstrate the IC 50 IC values ​​ranged from 3.8 to 29.6 nM; IC values ​​were measured in 7 of 8 cell lines. 50 <10 nM. Thus, although the aggregate level of all LGR expression is unknown for these cell lines, the data indicate that FcF2-MMAE is highly potent across this panel of ovarian cancer cell lines.

[0142] Determinants of cytotoxic selectivity RSPO1 is a dual-specific ligand. The Fu1 domain of RSPO1 binds to the ubiquitin ligase receptors ZNRF3 and RNF43, while the Fu2 domain binds to LGR4, LGR5, or LGR6. The relative contribution of each of these receptor types to the selectivity of FcF2-MMAE for OVCAR8 / EV and OVCAR8 / LGR5 cells was investigated by introducing mutations into one or the other Fu domain that have previously been shown to abolish binding to its corresponding receptor. The cytotoxicity of the mutant forms was tested in in vitro OVCAR8 / EV and OVCAR8 / LGR5 models. The growth inhibition curves presented in Figure 4 indicate that both domains contribute significantly to FcF2-MMAE selectivity. The Q71R mutation in Fu1 significantly reduced the IC 50 The ratio decreased from 19.5 to 5.1 (p=0.05), and the F106R-F110R mutation in Fu2 reduced the ratio to 2.1 (p=0.03), eliminating selectivity when both sets of mutations were present. Thus, in this model system, the ability to bind to both types of receptors is highly important for successful internalization of FcF2-MMAE and release of free MMAE.

[0143] Pharmacokinetics of FcF2-MMAE in mice BALB / c mice were given a bolus injection of 0.1 nmol / g (9 μg / g) FcF2-MMAE, and plasma samples were obtained from three mice at each sampling time point. FcF2-MMAE concentrations were measured using an ELISA with a different species of monoclonal capture antibody and a polyclonal detection antibody prepared by immunization with RSPO1 (LLQ 1.5 pmol / ml). Figure 5A presents the composite plasma decay curve; analysis using WinNonLin curve-fitting software yielded estimates of 4.47 h for the distribution phase half-life and 29.7 h for the terminal phase half-life. The initial half-life was shorter than expected. One mechanism by which FcF2-MMAE may be removed from the plasma compartment is through binding to red blood cells, white blood cells, or platelets. However, when the precipitated formed elements were reconstituted to their normal volume with FcF2-MMAE-spiked plasma, there was no significant removal of the drug from the plasma fraction over 27 h at 4°C (Fig. 5B), suggesting that the rapid initial half-life was primarily due to distribution to tissues.

[0144] Efficacy of FcF2-MMAE in a human ovarian cancer xenograft model We investigated both the in vivo efficacy and selectivity of FcF2-MMAE using xenografts established from isogenic pairs of OVCAR8 / EV and OCVAR8 / LGR5 cells. In the experiment shown in Figure 6A, mice were treated with four doses of 0.5 nmol / g (42 mg / kg) given by the IP route every four days as soon as tumors became palpable. FcF2-MMAE had a much greater effect on OVCAR8 / LGR5 tumors than on OVCAR8 / EV tumors. The difference in growth rate was evident by day 20 and persisted until day 60, at which point FcF2-MMAE-treated OVCAR8 / LGR5 tumors were, on average, only 35% of the size of vehicle-treated tumors. FcF2-MMAE was also effective in slowing the growth rate of OVCAR8 / EV cells, consistent with evidence that the cells express several combinations of LGR receptors; however, the curves for drug- and vehicle-treated tumors did not separate until 30 days, and at 60 days, FcF2-treated OVCAR8 / EV tumors were, on average, 63% the size of control tumors. Of note, the reduction in tumor growth rate was maintained for more than 1.5 months after the final dose of FcF2-MMAE for both OVCAR8 / EV and OCVAR8 / LGR5 tumors, an effect consistent with targeting stem cells in tumors whose growth rate is difficult to restore. The lack of dose-limiting toxicity prompted a second efficacy study with the same design but using a 1.5 nmol / g dose and a q7d x 4 schedule, which demonstrated the same large difference in FcF2-MMAE efficacy between the two types of xenografts (Figure 12A).

[0145] Figure 6B shows the change in average mouse weight during and after four injections of FcF2-MMAE at a dose of 0.5 nmol / g, and Figure 12B shows the same data for a dose of 1.5 nmol / g given on a q7d x 4 schedule. The first injection caused a transient weight loss on average, which rapidly recovered, and mice gained weight overall over the treatment period in both studies. No animals treated with 0.5 nmol / g died before being sacrificed due to tumor burden in either the control or treatment groups. 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.

[0146] The OVCAR8 / LGR5 model was used to investigate the efficacy and toxicity of FcF2-MMAE as a function of dose using a more clinically relevant 7-day schedule. Growth curves presented in Figure 7A demonstrate increasing efficacy as dose increased from 0.125 nmol / g to 1.0 nmol / g. Even a dose of 0.125 nmol / g (10.6 mg / kg) produced a significant reduction in growth rate (final tumor volume was 66% of untreated controls). As shown in Figure 7B, this dose did not produce weight loss. Efficacy and maximum weight loss increased with dose up to 0.75 nmol / g; no further increase in either parameter was observed at 1.0 nmol / g. This experiment suggests a therapeutic window spanning an 8-fold dose range, but refining this estimate will require FcF2-MMAE produced under GMP conditions.

[0147] Efficacy of FcF2-MMAE in a wild-type human ovarian cancer xenograft model The efficacy of FcF2-MMAE was investigated in three ovarian cancer xenograft models established from cell lines without any genetic modifications to increase LGR5 expression. A single dose of 1.0 nmol / g given IP once tumors became palpable produced long-lasting tumor growth delay in KF-28 and CAOV3 models without dose-limiting weight loss (Figure 8A / C). At a dose of 0.5 nmol / g, FcF2-MMAE was active against IGROV8 and CAOV3 xenografts on a q7d x 4 schedule without significant toxicity in the form of weight loss (Figure 8B / D). These data provide evidence for efficacy in three different human ovarian cancer models without molecular manipulation to increase LGR5 expression. Because it is currently impossible to precisely define the levels of LGR4, LGR5, and LGR6 expression in these models, sensitivity cannot be linked to the expression of any one of them. However, the data are consistent with the notion that stem-like cells in each of these tumors collectively express enough of these receptors to respond to FcF2-MMAE.

[0148] Selective targeting of stem cells in tumors may reduce or limit tumor spread and the metastatic potential of cancers. The rationale for using the receptor-binding Fu1-Fu2 domain of RSPO1 to achieve this goal is based on its high affinity for LGR5 and LGR6 (approximately 3 nM) (Carmon et al., 2011) and evidence that expression of these receptors marks stem cells in tumors similarly to their role in normal epithelium. This approach is particularly interesting in the case of ovarian cancer, because LGR5 and LGR6 mark stem cells in the ovarian surface and fallopian epithelium, from which ovarian cancer arises (Zhang et al., 2019), and because 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 in a bispecific manner to both LGR and either ZNRF3 or RNF43 favors specificity and reduces the risk of immunogenicity because the domain is part of a normal human protein.

[0149] FcF2-MMAE differs from the R1FF-MMAE molecule ( Yu et al., 2021 ), which consists solely of the Fu1-Fu2 domain of RSPO1 linked to MMAE through a cleavable linker. Although R1FF-MMAE exhibited LGR5-dependent cytotoxicity and in vivo activity, it did not possess optimal pharmaceutical properties. The modifications made to create FcF2-MMAE aimed to: (a) increase protein yield from transiently transfected cultures by exploiting the chaperone function of Fc to improve folding; (b) dimerize it so that it carries two MMAE molecules instead of one; (c) increase plasma half-life by including a mutant form of Fc with improved FcRn-binding characteristics (Lee et al., 2019); and (d) increase avidity of binding to LGR and ZNRF3 / RNF43 by including two copies of the Fu1-Fu2 domain instead of just one. Achievement of these goals was evidenced by significantly greater yields of the FcF2-His precursor, greater potency and selectivity when tested in isogenic OVCAR8 / EV and OVCAR8 / LGR5 cells, a six-fold increase in terminal plasma half-life, and improved efficacy in xenograft models. 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, and remained high.

[0150] FcF2-MMAE produces LGR5-dependent killing in vitro and differentially depletes cells with the highest levels of this receptor. 50The 76-fold mean difference in values ​​was sufficient to produce significantly greater in vivo efficacy against LGR5-enriched OVCAR8 / LGR5 cells. Cytotoxicity assay results indicate that engagement with both the LGR and ubiquitin ligase receptors is important for optimal selectivity. Given its terminal half-life of 27.4 hours, it was intriguing that efficacy was better when injected every 7 days rather than every 4 days. Consistent with its subnanomolar potency in vitro, in vivo activity was detected at a dose of only 0.125 nmol / g (10.6 mg / kg). Efficacy increased with dose, up to levels approaching the maximum tolerated dose, yielding an estimated 8-fold therapeutic window, significantly larger than the therapeutic window of many chemotherapeutic agents used to treat ovarian cancer. Most importantly, FcF2-MMAE demonstrated cytotoxicity at <10 nM in all but one member of a panel of ovarian cell lines and had in vivo activity in two additional ovarian xenograft models established from cells expressing only endogenous, unmanipulated levels of LGRs. While the sum of expression of each individual member of the LGR4-6 family cannot be precisely determined due to differences in the affinity of available antibodies and the affinity of each LGR for the RSPO1 Fu1-Fu2 domain in its dimeric form, this provides substantial assurance that the sum of expression will be high enough to render FcF2-MMAE effective. LGR5 and LGR6 are expressed in the stem cells of many other types of cancer, supporting the use of FcF2-MMAE in treating a wide variety of cancers that express LGR5 and / or LGR6.

[0151] Cancers, including ovarian and other cancers, can be tested for LGR5 / LGR6 expression. Expression may be higher in some cancers (e.g., malignant cells with full stem cell potential). Cancers with reduced stem cell activity but that continue to express sufficient LGR5 or LGR6 may be sensitive to and benefit from treatment with FcF2-MMAE. Not all cells that express LGR5 can function as stem cells (Azkanaz et al., 2022). LGR5 expression is not limited to epithelial cells; LGR5 is expressed in some mesenchymal cells, but it is currently unclear whether these cells possess stem cell properties (Kim et al., 2022). These results support the use of FcF2-MMAE to destroy stem cells in tumors, and it is expected that similar selective killing will be observed in malignant cells.

[0152] Ablation of LGR5-expressing cells in the mouse intestine does not disrupt the epithelial integrity, and recent studies suggest this is due to the plasticity of transiently amplifying cells, which regenerate cells with full stem cell potential (Tian et al., 2011; Azkanaz et al., 2022). However, the extent to which such plasticity is retained in ovarian cancer or other types of cancer remains unclear. Studies using organoid-derived colon cancer xenografts indicate that ablation of LGR5 can produce relatively long-lasting responses, and that LGR5-targeted ADCs produce good responses in colon cancer xenograft models (Junttila et al., 2015; Gong et al., 2016). Long-lasting growth inhibition in the KF-28 and CAOV3 models was observed using a single dose of FcF2-MMAE.

[0153] The activity of FcF2-MMAE was also tested in vivo using a xenograft mouse model of human colorectal cancer. Mice bearing human colorectal xenografts (LoVo) were administered FcF2-MMAE at a dose of 1 nmol / g every 7 days for a total of four doses per mouse, and no clinical toxicity was observed at this dosage. Nevertheless, FcF2-MMAE caused a decrease in the average weight of colorectal tumors without changing the body weight of the mice. The results are shown in Figure 21.

[0154] FcF2-MMAE surprisingly produced little toxicity in mice at doses that produced antitumor activity. The differential effects on tumor versus normal tissues may be due to multiple factors, in addition to possible differences in LGR5 and LGR6 expression by tumor versus normal stem cells. While not wishing to be bound by any theory, normal epithelium may be more resistant to the loss of LGR5-positive cells, as has been shown for the intestine (Tian et al., 2011; Junttila et al., 2015; Gong et al., 2016). In normal tissues, stem cells reside in a highly structured, protected niche surrounded by cells that provide support in the form of WNT, RSPO1, and cytokines. Due to differences in microanatomy and physiology, FcF2-MMAE may have much better access to tumor stem cells than to stem cells in normal epithelial niches. To the extent that LGRs and ZNRF3 / RNF43 are expressed on the luminal surface of stem cells, rather than the basal surface, when stem cells reside in a normal polarized niche, the ability of RSPO1 in plasma to access stem cells may be limited (de Vreede et al., 2022). However, polarization is lost after transformation, and FcF2-MMAE may have better access to receptors diffusively distributed on cancer stem cells once the drug reaches the stem cell environment. 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, favoring 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 part of the molecule to activate WNT signaling through a non-LGR-dependent pathway, a potential cause of toxicity, although it does not impair receptor binding ( Dubey et al., 2020 ).

[0155] At therapeutically effective doses, FcF2-MMAE produced few clinically observable adverse events in mice. It is currently unknown whether the Fu1-Fu2 domain may drive some degree of unwanted proliferation of both normal and tumor tissues. Systemic administration of large doses of full-length RSPO1 produced a rapid, but transient, upregulation of WNT signaling in the small intestine, as detected by increased expression of AXIN2 and Ki-67. The response was detectable at 3 hours, peaked at 24 hours, and largely resolved by 48 hours (Kim et al., 2005). These results were not observed to be associated with any adverse clinical outcomes. Repeated, large daily doses of endogenous RSPO1 can generate proliferative responses in stem cells of the jejunum (Zhou et al., 2013; Sun et al., 2021), liver (Sun et al., 2021), and skin (Weber et al., 2020), which are also well tolerated. RSPO1 can aid in recovery from both radiation-induced and chemical-induced enteritis (Zhao et al., 2007; Zhao et al., 2009; Zhou et al., 2013). These results support the idea that even if FcF2-MMAE produces increased WNT signaling in vivo, the response will likely be transient and unsustained when the drug is administered on a weekly schedule.

[0156] Historically, developing high-affinity antibodies selective for LGR5 and LGR6 has been extremely challenging. Nevertheless, several researchers have investigated 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). While favorable responses were observed, the magnitude of selectivity mediated by LGR5 was modest.

[0157] The data support the idea that RSPO targeting may have several important advantages over ADCs. ADC development has not progressed, and no anti-LGR5 ADCs have advanced to clinical trials. RSPO targeting may offer several advantages, including: first, FcF2-MMAE uses a natural ligand that binds with nanomolar affinity and can be rapidly internalized by endocytosis, resulting in intracellular delivery of its cargo. second, Fu1-Fu2 equipped with a cytotoxin has the potential to simultaneously target all three LGR family members (LGR4, LGR5, and LGR6) and two ubiquitin ligase receptors, ZNRF3 and RNF43, whereas ADCs can only target a single LGR at a time. Thus, the Fu1-Fu2 domain equipped with a cytotoxin has the potential to kill cells that express low levels of one type of LGR or ubiquitin ligase receptor but high levels of another. Third, the precision with which the sortase reaction conjugates MMAE results in a more homogeneous population of molecules. Fourth, to the extent that ZNRF3 / RNF43 are also expressed on CSCs, the bispecific binding of the Fu1-Fu2 domain favors selectivity and enhanced internalization rate and extent. Fifth, some tumors overexpress ZNRF3 and RNF43 independently of LGR. Because the Fu1 domain binds to these two receptors, it can also target this type of tumor. Sixth, the Fu1-Fu2 domain can simultaneously engage both LGR and ZNRF3 or RNF43. Essentially, this is equivalent to a bispecific ADC; this type of ADC is currently attracting significant interest due to its enhanced avidity, specificity, and ability to cluster receptors and mediate enhanced internalization (Shim, 2020). These results support both Fu1-Fu2 as a targeting ligand for delivering cytotoxic payloads to cancerous cells expressing LGR5 / LGR6, and also the use of FcF2-MMAE to treat cancers that may involve CSCs.

[0158] Example 2 material and method The following materials and methods were used in the experiments provided in Example 1.

[0159] 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 #MAB8078, R&D Systems; PE-conjugated anti-mouse IgG antibody, catalog #F0102B, R&D Systems. Ni-NTA resin was purchased from Qiagen, and SP-Sepharose resin and DEAE resin were purchased from GE Healthcare Life Sciences. Propidium iodide was purchased from ThermoFisher (catalog #P3566). All ovarian cancer cell lines were obtained from either ATCC or US laboratories; all cell lines were ATCC STR-certified. Sortase plasmid vector pet30b-7M SrtA was purchased from Addgene. Sortase-6xHis containing the mutations P94R, E105K, E108Q, D160N, D165A, K190E, and K196T was produced in the Escherichia coli (E. coli) strain Rosetta and purified using Ni-NTA resin chromatography as previously reported in Yu et al. 2021. (Gly)3-vc-PAB-MMAE was synthesized by Levena Biopharma. Plasma levels of FcF2-MMAE were determined using an ELISA kit (DY4645-05) from R&D Systems.

[0160] Synthesis and Purification of FcF2-His: FcF2-His was produced by transient transfection of HEK293E cells with a pcDNA3.1 vector containing an insert encoding the Fu1-Fu2 domains of RSPO1. Cells were grown in 300 ml of HEK293E culture medium consisting of 150 mL of Gibco FreeStyle 293 medium (catalog #12338-026, Thermofisher), 150 mL of HyClone SFM4HEK293 medium (catalog #82003-356), 6 mL of fetal bovine serum (catalog #26140-079, ThermoFisher), 333 μL of G418 sulfate (catalog #G8168, Sigma), and 333 μL of anticoagulant (catalog #0010057AE, ThermoFisher) in a 1 L flask on a rotating platform at 130 rpm. The cell supernatant was collected after 5 days of culture, centrifuged to sediment debris, and then loaded onto a column containing Ni-NTA resin (Cat. No. 30250, Qiagen) equilibrated with a buffer containing 150 mM NaCl and 20 mM Tris at 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 at pH 7.6. The eluate was diluted 1:3 with 20 mM Tris at pH 7.6 and then loaded onto a DEAE column. The concentration of the flow-through from the DEAE column was quantified by reverse-phase HPLC analysis using a C4 column.

[0161] Conjugation of FcF2-His to MMAE using sortase: FcF2-His was immobilized on SP-Sepharose resin (catalog #17072901, Cytiva), and the sortase reaction was carried out at 37°C for 4 hours. The reaction mixture contained (Gly)3-vc-PAB-MMAE and FcF2-His at a molar ratio of 20:1, and sortase-6xHis and FcF2-His at a molar ratio of 1:4. After washing the SP-Sepharose to remove sortase-6xHis and unreacted (Gly)3-vc-PAB-MMAE, the purified FcF2-MMAE was eluted with phosphate buffer containing 1M NaCl. The FcF2-MMAE was diluted to a NaCl concentration of 200 mM, sterilized through a 0.22 μm filter, and stored frozen at -80°C.

[0162] Flow cytometry analysis: LGR5 expression in live cells was determined by flow cytometry after staining with anti-hLGR5 / GPR49 antibody at a final concentration of 12.5 μg / mL for 30 minutes at 25°C protected from light. Excess anti-hLGR5 / GPR49 antibody was removed with two rounds of PBS washing, and the cells were incubated with PE-conjugated anti-mouse IgG antibody diluted 1:20 for 30 minutes at 25°C protected from light. Excess anti-mouse IgG antibody was removed with two rounds of PBS washing, 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.

[0163] Growth rate inhibition assay: The effect of FcF2-MMAE on cell growth rate in vitro was determined using CCK8 reagent (Dojindo, Inc.). Cells were seeded in triplicate wells for each drug concentration at a density sufficient to obtain an OD450 of >1.5 in control wells after subtracting the OD determined at the start of drug exposure. Viability was calculated as the percent reduction between T=0 and the time the assay was stopped (Hafner et al., 2016). All data points represent the mean ± SEM of triplicate cultures for each concentration of drug tested.

[0164] Pharmacokinetic studies: BALB / c mice were injected intravenously with FcF2-MMAE at a dose of 0.1 nmol / g, and blood was collected at regular intervals from the buccal or tail vein into EDTA-coated tubes. Plasma FcF2-MMAE concentrations were determined by ELISA using capture and detection antibodies specific for human RSPO1, which recognize the Fu1 and Fu2 subdomains. Pharmacokinetic parameters were evaluated using Phoenix WinNonlin version 8.1 (Certara Inc., Princeton, NJ, USA).

[0165] Efficacy studies: BALB / c nu / nu mice were obtained from the UCSD breeding colony and inoculated SC with tumor cells harvested from culture and mixed 2:1 v / v with Matrigel before injecting 150 μl of the mixture. The number of cells inoculated varied by tumor type, with OVCAR8 / EV and OVCAR8 / LGR5 at 2.5×10 6 / site; KF-28 is 2.5 × 10 6 / site; CAOV3: 5 × 10 6 / site. Formula V = (w 2 Tumor growth rates were determined from orthogonal diameter measurements taken once or twice weekly using a 2×L) / 2. Control mice received vehicle alone (phosphate-buffered saline with 0.02% Tween-20) on the same schedule.

[0166] Example 3 Inclusion of multiple FuFu domains in therapeutic compounds Based on the hypothesis that RSPO1 can be used to selectively target cytotoxic drugs to CSCs, we created a compound (R1FF-MMAE) containing two binding domains and equipped with the cytotoxin monomethylaurostatin. Because the affinity of a ligand can be affected by the number of binding sites, we conducted experiments to determine whether 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 linking MMAE to the polypeptide using a sortase reaction, we characterized the drug (FcST4-MMAE) using gel electrophoresis, HPLC analysis on ion exchange and size exclusion columns, Western blot, and cytotoxicity assays. The results showed that FcST4-MMAE could be purified to high purity and that the compound was remarkably stable at 4°C and a low pH of 3.0. Cytotoxicity studies revealed that increasing the binding domain did not increase potency, possibly due to increased ligand size or conformational changes mediated by interdomain interactions that may have hindered access to the LGR5 / LGR6 receptor.

[0167] The vector construct for the FcST4 molecule was designed to contain an IgG leader sequence at the N-terminus, followed by a mutated Fc domain with two modified receptor-binding domains (STs), connected in tandem by a linker sequence between the two ST domains. A second spacer sequence was inserted immediately upstream of the LPETGG sortase recognition (donor) motif, and an 8xHis tag was placed at the C-terminus. Figure 17 shows a schematic diagram of the steps used to create the FcST4 vector using overlapping PCR technology and the amino acid sequence of FcST4. As shown in Figure 17, this construct contains two LGR-binding domains separated by a G4S linker. The nucleotide sequence of FcST4 is provided (SEQ ID NO: 103). Using double restriction enzyme digestion, two DNA fragments were created from an existing FcST2 vector, both of which shared an overlap in the linker sequence that allowed the two fragments to anneal. Appropriate primers were designed to amplify both fragments separately, and then the two fragments were annealed at 70°C to generate the final product. The final product was ligated into pcDNA3.1. Sanger sequencing of the maxipreps was used to confirm that the sequence matched the planned sequence 100%, and the ligated vector was then stored at -80°C for use in transfection.

[0168] Full-length proteins were produced in HEK293e cells. The FcST4.pcDNA3.1 vector was transiently transfected into HEK293e cells. HEK293e cells were grown in Freestyle 293 medium for 5 days before use in transfection. On the day of transfection, the cell medium was changed, and a mix 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 and 5% CO2 for 4 hours. Four hours after transfection, the flask was supplemented with 150 mL of HySFM293 medium and 30 mL of Freestyle 293 medium to a final volume of 300 mL. The cells were allowed to grow. Approximately 24 hours after transfection, valproic acid and 1:1000 v / v of an anticoagulant were added to the flask containing the transfected HEK293E cells. Cells were harvested on day 5. The average cell viability on the day of harvest of the seven batches of FcST4 produced was 79%.

[0169] After harvesting, the cell supernatant was processed through the initial steps of purification. The first purification step was based on the ability of the 8xHis tag to bind to Ni resin. The cell supernatant was removed by high-speed centrifugation to remove particulate matter and then loaded onto a gravity-flow column packed with pre-packed Ni-NTA resin. FcST4-His was eluted using 300 mM imidazole, and the protein was then passed through a DEAE column. The flow-through was characterized using HPLC reverse-phase C4 column and non-reducing SDS / PAGE gel analysis.

[0170] The results of the initial Ni purification step were examined in more detail by collecting and analyzing fractions from sequential wash and elution steps with 300 mM imidazole, as well as fractions from the successive flow-through and wash steps of the DEAE column. The band size ratio did not vary with the sequential elutions, indicating that selective purification of the 120 kD FcST4-His dimer could not be achieved using this strategy. The DEAE step was successful in removing some of the HMW form; on average, 28% of FcST4-His was lost in this step. However, this loss was primarily the result of the HMW form being successfully retained on the DEAE resin. The use of sequential Ni-NTA and DEAE steps resulted in a fairly pure form of FcST4 that subsequently served as input to the sortase reaction, which conjugates MMAE onto the molecule. The first step, Ni-NTA purification, and the second step, DEAE purification, yielded a fairly pure FcST4-His protein, as evidenced by the presence of a single peak in the HPLC-C4 profile. The protein was successfully captured from the cell supernatant by Protein A resin, and it was successfully eluted with PBS containing 1 M NaCl, as evidenced by a band of the correct size on an SDS / PAGE gel.

[0171] After Ni-NTA and DEAE purification, FcST4-His was conjugated to the cytotoxin monomethylaurostatin (MMAE) using a sortase reaction. The sortase enzyme cleaves between the threonine and glycine in the LPETGG tag, removing the GG-8xHis tag. The sortase reaction was performed using precursor FcST4-His loaded onto the cation exchange resin SP-Sepharose. This allowed for washing away excess G3-val-cit-PAB-MMAE and sortase enzyme before elution of the SP-Sepharose with 1M NaCl. The efficiency of the sortase reaction across all batches of FcST4-His was 100 ± 37% (SD). However, the average percent yield of the final product after the sortase reaction was 90 ± 22% (SD).

[0172] To assess the stability of the FcST4-MMAE molecule, samples were subjected to multiple freeze-thaw cycles. Even after four cycles, there was no significant change in the SEC130 trace of the FcST4-MMAE molecule.

[0173] After generating seven batches of FcST4-MMAE, batches 2–6 were used for cytotoxicity assays. HEK293e suspension cells were grown and treated with drug at a range of concentrations starting from 0 mM to 400 mM in 96-well plates. On day 6, CCK8 reagent was added, and the OD450 of the experimental plates was read. Consistently, differences in target killing were observed between HEK293e empty vector cells and cells overexpressing the LGR5 receptor for batches 2–6 of FcST4-MMAE, although the differences were not statistically significant. Cytotoxicity assays for FcST4-MMAE were also performed using the OVCAR8 cell line. Drug concentrations were tested starting from 400 nM to 0 mM. Similar to the results with HEK293e cells, differences in target killing were observed between OVCAR8 cells expressing only the empty vector and cells engineered to overexpress the LGR5 receptor, although the differences were not statistically significant. The IC50 values ​​ranged from 1.9 to 3.8 nM. The efficacy of FcST2-MMAE was tested against eight human ovarian cancer cell lines. The results are shown in Figure 18. FcST2-MMAE was remarkably effective in killing these cells, with IC 50 The IC values ​​ranged from 3.8 to 29 nM, and all but one cell line had IC values ​​below 20 nM. 50 had the following characteristics:

[0174] Example 4 Linker-less FcF2-MMAE The FcF2-MMAE compound was made as described in the previous example, with the modification that the G4S linker was not included in the polypeptide. A diagram of the linkerless FcF2 polypeptide portion of the compound (FcF2Δlinker-His) is shown in Figure 19.

[0175] The molecular weight of the resulting monomer was reduced by 1,279, from 42,642 to 41,363, a roughly 3% reduction in size. The FcF2Δ linker-His polypeptide was produced using ExpiCHO cells. Ni-NTA and DEAE purification steps were performed. The production and purification of the FcF2Δ linker-His polypeptide were consistent with the yield and purity of the FcF2-His polypeptide based on SDS page experiments and HPLC reverse-phase C4 profiles. MMAE was covalently tethered to the FcF2Δ linker using sortase as described in the previous examples.

[0176] Cytotoxicity experiments were performed using FcF2-MMAE and FcF2Δlinker-MMAE against OVCAR8 cancer cells (OVCAR8 / EV) and OVCAR8 cancer cells overexpressing the LGR5 receptor (OVCAR8 / LGR5). Both FcF2-His and FcF2Δlinker-His, from which MMAE is derived, were generated in ExpiCHO. MMAE was ligated using the sortase reaction described above. As shown in Figure 20, both FcF2-MMAE and FcF2Δlinker-MMAE resulted in cancer cell killing, and increased cancer cell death was observed in cancer cells overexpressing the LGR5 receptor (OVCAR8 / LGR5). Overall, IC 50 No clear differences were observed in either the level or the degree of selectivity. FcF2Δlinker-MMAE appeared to be less potent against both LGR5-poor and LGR5-rich cells. These results indicate that therapeutic cancer-killing effects can be observed in FcF2-MMAE constructs in which the linker is removed from the polypeptide portion of the therapeutic molecule.

[0177] Example 5 Efficacy of FcF2-MMAE in vivo FcF2-MMAE was tested for efficacy in additional human tumor xenograft models in which tumors were grown subcutaneously from previously established tumor cell lines. The LoVo cell line expresses KRAS G13DThis is a colorectal line containing a mutation and high levels of WNT signaling. LoVo cells were inoculated SC into nu / nu mice; there were a total of 20 tumors in each group. FcF2-MMAE administration began on the first day any tumors were detectable. The control group received saline only; the experimental group received FcF2-MMAE IP injected q7d x 4 at a dose of 1 nmol per gram of body weight, after which further administration was discontinued. As shown in Figure 22, FcF2-MMAE delayed the onset of tumor growth compared to the untreated control group and slowed the rate of growth once established. There was no significant weight loss in either the control or experimental groups of mice. These results demonstrate that FcF2-MMAE was active in this model. The results are shown in Figures 21 and 22.

[0178] FcF2-MMAE was further tested for efficacy using tumors grown from the AGS human gastric cancer cell line. This line was selected for testing because it possesses high levels of WNT pathway signaling and represents a tumor type with unmet medical need. AGS cells were inoculated SC into nu / nu mice; a total of 20 tumors were present in each group. FcF2-MMAE administration began on the first day any tumors were detectable. The control group received saline alone; the experimental group received FcF2-MMAE IP injections q7d x 4 at a dose of 1 nmol per gram of body weight, after which further administration was discontinued. As shown in Figure 23, FcF2-MMAE significantly suppressed tumor growth in the experimental group. Tumors developed in 11 of 20 inoculation sites in the control group; none of 20 inoculation sites in the experimental group developed tumors (p<0.0037 chi-square). In the experimental group, no tumors appeared until 160 days after inoculation. There was no significant weight loss in either the control or experimental groups of mice. These results demonstrate that FcF2-MMAE was curative in these mice that received FcF2-MMAE. The results are shown in Figure 23.

[0179] FcF2-MMAE was also tested for efficacy using tumors grown from the SKNAS neuroblastoma cell line. This line was chosen for testing because it has both high levels of WNT pathway signaling and is a highly aggressive tumor. SKNAS cells were inoculated SC into nu / nu mice; there were a total of 20 tumors in each group. FcF2-MMAE administration began on the first day any tumors were detectable (day 7 post-inoculation). The control group received saline only; the experimental group received FcF2-MMAE IP injected q7d x 4 at a dose of 1 nmol per gram of body weight, after which further administration was discontinued. As shown in Figure 24, FcF2-MMAE increased the time to first detection and the time to the onset of explosive tumor growth from 15 to 38 days. There was no significant weight loss in either the control or experimental groups of mice. These results demonstrate that FcF2-MMAE was highly active in this model. The results are shown in Figure 24.

[0180] Example 6 Conjugation of FcF2-His with cytotoxins As shown in this example, FcF2-His can be conjugated to a variety of cytotoxins. Monomethylaurostatin (MMAE) is a highly potent cytotoxin, but 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 other classes of warheads to FcF2-His. The same sortase A reaction conditions described above for coupling (Gly)3-val / cit-PAB-MMAE were used to conjugate PNU159682 and deruxtecan.

[0181] Figure 25A shows SDS-PAGE analysis of FcF2-His conjugated with PNU159682, an anthracycline derivative. Conjugation with this molecule did not alter the structural characteristics of FcF2-His, as detected by SDS-PAGE. FcF2-PNU159682 was tested on OVCAR8 / EV cells, which are poor in LGR5, and on OVCAR8 / LGR5 cells, which express approximately 10-fold more of the LGR5 receptor. IC against OVCAR8 / LGR5 cells 50 The IC for OVCAR8 / EV was 300 pM, whereas the IC for OVCAR8 / EV was >3,000 pM. 50 The ratio was approximately 10 (Figure 25B). Figure 25C shows the structure of PNU-159682.

[0182] Deruxtecan has demonstrated high efficacy in breast cancer patients as a cytotoxin contained in the antibody-drug conjugate Fam-trastuzumab deruxtecan-nxki. To further demonstrate the feasibility of conjugating FcF2-His to other types of warheads, deruxtecan was conjugated to FcF2-His using the sortase A reaction. The first step was to construct a linker for deruxtecan that would serve as a substrate for the sortase A reaction. To do this, conditions were explored for the reaction of the peptide GGGC with commercially available maleimide-GGFG-deruxtecan to generate the GGGC-MA-GGFG-deruxtecan linker. This required optimizing the conditions so that the linker remained soluble in aqueous buffer. Figure 26A shows a schematic of the reaction, and Figure 26B shows a reverse-phase HPLC analysis of the GGGC-MA-GGFG-deruxtecan linker, demonstrating its purity.

[0183] The GGGC-MA-GGFG-deluxtecan linker was conjugated to FcF2-LEPTGG-His using a sortase reaction. Figure 27A shows the reversed-phase HPLC (C4 column) analysis of FcF2-deluxtecan, demonstrating its purity and the absence of unreacted substrate. The ability of FcF2-deluxtecan to inhibit the growth of LGR5-poor OVCAR8 / EV cells versus LGR5-rich OVCAR8 / LGR5 cells was determined using a standard CCK8 cytotoxicity assay and 120 hours of exposure. As shown in Figure 27B, this assay revealed that FcF2-deluxtecan was highly potent, with IC 1000 for OVCAR8 / LGR5 cells and OVCAR8 / EV cells. 50 values ​​of 0.7532 nM and 9.045 nM. Most importantly, FcF2-deltecan retained 12-fold selectivity in favor of LGR5-enriched cells.

[0184] Overall, these results demonstrate that FcF2-His can be successfully conjugated to a wide variety of different types of cytotoxic agents without obvious structural damage or loss of selectivity for the LGR5 stem cell receptor.

[0185] Example 7 A mutant form of FcF2-MMAE with an extended plasma half-life All protein therapeutics are gradually degraded by plasma proteases and cellular clearance mechanisms while circulating in the bloodstream. Western blot analysis of FcF2-MMAE incubated in human plasma at 37°C identified a low-molecular-weight fragment suggesting partial truncation of the first part of the FcF2-MMAE Fu1 domain. This was the region previously suggested to be truncated by enterokinase. Vectors expressing mutant forms of FcF2-LPETGG-His containing four short deletions in this region were constructed, and the proteins were produced and tested for degradation rates in human plasma. This approach highlighted a particularly interesting subregion. A series of additional FcF2-LPETGG-His vectors containing single alanine substitution mutants were constructed, and their protein products were produced and tested. This screening identified the mutations R28A and R30A as particularly interesting, although several other single-point mutants also delayed plasma degradation.

[0186] The FcF2(R28A) mutant was generated using the following nucleotide sequence: TIFF2026503181000024.tif160161

[0187] FcF2(R28A) mutant as amino acid sequence: TIFF2026503181000025.tif54160

[0188] The Fu1-Fu2(R28A) mutant has the following amino acid sequence: TIFF2026503181000026.tif19160

[0189] The FcF2(R30A) mutant was generated using the following nucleotide sequence: TIFF2026503181000027.tif160161

[0190] The FcF2(R30A) mutant has the following amino acid sequence: TIFF2026503181000028.tif54160

[0191] The Fu1-Fu2(R30A) variant has the following nucleotide sequence: TIFF2026503181000029.tif47160

[0192] Variants of the FcF2 construct were generated using the Fu1-Fu2 region containing additional deletion mutations as follows: Additional deletion mutants of the Fu1-Fu2 region were generated: R22-R31 (RSPO1 nucleotides 64-93), K25-R31 (RSPO1 nucleotides 73-93), R28-R31 (RSPO1 nucleotides 82-93), and R22-K27 (RSPO1 nucleotides 64-81). These deletion mutants can be used in a similar manner as other Fu1-Fu2 regions to bind to LGR. The deletion mutants have the following sequence: Fu1-Fu2 (R22-R31 deletion) TIFF2026503181000030.tif18159, Fu1-Fu2 (K25-R31 deletion) TIFF2026503181000031.tif18160, Fu1-Fu2 (R28-R31 deletion) TIFF2026503181000032.tif18160, Fu1-Fu2 (R22~K27 deletion) TIFF2026503181000033.tif18160, and Fu1-Fu2 (S21-Q38 deletion) TIFF2026503181000034.tif11159. These deletion mutants may result in increased plasma half-life.

[0193] FcF2-MMAE (WT), FcF2-R28A-MMAE, and FcF2-R30A-MMAE were tested for potency and selectivity using OVCAR8 / EV cells versus OVCAR8 / LGR5 cells. Figure 28 shows the concentration-survival curve data, and it was observed that neither the R28A nor the R30A mutation altered the potency of selectivity for LGR5-enriched cells.

[0194] Pharmacokinetic studies were then performed in BALB / c mice. Mice were given intravenous (IV) injections of either FcF2-MMAE (WT), FcF2-R28A-MMAE, or FcF2-R30A-MMAE, and the plasma concentrations of each were determined in timed samples by ELISA using capture and detection antibodies against different portions of the molecule. Figure 29 shows that the R28A mutation prolongs the initial half-life of the molecule and increases the AUC 0-120 The results show that the R28A mutation increased the half-life of FcF2 constructs, including cytotoxic constructs, by 3.4-fold, whereas the R30A mutation did not. Of particular importance, during the early phase of the plasma decay curve, when drug penetration into tumors is likely occurring at its fastest rate, the concentration of FcF2-R28A-MMAE was more than two orders of magnitude higher than that of FcF2-MAME. These results demonstrate that the R28A mutation can increase the half-life of FcF2 constructs, including cytotoxic constructs.

[0195] The plasma half-life of the FcF2-MMAE construct was compared to that of the R1FF-MMAE ("RSPO1-MMAE") construct described in Yu et al. (2021) using BALB / C female mice. The in vivo distribution phase half-life of FcF2-MMAE was observed to be approximately 15-fold longer than that of R1FF-MMAE. The results are shown in Figure 34.

[0196] Example 8 Increasing MMAE loading onto FcF2-His using sortase E Several studies using antibody-drug conjugates have supported the idea that, in some cases, increasing the number of cytotoxins per antibody molecule (DAR) may increase potency and efficacy. In the case of FcF2-LPETGG-His, MMAE warheads can be placed at both ends of the molecule via a sortase A reaction. Because the sortase A reaction is reversible, the main challenge in the process of adding a second MMAE to the N-terminus is that the MMAE already installed at the C-terminus may be removed by the reaction. The inventors then worked to develop further methods for linking additional cytotoxic moieties to FcF2.

[0197] Figure 30 presents a schematic of this approach. The recently isolated sortase E ligates substrates containing an N-terminal GG motif to the sequence LAHTGG (SEQ ID NO:106) on the C-terminal end of other proteins or peptides. Because sortase A and sortase E have high specificity for different recognition sequences (LPETGGG vs. LAHTGG), they could potentially work together to equip both ends of a variant of the FcF2 molecule containing a diglycine at the N-terminus and an LPETGG sequence at the C-terminus with MMAE.

[0198] A vector expressing GG-FcF2-LEPTGG-His was constructed, produced in HEK293 cells, and purified; studies showed that the addition of two glycines at the N-terminus did not alter potency or selectivity when loaded with MMAE using sortase A.

[0199] A gene containing the sortase E sequence was constructed and cloned into a bacterial expression vector, and the protein was synthesized in the Rosetta strain of E. coli and purified using Ni-NTA chromatography. To facilitate testing of sortase E, we constructed a version of the FcF2-LPETGG-His vector 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, we prepared a linker consisting of the peptide GGGC conjugated to Dye650, a maleimide-containing fluorescent molecule. Figure 31 shows that sortase E was able to load GGGC-MA-Dye650 onto FcF2-LAHTGG-His, thus establishing that both sortase A and sortase E can be used to load substrates with N-terminal glycines onto FcF2 containing the appropriate sortase recognition sequence. Repetition of these experiments will further corroborate these results and can be used to perform statistical analysis on these results, if desired.

[0200] Example 9 Increased loading of MMAE onto FcF2-His using partial reduction of the disulfide The FcF2-LEPTGG-His molecule contains three disulfide bonds in the immunoglobulin Fc domain and eight disulfide bonds in the Fu1-Fu2 domain. Antibody-drug conjugates are often loaded with cytotoxins by partially reducing the disulfide bonds in their Fc domains and subsequently reacting them with a thiol-reactive maleimide-containing linker precoupled to the cytotoxin. This allows loading of two to eight or more cytotoxins per molecule, but excessive loading must be individualized because it can distort the protein structure and reduce plasma half-life. To determine whether this same approach could be used to load MMAE onto FcF2-His without using a sortase reaction, we performed a series of experiments evaluating loading capacity as a function of TCEP concentration.

[0201] Aliquots of FcF2-His were exposed to TCEP at concentrations ranging from 0.0005 to 5000 μM. After 25 minutes, maleimide-val / cit-PAB-MMAE (MA-MMAE) was added to the reaction at a ratio of either 1:2 or 1:8 (protein:MMAE). The reaction was then allowed to continue overnight. Figure 32 shows Western blot analysis of these samples probed with anti-RSPO1 and anti-MMAE. In the absence of MA-MMAE, no MMAE signal was detectable; however, increasing the concentration of TCEP resulted in progressively greater amounts of MMAE being loaded onto FcF2-His. Higher protein:MA-MMAE ratios resulted in greater loading. A TCEP concentration of 5000 μM reduced all disulfide bonds, resulting in only visible FcF2-His monomers, but significantly greater amounts of MMAE were loaded when all disulfides were reduced.

[0202] This data demonstrates that MMAE can be loaded onto FcF2-His using a partial disulfide reduction approach. Additional studies can be performed to determine the number of MMAE molecules loaded and how efficacy, selectivity, and plasma half-life vary with loading. Increasing the loading of a cytotoxic moiety (e.g., MMAE) may result in increased potency or enhanced killing of the construct due to the additional molecules of cytotoxic moiety. As shown above, additional cytotoxic moieties can be covalently attached to the construct (e.g., at the N- and C-terminal ends of FcF2) via a variety of techniques, including partial disulfide reduction and covalent linkage using sortases (e.g., sortase A and sortase E).

[0203] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods, and to the method steps or to the sequence of method steps described herein, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes 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.

[0204] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2026503181000035.tif186161TIFF2026503181000036.tif205161TIFF2026503181 000037.tif219161TIFF2026503181000038.tif206161TIFF2026503181000039.tif30161

[0205] array FcF2 sequence - without leader sequence or 8x His (SEQ ID NO:1) TIFF2026503181000040.tif46161 FcF2 sequence - including leader sequence (SEQ ID NO:2) TIFF2026503181000041.tif46161 FcF2 sequence - containing 8x His (SEQ ID NO:3) TIFF2026503181000042.tif46161 Fu1-Fu2 (FuFu) sequence (SEQ ID NO:4) TIFF2026503181000043.tif25161 (G 4 S) 2 Linker (SEQ ID NO:5) GGGGSGGGGS LPETGG sortase recognition site (SEQ ID NO:6) LPETGG 8xHis sequence (SEQ ID NO:7) HHHHHHHH IgGk leader sequence (SEQ ID NO:8) TIFF2026503181000044.tif3128 Wild-type IgG1-Fc domain: (SEQ ID NO: 9) TIFF2026503181000045.tif25160 Wild-type IgG2-Fc domain (SEQ ID NO:10) TIFF2026503181000046.tif25159 Wild-type IgG3-Fc domain (SEQ ID NO:11) TIFF2026503181000047.tif33160 Wild-type IgG4-Fc domain (SEQ ID NO: 12) TIFF2026503181000048.tif25160 >DHS-IgG1 (SEQ ID NO: 13) TIFF2026503181000049.tif25161 >DHS-IgG2 (SEQ ID NO: 14) TIFF2026503181000050.tif25159 >DHS-IgG3 (SEQ ID NO: 15) TIFF2026503181000051.tif33160 >DHS-IgG4 (SEQ ID NO: 16) TIFF2026503181000052.tif25160 FuFu (N137A mutant) amino acid (SEQ ID NO: 17) TIFF2026503181000053.tif46161 G 4 S linker (SEQ ID NO: 18) GGGGS FcF2 sequence - including leader and 8x His (SEQ ID NO:19) TIFF2026503181000054.tif46161 DHSFc-(FuFu) 2 Sequence (SEQ ID NO:20) TIFF2026503181000055.tif189161 EDHS: (SEQ ID NO: 38) TIFF2026503181000056.tif25160 EDHY: (SEQ ID NO: 39) TIFF2026503181000057.tif25160 DHY (SEQ ID NO: 40): TIFF2026503181000058.tif167161TIFF2026503181000059.tif132128 FcF2 sequence - after ligation with sortase (SEQ ID NO:76) TIFF2026503181000060.tif46161 FcF2 sequence - after ligation by sortase, including the cleavable bond (SEQ ID NO:77) TIFF2026503181000061.tif46161 RSPO2 - Fu1(33~84) - (SEQ ID NO:78) TIFF2026503181000062.tif4140 RSPO2 - Fu2(90~134) - (SEQ ID NO:79) TIFF2026503181000063.tif3128 RSPO3 - Fu1(35~86) - (SEQ ID NO:80) TIFF2026503181000064.tif4140 RSPO3 - Fu2(92~135) - (SEQ ID NO:81) TIFF2026503181000065.tif3128 RSPO4 - Fu1(28~81) - (SEQ ID NO:82) TIFF2026503181000066.tif4141 RSPO4 - Fu2(85~128) - (SEQ ID NO:83) TIFF2026503181000067.tif4128 (EAAAK) 3 (SEQ ID NO:84) TIFF2026503181000068.tif3128 RSPO2-Fu1-Fu2: (SEQ ID NO: 85) TIFF2026503181000069.tif11158 RSPO3-Fu1-Fu2: (SEQ ID NO: 86) TIFF2026503181000070.tif11158 RSPO4-Fu1-Fu2: (SEQ ID NO: 87) TIFF2026503181000071.tif11160 RSPO1-Fu1: (SEQ ID NO: 88) TIFF2026503181000072.tif4138 RSPO1-Fu2: (SEQ ID NO: 89) TIFF2026503181000073.tif3128 FcF2(R28A) mutant - nucleotide sequence (SEQ ID NO:97) TIFF2026503181000074.tif160161 FcF2(R28A) mutant - amino acid sequence (SEQ ID NO:98) TIFF2026503181000075.tif53160 Fu1-Fu2(R28A) mutant - amino acid sequence (SEQ ID NO:90) TIFF2026503181000076.tif18160 FcF2(R30A) mutant - nucleotide sequence (SEQ ID NO:101) TIFF2026503181000077.tif160161 FcF2(R30A) mutant - amino acid sequence (SEQ ID NO:102) TIFF2026503181000078.tif53160 Fu1-Fu2(R30A) mutant - nucleotide sequence (SEQ ID NO: 100) TIFF2026503181000079.tif46160 Fu1-Fu2 (R22-R31 deletion) (SEQ ID NO: 92) TIFF2026503181000080.tif10158 Fu1-Fu2 (K25 to R31 deletion) (SEQ ID NO: 93) TIFF2026503181000081.tif10160 Fu1-Fu2 (R28-R31 deletion) (SEQ ID NO: 94) TIFF2026503181000082.tif17160 Fu1-Fu2 (R22 to K27 deletion) (SEQ ID NO: 95) TIFF2026503181000083.tif10160 Fu1-Fu2 (S21-Q38 deletion) (SEQ ID NO: 96) TIFF2026503181000084.tif10158 Amino acid sequence of FcST4 (SEQ ID NO:105) TIFF2026503181000085.tif67161 Nucleotide sequence of FcST4 (SEQ ID NO: 103) TIFF2026503181000086.tif160161

Claims

1. 1. A compound comprising one or more cytotoxic agents conjugated to a polypeptide comprising one or more LGR binding domains, (i) the polypeptide further comprises an Fc region, and / or (ii) the polypeptide comprises at least two copies of the LGR-binding domain; and 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 NOs: 4, 78-83, 85-89, 90-96, 98, or 102; The compound.

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

87.

3. 2. 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 comprises an amino acid sequence independently 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 at R30, numbered according to Kabat.

6. 6. The compound of claim 5, wherein the substitution mutation is arginine to 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 a Fu1-Fu2(R30A) mutant (SEQ ID NO:91) or a 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. 2. The compound of claim 1, wherein the LGR binding domain comprises Fu1-Fu2 (R22-R31 deleted) (SEQ ID NO:92), Fu1-Fu2 (K25-R31 deleted) (SEQ ID NO:93), Fu1-Fu2 (R28-R31 deleted) (SEQ ID NO:94), or Fu1-Fu2 (R22-K27 deleted) (SEQ ID NO:95).

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

12. The compound of claim 1, wherein the Fc region is N-terminal to the LGR binding domain and / or the polypeptide comprises the Fc region and the LGR binding domain in an N to C direction.

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

14. 14. The compound of 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 of claim 1, wherein the polypeptide comprises SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, or SEQ ID NO:88; and 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 of claim 1, wherein the polypeptide comprises SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, or SEQ ID NO:89; and the polypeptide does not comprise SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, or SEQ ID NO:

88.

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

13. The compound of claim 12.

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

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

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

13.

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

23. 23. The compound of claim 22, wherein the Fc domain has essentially the same binding affinity to an FcγR as compared to a wild-type Fc domain.

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

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

26. 26. The compound of claim 25, wherein the Fc domain exhibits (i) enhanced binding at pH 5.8, and (ii) reduced or no detectable binding at pH 7.4 for FcRn compared to wild-type.

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

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

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

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

31. the Fc domain comprising the following substitution mutation: (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) 29. The compound of any one of claims 17 to 28, comprising:

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

33. The compound of any one of claims 12 to 32, which is dimerized via a disulfide bond formed in the Fc domain.

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

35. The linker is G 4 S(SEQ ID NO:18) or (G 4 S) 2 35. The compound of claim 34, comprising (SEQ ID NO:5).

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

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

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

39. Two copies of FuFu (SEQ ID NO:4) or FuFu N137A (SEQ ID NO:17) are inserted between the linker, preferably G 4 S linker (SEQ ID NO: 18) or (G 4 S) 2 39. The compound of claim 38, separated via a linker (SEQ ID NO:5).

40. 40. The compound of claim 39, comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:

19.

41. 41. The compound of any one of claims 1 to 40, comprising SEQ ID NO:

1.

42. 2. The compound of claim 1, comprising SEQ ID NO:

20.

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

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

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

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

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

48. 48. The compound of claim 47, wherein the drug is a maytansinoid, auristatin, amanitin, calicheamicin, psymberin, duocarmycin, anthracycline, camptothecin, doxorubicin, taxol, tubulysin, eribruin, or a pyrrolobenzodiazepine.

49. 49. The compound of claim 48, wherein the drug is an auristatin.

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

51. 50. The compound of claim 49, wherein the auristatin is monomethylauristatin E (MMAE).

52. 48. The compound of claim 47, wherein the drug is a camptothecin analog.

53. 53. The compound of claim 52, wherein the camptothecin analog is topotecan, irinotecan, belotecan, or deruxtecan.

54. 48. The compound of claim 47, wherein the drug is an anthracycline analog.

55. 55. The compound of claim 54, wherein the anthracycline analog is PNU-159682 (CAS number: 202350-68-3).

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

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

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

59. 59. The compound of any one of claims 1 to 58, comprising at least one spacer positioned between the cytotoxic agent and the LGR binding domain.

60. 60. The compound of claim 59, comprising two linkers.

61. The linker is G 4 S(SEQ ID NO:18) or (G 4 S) 2 61. The compound of any one of claims 59-60, comprising (SEQ ID NO:5).

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

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

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

65. 2. The compound of claim 1, comprising SEQ ID NO:1 covalently linked to monomethyl auristatin E (MMAE).

66. 10. The compound of claim 1, comprising SEQ ID NO:2 covalently linked to monomethyl auristatin E (MMAE).

67. 67. The compound of any one of claims 12 to 66, which is dimerized via a disulfide bond in the Fc domain.

68. 68. The compound of any one of claims 1-67, wherein the cytotoxic agent is covalently attached to the polypeptide via a sortase linker.

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

70. 68. The compound of any one of claims 1-67, comprising a sortase linker between the LGR binding domain and the cytotoxic agent.

71. 71. The compound of claim 70, wherein the cytotoxic agent is covalently attached to the polypeptide via a sortase.

72. 72. The compound of claim 71, wherein the sortase is sortase A or sortase E.

73. The sortase linker has the sequence LPXT(G) n or LAHTGG (SEQ ID NO: 106), where n=1-10.

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

75. 75. The compound of any one of claims 1-74, further comprising a second cytotoxic agent.

76. 76. The compound of claim 75, wherein the first cytotoxic agent and the second cytotoxic agent are each independently selected from the cytotoxic agents of claims 47-64.

77. 77. The compound of any one of claims 75-76, wherein the first cytotoxic agent and the second cytotoxic agent are covalently attached to said polypeptide via a sortase linker.

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

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

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

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

82. 81. The compound of any one of claims 78-80, wherein the first cytotoxic moiety is covalently attached to the first sortase linker using sortase A, and the second cytotoxic moiety is covalently attached to the second sortase linker using sortase E.

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

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

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

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

87. 77. The compound of 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 the disulfide bond is present in the Fc region or the LGR binding domain.

88. 88. The compound of claim 87, wherein the disulfide bond is contained in a maleimide group.

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

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

91. The first cytotoxic agent has the sequence LPXT(G) n or LAHTGG (SEQ ID NO: 106), where n=1-10; and a second cytotoxic agent is linked to the polypeptide via a disulfide bond; 88. The compound of claim 87.

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

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

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

76.

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

77.

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

97. 97. The compound of any one of claims 1 to 96, in a pharmaceutical composition.

98. 98. A pharmaceutical composition comprising a compound of any one of claims 1-97.

99. 99. The pharmaceutical composition of claim 98, formulated for intravenous, intraperitoneal, subcutaneous, intratumoral, intrathecal, inhalation, intraarterial, or intrapleural administration.

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

101. 101. A host cell comprising the nucleic acid of claim 100.

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

103. 102. The host cell of claim 101, wherein the cell is a eukaryotic cell.

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

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

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

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

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

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

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

111. 111. The method of any one of claims 106-110, 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 C-terminal sortase donor sequence is LPXT(G). n 112. The method of claim 111, wherein n=1 to 10.

113. 113. The method of claim 112, wherein the C-terminal sortase donor sequence is LPETGG (SEQ ID NO:6).

114. The sortase linker is -(His) n -, wherein n=1 to 10.

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

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

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

118. 117. The method of claim 116, wherein the drug is a maytansinoid, auristatin, amanitin, calicheamicin, psymberin, duocarmycin, anthracycline, camptothecin, doxorubicin, taxol, tubulysin, eribruin, or a pyrrolobenzodiazepine.

119. 119. The method of claim 118, wherein the drug is an auristatin.

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

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

122. 117. The method of claim 116, wherein the drug is a camptothecin analog.

123. 123. The method of claim 122, wherein the camptothecin analog is topotecan, irinotecan, belotecan, or deruxtecan.

124. 117. The method of claim 116, wherein the drug is an anthracycline analog.

125. The method of claim 124, wherein the anthracycline analog is PNU-159682 (CAS number: 202350-68-3).

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

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

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

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

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

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

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

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

134. (d) Below: (i) contacting a second cytotoxic agent and the polypeptide with a second transpeptidase; or (ii) covalently attaching a second cytotoxic agent to the polypeptide via a partial disulfide reaction to form a disulfide bond. covalently attaching a second cytotoxic compound to the polypeptide by either 134. The method of any one of claims 106-133, further comprising:

135. The method of claim 134, wherein the second cytotoxic agent is attached to the Fc region or the LGR binding domain by a partial disulfide reaction.

136. 136. The method of claim 135, wherein the second cytotoxic compound comprises a linker, and the linker is attached to the polypeptide by a partial disulfide reaction.

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

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

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

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

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

142. 142. The method of any one of claims 134 to 141, wherein the second cytotoxic agent is a 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 the steps of: (a) expressing the nucleic acid of claim 100 in a cell under conditions that produce the encoded polypeptide; and (b) purifying the polypeptide from the cells.

144. 100. A method of treating a subject having a cell proliferative disorder, comprising administering to the subject an effective amount of a compound of any one of claims 1-97 and / or a pharmaceutical composition of claim 98 or 99.

145. The method of claim 144, wherein the cell proliferative disorder is an autoimmune disease.

146. 145. The method of claim 144, wherein the cell proliferative disorder is cancer or a precancerous condition.

147. 147. The method of claim 146, wherein the cancer or precancerous condition is characterized by the presence of cancer stem cells.

148. The method of claim 147, wherein the cancer stem cells display the LGR on their surface.

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

150. 147. The method of 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. 147. The method of claim 146, wherein the cancer is breast cancer, ovarian cancer, endometrial cancer, colon cancer, gastric cancer, bile duct cancer, lung cancer, liver cancer, skin cancer, neuroblastoma, or leukemia.

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

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

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

155. 155. The method of claim 154, wherein the second anticancer therapy is surgery, chemotherapy, radiation therapy, gene therapy, or immunotherapy.

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

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

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

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

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