WO2025085647A2 - Fc fragments, conjugates, compositions, and methods of use - Google Patents

Abstract

Monomeric Fc (mFc) fragments are provided. In addition, conjugates and compositions comprising such mFc fragments wherein the conjugates provided comprise a folate moiety, a linker, and two haptens are also provided as are methods of use thereof.

Description

70404-02 Fc FRAGMENTS, CONJUGATES, COMPOSITIONS, AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority from Appl. No. 63/590,994, filed October 17, 2023, which is incorporated by reference as if fully set forth herein. TECHNICAL FIELD [0002] The present disclosure relates to Fc (fragment crystallizable domain) fragments, conjugates comprising them, compositions, and methods of use. BACKGROUND [0003] The folate receptor (FR) is a proven target for a variety of tumors and activated macrophages in humans. The FR binds to folic acid conjugates with high affinity and internalizes rapidly, which are ideal attributes for receptor-mediated drug delivery. [0004] Antibodies, through their Fragment crystallizable region (“Fc”), interact with cells or other proteins in the body and it is through this region, that the immune system is activated. The Fc region is typically more conserved between antibodies compared to the other, Fab, region of antibodies. The disclosure describes both novel Fc antibody fragments as well as conjugates of novel Fc fragments and folate targeting ligands that may be, in turn, for example, used in the treatment of cancer. SUMMARY [0005] A monomeric, fragment crystallizable domain (mFc) fragment of immunoglobulin G1 (IgG1 or hIgG1) is provided. In many embodiments, the mFc fragment has (i) a molecular weight of less than 40 kD, (ii) comprises a C_H2 domain and a C_H3 domain, and (iii) binds to the FcRn DQG^DW^OHDVW^RQH^)FȖ^UHFHSWRU. [0006] The mFc fragment can comprise an amino acid sequence of SEQ ID NO:1. The mFc fragment can comprise an amino acid sequence of SEQ ID NO:2. The mFc fragment can comprise an amino acid sequence of SEQ ID NO:3. The mFc fragment can comprise an amino acid sequence of SEQ ID NO:4. The mFc fragment can comprise an amino acid sequence of SEQ ID NO:5. The mFc fragment can comprise an amino acid sequence of SEQ ID NO:6. [0007] At OHDVW^ RQH^ )FȖ^ UHFHSWRU^ can be )FȖ5,^^ )FȖ5,,D^^ )FȖ5,,E^^ DQG^RU^ )FȖ5,,,^^The mFc fragment can comprise DW^OHDVW^RQH^PXWDWLRQ^WKDW^LQFUHDVHV^DIILQLW\^WR^DW^OHDVW^RQH^)FȖ^UHFHSWRU^^In certain embodiments, the at least one mutation is at least one of S239D, A330L, and I332E. 70404-02 [0008] The DW^OHDVW^RQH^)FȖ^UHFHSWRU^can be )FȖ5,,,^^ [0009] The mFc fragment can comprise at least one mutation that increases affinity to the FcRn receptor comparative to wild-type. In certain embodiments, the at least one mutation that increases affinity to the FcRn receptor is selected from the group consisting of H433K/N434F/Y436H and H433K/N434F. [0010] The mFc fragment can comprise at least one mutation that increases in vivo stability of the fragment. In certain embodiments, the at least one mutation that increases in vivo stability of the fragment is at least one of L242C, K334C, P343C, and A431C. [0011] The mFc fragment can comprise at least one mutation that decreases or eliminates aggregation of the fragment. In certain embodiments, the at least one mutation that decreases or eliminates aggregation of the fragment is at least one of L351S, T366R, L368H, and P395K. [0012] The mFc fragment can comprise at least one site for conjugation. The at least one site for conjugation can be at least one of a cysteine, a serine, a lysine, a tyrosine, and an unnatural amino acid. [0013] In many aspects of the disclosure, conjugates of the Formula I, or pharmaceutically acceptable salts, are provided: mFc-L-G (Formula I) wherein: mFc is a monomeric Fc (mFc) fragment of immunoglobulin G1 (IgG1) G is a folate moiety; and L comprises a linker that is covalently bound to G and mFc. [0014] A composition comprising any of the conjugates described herein is also provided, wherein the composition comprises the conjugate and a pharmaceutically acceptable carrier. [0015] Methods for delivering an effective amount of a conjugate hereof or a composition comprising the conjugate and a pharmaceutically acceptable carrier are also described herein. [0016] Methods for treating a subject having cancer are also provided. In certain embodiments, the method for treating a subject having cancer comprises administering to the subject an effective amount of a conjugate described herein or a composition comprising the conjugate and a pharmaceutically acceptable carrier. 70404-02 BRIEF DESCRIPTION OF THE DRAWINGS [0017] The disclosed embodiments and other features, advantages, and aspects contained herein, and the matter of attaining them, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure. Such detailed description will be better understood when taken in conjunction with the accompanying drawings. [0018] FIG. 1 is a schematic showing the design of monomeric Fc fragments with mutations for site-specific conjugation. [0019] FIG. 2 is a MALDI-MS spectrum characterizing the Fc fragments of SEQ ID NO:1 and SEQ ID NO:2 following expression. [0020] FIG.3 is results of Elman’s test for folate conjugates comprising Fc fragments comprising SEQ ID NO:1 and SEQ ID NO:2 indicating successful conjugation of folate to the Fc fragments. [0021] FIG. 4A is a Ponceau stained blot confirming Folate-mFc conjugation. [0022] FIG. 4B is a near-IR (NIR) view of a blot also confirming Folate-mFc conjugation. [0023] FIG. 5 is a plot showing in vivo efficacy evaluation of the folate conjugates comprising the Fc fragments of SEQ ID NO:1 and SEQ ID NO:2 in the M109 lung cancer model. [0024] FIG. 6 is a plot showing in vivo efficacy evaluation of the folate conjugates comprising the Fc fragments of SEQ ID NO:1 and SEQ ID NO:2 in the CT26 colorectal model. [0025] While the present disclosure is susceptible to various modifications and alternative forms, exemplary embodiments thereof are shown by way of example in the drawings and are herein described in detail. DETAILED DESCRIPTION [0026] While the concepts of the present disclosure are illustrated and described in detail in the description herein, results in the description are to be considered as exemplary and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected. [0027] The present disclosure provides materials and methods for delivering an immune effector to a tumor in a subject. More specifically, the present disclosure is directed to the design of Fc (fragment crystallizable domain) fragments, in particular monomeric C_H2 and monomeric C_H2- C_H3 fragments. The monomeric Fc (mFc) fragments hereof are polypeptides of immunoglobulin G1 (IgG1 or hIgG1), which engage immune cells, and penetrate tissues, including solid tumors. 70404-02 Conjugates are also provided that comprise the Fc monomeric fragments, as are pharmaceutical compositions. Methods of making and using such molecules, conjugates, and compositions are also provided. [0028] Monoclonal antibodies (mAbs) are widely used for therapeutic applications. While mAbs represent the largest class of biological drugs, their large size results in poor tissue penetration. Consequently, various small antibody formats have been developed, such as fragment antigen- binding regions (Fabs), variable domains (Fv), single-chain variable fragments (scFv), variable region of the heavy chain (V_H) and nanobodies (VHHs). While the small antibody formats allow for better tissue penetration (comparative to mAbs), they have relatively short half-lives. [0029] The wild-type Fc is homodimeric in nature and this feature is driven by the strong, high- affinity interaction that exists between the two C_H3 domains. Although the term “Fc” is typically thought of as a homodimer of polypeptides, the term as used herein, will also include monomeric polypeptides which comprise a sequence of amino acids corresponding to the Fc portion of the heavy chain (e.g., containing a C_H2 and C_H3 domain). [0030] In many aspects of the disclosure, conjugates of the Formula I, or pharmaceutically acceptable salts, are provided: mFc-L-G (Formula I) wherein: mFc is a monomeric Fc (mFc) fragment of immunoglobulin G1 (IgG1 or hIgG1); G is a folate moiety; and L comprises a linker that is covalently bound to G and mFc. mFc Fragments of IgG1

[0031] In view of an mFc fragment of immunoglobulin G1 (IgG1 or IgG1) is provided. The mFc fragment is a polypeptide and, more specifically, mFc fragments of human IgG1 are portions of the antibody molecule that consist of the constant fragment of the heavy chain (Fc) region of a single IgG1 molecule. The Fc region of IgG1 is responsible for many of the antibody’s effector functions, such as binding to Fc receptors on immune cells, activating complement cascade, and facilitating antibody-dependent cell-mediated cytotoxicity (ADCC). The Fc region is also involved in the regulation of the immune response and the transport of the IgG1 across cell membranes. 70404-02 [0032] In certain embodiments, the mFc fragment has a molecular weight of less than about 40 kDa (e.g., less than 40 kDa). The mFc fragment can have a molecular weight of at or about 25 kDa (e.g., 25 kDa). The mFc fragment can have a molecular weight of at or about 26 kDa (e.g., 26 kDa). The mFc fragment can have a molecular weight of at or about at or about 30 kDa (e.g., 30 kDa). The mFc fragment can have a molecular weight of at or about 35 kDa (e.g., 35 kDa). [0033] A “variant” of a polypeptide comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from, and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants hereof can include those comprising a variant C_H2 or C_H3 domain. In certain embodiments, a variant comprises one or more mutations that, when present in an Fc molecule, increase affinity for the polypeptide to one or more )FȖ^ receptors and/or FcRns. [0034] 7KH^P)F^IUDJPHQW^FDQ^LQWHUDFW^ELQG^WR^WKH^)F5Q^DQG^DW^OHDVW^RQH^)FȖ^UHFHSWRU^(e.g.^^D^)FȖ^ receptor on the surface of an immune cell in a subject and/or the FcRn on the surface of an endothelial cell or macrophage of a subject). 7KH^)FȖ^UHFHSWRU^FDQ^EH^)FȖ5, (CD64)^^)FȖ5,,D (CD32)^^)FȖ5,,E (CD32B)^^DQG^RU^)FȖ5,,, (CD16) (e.g.^^)FȖ5,,,D^DQG^RU^)FȖ5,,,E^. Binding to )FȖ5^^FDQ^KHOS^SKDJRF\WRVLV^E\^DFWLYDWHG^PDFURSKDJHV^^^%LQGLQJ^WR^)FȖ5^^FDQ^KHOS^NLOO^QDWXUDO^ killer (NK) cells. [0035] A monomeric, fragment crystallizable domain (mFc) fragment of immunoglobulin G1 (IgG1 or hIgG1) is provided. In many embodiments, the mFc fragment has (i) a molecular weight of less than 40 kD, (ii) comprises a C_H2 domain and a C_H3 domain, and (iii) bind to the FcRn and DW^OHDVW^RQH^)FȖ^The mFc fragment can comprise the amino acid sequence of SEQ ID NO:1. The mFc can comprise the amino acid sequence of SEQ ID NO:2, 3, 4, 5, or 6. The mFc can have at least 60% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, or 6. In a particular embodiment, the mFc comprises at least 65%, 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 98%, or at least 99% identity to SEQ ID NO:2, 3, 4, 5, or 6. [0036] “Sequence identity” or “percent identity” herein describes the extent to which two nucleotide or amino acid sequences are invariant in an alignment of sequences. “Sequence alignment” means the process of lining up two or more sequences to achieve maximal levels of identity (and, in the case of amino acid sequences, conservation) for the purpose of assessing the degree of similarity. An alignment of sequences can be created by manually aligning at least two sequences, for example, a stated sequence as a reference and another sequence, to produce the 70404-02 highest number of matching elements (e.g., individual nucleotides or amino acids) while allowing for the introduction of gaps into either sequence. An “identity fraction” for a sequence aligned with a reference sequence is the number of matching elements, divided by the full length of the reference sequence, not including gaps introduced by the alignment process into the reference sequence. “Percent identity” as used herein is the identity fraction times 100. [0037] The mFc fragment can comprise at least one mutation that increases affinity to at least one )FȖ^UHFHSWRU (e.g., comparative to wild-type or the mFc fragment without the mutation), at least one mutation that increases affinity to the FcRn receptor (e.g., comparative to wild-type or the mFc fragment without the mutation), at least one mutation that increases in vivo stability of the fragment (e.g., comparative to wild-type or the mFc fragment without the mutation), and/or at least one mutation that decreases or eliminates aggregation of the fragment. While these mutations can comprise one or more conservative amino acid substitutions (see, e.g., Table 1 and described above), such mutations also can include non-conservative amino acid substitutions. Examples of non-conservative amino acid substitutions include substitution of a hydrophilic residue (e.g., Ser or Thr) with a hydrophobic residue (e.g., Leu, Ile, Phe, Val or Ala) or vice versa, substitution of a Cys or Pro with any other residue or vice versa, substitution of an amino acid having an electropositive side chain (e.g., Lys, Arg, or His) with an amino acid having an electronegative side chain (e.g., Glu or Asp) or vice versa, or substitution of an amino acid having a bulky side chain (e.g., Phe) with an amino acid not having a bulky side chain (e.g., Gly). [0038] The mFc fragment can comprise at least one mutation that increases affinity to at least one )FȖ^UHFHSWRU^^e.g^^^)FȖ5,,,^^ 7KH^DW^OHDVW^RQH^PXWDWLRQ^WKDW^LQFUHDVHV^DIILQLW\^WR^DW^OHDVW^RQH^)FȖ^ receptor can be at least one of S239D, A330L, and I332E. [0039] The mFc fragment can comprise at least one mutation that increases in vivo stability of the fragment. The at least one mutation that increases in vivo stability of the fragment can be at least one of L242C, K334C, P343C, and A431C. The mFc fragment can comprise at least one mutation that decreases or eliminates aggregation of the fragment (e.g., in vivo). The at least one mutation that decreases or eliminates aggregation of the fragment can be at least one of L351S, T366R, L368H, and P395K. The at least one mutation that decreases or eliminates aggregation of the fragment can be a mutation that increase the thermal stability of the fragment and/or reduce exposure to hydrophobic patches that promote protein-protein interactions. 70404-02 [0040] The mFc fragment can be glycosylated or unglycosylated. For example, a recombinant mFc fragment can be expressed in a mammalian that glycosylates the expressed mFc fragment. [0041] In general, the IgG1 C_H2, C_H3 and C_H2-C_H3 domain wild-type and mutants can be synthesized or cloned. The sequences can be synthesized by any suitable method known in the art, such as the phosphotriester method, the phosphodiester method, the diethylphosphoramidite method, the solid phase phosphoramidite triester method, and the solid support method of U.S. Patent No. 4,458,066. Narang et al., Improved phosphotriester method for the synthesis of gene fragments, Methods in Enzymology 68: 90-99 (1979); Brown et al., Chemical synthesis and cloning of a tyrosine tRNA gene, Methods in Enzymology 68: 109-151 (1979); Beaucage et al., Deoxynucleoside phosphoramidites – a new class of key intermediates for deoxypolynucleotide synthesis, Tetrahedron Letters 22: 1859-1862 (1981). [0042] The sequences can be cloned by any suitable method known in the art. See, e.g., Molecular Cloning: A Laboratory Manual, Green et al., 4^th ed. (2012); Berger & Kimmel, Methods in Enzymology, vol. 152: Guide to Molecular Cloning Techniques, 1^st ed. (1987); Short Protocols in Molecular Biology, Ausubel et al., eds. 4^th ed. (1999). In some embodiments, nucleic acid molecules which encode a polypeptide (e.g., mFc fragment) set forth in any of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 are provided. These nucleic acid molecules can be isolated using standard molecular biology techniques and the sequence information provided herein. Nucleic acids can be prepared by amplification, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification system (AS), or the self-sustained sequence replication system (3SR). Furthermore, oligonucleotides corresponding to nucleotide sequences can be prepared by standard synthetic techniques. [0043] The IgG1 C_H2, C_H3 and C_H2-C_H3 domain wild-type and mutants then can be subcloned into a vector in operable linkage with a regulatory sequence such as a promoter. Expression vectors include, but are not limited to, plasmids, phages, viruses, adenoviruses, bacterial artificial chromosomes, yeast artificial chromosomes, and other vehicles suitable for expressing a polypeptide. Any vector that transduces genetic material into a cell and, if replication is desired, which is replicable and viable in the relevant host can be used. [0044] The term “vector” or “expression vector” means the vehicle by which a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence (e.g., a foreign gene) can be introduced into a host cell so as to transform the host and promote expression (e.g., transcription and translation) of 70404-02 the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein (e.g., a mFc fragment thereof) is inserted by restriction enzyme technology or the like. A common type of vector is a plasmid, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can be readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example by producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein or polypeptide (e.g., a mFc fragment hereof). The expression product itself, e.g., the resulting protein, can also be said to be “expressed” by the cell. A polypeptide can be expressed recombinantly, for example, when it is expressed or produced in a foreign cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter. [0045] The regulatory sequence can comprise a promoter operably linked to the sequence. The promoter can be constitutive or inducible. The promoter can optionally include distal elements, such as enhancer or repressor elements. Other elements include those that control promoter- dependent gene expression by cell or tissue type or induction by an external agent or signal. An example of a promoter is the cytomegalovirus (CMV) promoter. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available. [0046] The vector can be introduced into a host cell for replication and expression of the targeted polypeptide or protein. Examples of host cells include microbial, yeast, insect, and mammalian organisms and cell lines (e.g., COS, CHO, HeLa and myeloma). The vector can be introduced using any suitable method known in the art. Examples of methods include transformation, transfection, microinjection, electroporation, and liposomes. [0047] Recombinantly expressed polypeptides can be carried out by conventional means, such as preparative chromatography and immunological separations. Purification can be accomplished using standard procedures, such as ammonium sulfate precipitation, affinity columns, column chromatography, and the like. 70404-02 [0048] Alternatively, such polypeptides can be synthesized, in whole or in part, using standard peptide synthesis. See, e.g., Barany & Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special methods in Peptide Synthesis, Part A. pp.3-284; Merrifield et al., JACS 85: 2149-2156 (1963); and Stewart et al., Solid Phase Peptide Synthesis, 2^nd ed., Pierce Chem. Co., Rockford, IL (1984). [0049] Conjugates [0050] A conjugate of Formula I is also provided: mFc-L-G (Formula I), or a pharmaceutically acceptable salt thereof, wherein: mFc is a monomeric Fc (mFc) fragment of immunoglobulin G1 (IgG1) G comprises a folate moiety; and L comprises a linker that is covalently bound to G and mFc. [0051] In many embodiments, the mFc is of human IgG1, has (i) a molecular weight of less than 40 kD, (ii) comprises a C_H2 domain and a C_H3 domain, and (iii) binds to the FcRn and at least one )FȖ^UHFHSWRU. In certain embodiments, the mFc comprises the amino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, or 6. In certain embodiments, the mFc comprises an amino acid sequence with at least 60% sequence identity to SEQ ID NO:1, 2, 3, 4, 5, or 6. [0052] G comprises a folate moiety which includes folate analogs such as radicals of methotrexate such as:

_. [0053] In many embodiments, G can be, for example, any of the following folate moieties:

70404-02

[0054] In many embodiments, L can comprise a chain of atoms from 3 atoms to 60 atoms in OHQJWK^^,Q^PDQ\^HPERGLPHQWV^^/^FDQ^FRPSULVH^D^FKDLQ^RI^DWRPV^IURP^DERXW^^^Ⴒ^WR^DERXW^^^^ Ⴒ^LQ^OHQJWK^^7KH^FKDLQ^RI^DWRPV^DUH^SDUW^RI^WKH^EDFNERQH^RI^WKH^FRQMXJDWH^RI^)RUPXOD^,^ The “backbone” of the linker L is the shortest chain of contiguous atoms forming a covalently bonded connection between mFc and L on one side of the conjugate mFc-L-G, and L and G on the other side of the conjugate mFc-L-G. [0055] In many embodiments, L comprises one or more peptide residues. For example, L comprises one or more phenylalanine residues, each of which is independently optionally substituted. L comprises at least one phenylalanyl-phenylalanyl residue, in which at least one phenyl is independently optionally substituted. In these and other embodiments, L comprises a polyethylene glycol_n (PEG_n) moiety, wherein n = 1-36. [0056] In many embodiments, L comprises one or more linker groups, each linker group may comprise one or more of a polyethylene glycol (PEG) moiety, an alkyl group, a sugar moiety, and/or a peptide moiety. In some embodiments, the linker is a polyethylene glycol- (PEG-) (e.g., pegylated-), alkyl-, sugar-, and peptide-based linker. [0057] In some embodiments, L is a non-releasable linker covalently to both mFc and to G. [0058] In some embodiments, the linker comprises a hydrophilic substituent, such as a substituent that comprises one or more amino acid residues (which are the same or different), an alkyl chain, a polyethylene glycol (PEG) moiety, or a combination of an any of the foregoing. in some embodiments, the linker comprises an oligomer of peptidoglycan residues, glycan residues, or anion of amino acids. [0059] For a linker that comprises one or more PEG moieties, all carbon and oxygen atoms of the one or more PEG moieties are part of the backbone of the linker L unless otherwise specified. [0060] In many embodiments, L has a chain of atoms at least 3 atoms in length, at least 7 atoms in length, at least 10 atoms in length, at least 14 atoms in length, or at least 20 atoms in 70404-02 length. In many embodiments, L has a chain of between 3 and 7 atoms in length, between 7 and 10 atoms in length, between 10 and 14 atoms in length, between 14 and 20 atoms in length, between 20 and 30 atoms in length, between 30 and 40 atoms in length, between 40 and 50 atoms in length, or between 50 and 60 atoms in length. In many embodiments, L has a chain of at 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 atoms in length. The use of the word “between” is inclusive of the endpoints meaning that between 3 and 5 atoms in a chain length includes 3 atoms and includes 5 atoms. [0061] In some embodiments, L is an amino acid residue of the formula:

with three carbon atoms in the linker backbone as presented wherein R can be H, alkyl, arylalkyl, -alkyl-S-alkyl or arylalkyl or the side-chain of any naturally- or non-naturally occurring amino acid.. Examples of R include H (making the residue a, glycine residue), alkyl (such that residue may be a residue of alanine, valine, isoleucine, and leucine among others), -alkyl-S-alkyl (such that the residue may be methionine among others), arylalkyl (such that the residue may be phenylalanine, tyrosine, and tryptophan among others).The carbon atom to which R is attached can be chiral and can have any suitable relative configuration, such as a D- or L-configuration. [0062] In many embodiments, the atoms used in forming L comprise one or more chains of carbon atoms forming alkylene groups, one or more chains of carbon and oxygen atoms. It is to be understood that the bonds connecting atoms in the chain can be either saturated or unsaturated, such that for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, and the like can be divalent radicals that are included in L. In each of the foregoing , the atoms of L in the chain can be substituted or unsubstituted. In many embodiments the substitutions are: 70404-02 ,

[0063] In addition, it is to be understood that the atoms forming the linker may also be cyclized to form saturated or unsaturated divalent cyclic radicals in the linker, such as radicals of the formulae:

wherein each X¹ is independently CH₂, NR’, or O wherein R’ is alkyl or hydrogen and each X² is independently S, O, N, NH, CR” wherein R” is alkyl or hydrogen. Examples of such radicals include:

[0064] In some embodiments, L comprises suitable substituents that change the hydrophobicity or hydrophilicity of L. Thus, for example, L can have hydrophobic side chain group, such as an alkyl, cycloalkyl, aryl, arylalkyl, or like group, each of which is optionally substituted. In some embodiments, L comprises one or more amino acid residues, such as hydrophobic amino acid side chains, including phenylalanine (Phe) and tyrosine (Tyr) residues, and further including substituted variants thereof, and analogs and derivatives of 70404-02 such side chains. Other examples of L include alkylene-amino-alkylenecarbonyl, alkylene- thio-(carbonylalkylsuccinimid-3-yl) moieties, as further illustrated by the following formulae:

wherein x and y are each independently 1, 2, 3, 4, or 5 , where the asterisk identifies points of attachment either to other linker moieties or to G or mFc. [0065] In many embodiments, the backbone atoms in the linker may contain the following atoms – carbon, nitrogen, oxygen, phosphorous, and sulfur. These atoms may be substituted with a number of different substituents including -CH₂-, -C(O)--N(H)-, -N(R¹)= wherein R¹ is, for example, H, alkyl, and alkylaryl, and -O-P(O)(OH)O-). [0066] In these and other embodiments, L comprises one or more of chains of carbon atoms forming alkyl groups, chains of carbon and oxygen atoms forming polyoxyalkyl groups, chains of carbon and nitrogen atoms forming polyamines, and others, including rings, such as those that form aryl and heterocyclyl groups (e.g., triazoles, and oxazoles). In addition, the bonds connecting atoms in the backbone of L can be either saturated or unsaturated, such that for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, can be divalent radicals that are included in L. Further, such atoms in the chain of L may be substituted or unsubstituted. [0067] Additional examples of radical groups which may be present in L include 1- alkylsuccinimid-3-yl, carbonyl, thionocarbonyl, alkyl, cycloalkyl, alkylcycloalkyl, alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-alkylsuccinimid-3-yl, 1- (carbonylalkyl)succinimid-3-yl, alkylsulfoxyl, sulfonylalkyl, alkylsulfoxylalkyl, alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1- (carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and 1- (carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each group can be substituted or unsubstituted. Any of the aforementioned groups may be the linker L or may be included as a portion of L. In some embodiments, one or more of the aforementioned groups can be used in combination (or more than once) (e.g., -alkyl-C(O)-alkyl) and may further comprise an additional nitrogen (e.g., alkyl-C(O)-NH-, -NH-alkyl-C(O)- or -NH-alkyl-), oxygen (e.g., - alkyl-O-alkyl-) or sulfur (e.g., -alkyl-S-alkyl-). Examples of such L groups are alkylcarbonyl, 70404-02 cycloalkylcarbonyl, carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, and succinimid-3-ylthiol, wherein each group can be substituted or unsubstituted. [0068] In some embodiments, L can be formed via click chemistry/click chemistry-derived. For example, L can be derived from copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain promoted azide-alkyne cycloaddition (SPAAC), inverse electron demand Diels-Alder reaction (IEDDA), and Staudinger ligation (SL). For example, G can be a moiety of the formula G-N3. G-N3 can then be reacted with an alkyne in order to make a triazole bound on one side to G and on the other side to a dual hapten linker construct, wherein the triazole may be considered part of the linker, if the linker is click-chemistry derived. [0069] In many embodiments, L comprises pegylated-, alkyl-, sugar-, and peptide- based linker; bivalently covalently attached to G and mFc. ,

wherein x is an integer from 0 to 10 and y is an integer from 3 to 24 [0070] In other exmaples, the linker L can comprise:

wherein each of R² and R³ is independently H or C_1-6alkyl; and z is an integer from 1 to 8. [0071] In some embodiments, L comprises one or more spacer linkers wherein the spacer linker is bound to a linker backbone atom. In some embodiments, spacer linkers are hydrophilic spacer linkers comprising a plurality of hydroxyl functional groups. A spacer linker may comprise any stable arrangement of atoms. For example, a spacer linker may comprise one or more L’ where each L’ is independently selected from a radical group consisting an amide, ester, urea, carbonate, carbamate, disulfide, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl (e.g., polyethylene glycol), cycloalkyl, aryl, 70404-02 heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof. In some embodiments, a spacer linker comprises any one or more of the following units: an amide, ester, urea, carbonate, carbamate, disulfide, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl, cycloalkyl, aryl, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof. In some embodiments, a spacer linker comprises a glycosylated amino acid. In some embodiments, a spacer comprises one or more monosaccharide, disaccharide, polysaccharide, glycan, or peptidoglycan. In some embodiments, a spacer comprises one or more units such as ethylene (e.g., polyethylene), ethylene glycol (e.g., PEG), ethanolamine, ethylenediamine, and the like (e.g., propylene glycol, propanolamine, or propylenediamine). In some embodiments, a spacer linker comprises an oligopeptide, polypeptide, a rigid functionality, peptidoglycan, oligoproline, oligopiperidine, or any combination thereof. In some embodiments, a spacer linker comprises an oligoethylene glycol or a PEG. In some embodiments, a spacer linker comprises an oligoethylene glycol. In some embodiments, a spacer linker comprises a PEG. In some embodiments, a spacer linker comprises an oligopeptide or polypeptide. In some embodiments, a spacer linker comprises an oligopeptide. In some embodiments, a spacer linker comprises a polypeptide. In some embodiments, a spacer linker comprises a peptidoglycan. In some embodiments, a spacer linker does not comprise a glycan. In some embodiments, a spacer linker does not comprise a sugar. [0072] In some embodiments, a rigid functionality is an oligoproline or oligopiperidine. In some embodiments, a rigid functionality is an oligoproline. In some embodiments, a rigid functionality is an oligopiperidine. In some embodiments, a rigid functionality is an oligophenyl. In some embodiments, a rigid functionality is an oligoalkyne. [0073] In some embodiments, an oligoproline or oligopiperidine has about two up to and including about fifty, about two to about forty, about two to about thirty, about two to about twenty, about two to about fifteen, about two to about ten, or about two to about six repeating units (e.g., prolines or piperidines). [0074] The conjugates can be presented as a pharmaceutically acceptable salt. Similarly, “pharmaceutically acceptable salt” refers to those salts with counter ions, which can be used in pharmaceuticals. More specifically with respect to the present disclosure, the terms “salts” and 70404-02 “pharmaceutically acceptable salts” as used herein refer to derivatives of the disclosed conjugates wherein the parent conjugate is modified by making acid or base salts thereof. [0075] The conjugates hereof can be “deuterated,” meaning one or more hydrogen atoms can be replaced with deuterium. As deuterium and hydrogen have nearly the same physical properties, deuterium substitution is the smallest structural change that can be made. Deuteration is well known to those of ordinary skill in the art. [0076] The conjugates, in some embodiments, can contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R)- or (S)-. In certain embodiments, the conjugate is of R- configuration. In certain embodiments, the conjugate is of S-configuration. Unless stated otherwise, it is intended that all stereoisomeric forms of the conjugates are contemplated. When the conjugates contain alkene double bonds, and unless specified otherwise, it is intended that both E and Z geometric isomers (e.g., cis or trans) and/or optical isomers are included. In certain embodiments, for example, D and A of a conjugate are arranged in a relative cis orientation. In certain embodiments, D and A of a conjugate are arranged in a relative trans orientation. Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included. The term “geometric isomer” refers to E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. The term “positional isomer” refers to structural isomers around a central ring, such as ortho-, meta-, and para- isomers around a benzene ring. [0077] Further, in each of the foregoing and following embodiments, it is to be understood that the formulae include and represent not only all pharmaceutically acceptable salts of the conjugates, but also includes hydrates and/or solvates of the conjugate formulae or salts thereof. Indeed, hydrates, solvates, of the conjugates are also contemplated. The term “solvate” means a conjugate, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate. [0078] Compositions [0079] Further provided is a composition. The composition comprises an above-described a conjugate of Formula I or the pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients or carriers. The term “composition” generally refers to any product comprising more than one ingredient, including the conjugate. 70404-02 [0080] Compositions contain an effective amount of conjugates of Formula I as described herein and optionally one or more other therapeutic agents included in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a subject. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the compositions also can be commingled with the conjugates of Formula I in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. [0081] The compositions hereof can comprise one or more pharmaceutically acceptable carriers, adjuvants, diluents, excipients, and/or [0082] The conjugates of Formula I can each be formulated as compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route of administration. The compositions can be formulated, e.g., for a given route of administration, and manufactured in accordance with methods in the art and described, for example, in Remington, The Science and Practice of Pharmacy, 22^nd edition (2012). [0083] Methods [0084] Still further provided is a method of treating cancer in a subject. The method can comprise administering, such as orally or intravenously, to the subject: a therapeutically effective amount of the above-described conjugates of Formula I or pharmaceutically acceptable salt thereof, or an above-described composition comprising one or more conjugates of Formula I. The subject can be an animal, such as a mammal, e.g., a human. [0085] The terms “treat,” “treating,” “treated,” or “treatment” (with respect to a disease or condition) is an approach for obtaining beneficial or desired results including and preferably clinical results and includes, but is not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, prolonging survival and/or prophylactic or preventative treatment. [0086] An “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. [0087] Certain Definitions 70404-02 [0088] As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art. [0089] The term “about,” when referring to a number or a numerical value or range (including, for example, whole numbers, fractions, and percentages), means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g., +/- 5 % to 15% of the recited value) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). The term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range. [0090] The terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods and/or steps of the type, which are described herein and/or which will become apparent to those ordinarily skilled in the art upon reading the disclosure. [0091] The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. [0092] An “alkyl group” is a saturated, partially saturated, or unsaturated straight chain or branched non-cyclic hydrocarbon having from 1 to 10 carbon atoms (C₁-C₁₀ alkyl), from 1 to 8 carbons (C₁-C₈ alkyl), from 1 to 6 (C₁-C₆ alkyl), 1 to 4 (C₁-C₄ alkyl), 1 to 3 (C₁-C₃ alkyl), or 2 to 6 (C₂-C₆ alkyl) carbon atoms. In some embodiments, the alkyl group has monovalency. Examples of alkyl groups with monovalency include -CH₃, -CH₂CH₃, and the like. Monovalent alkyls may be found on substitutions in the chain of linker, L, for example. In some embodiments, the alkyl group has bivalency, such as when found in the chain of the linker, L. Examples of alkyl groups with bivalency include, but are not limited to, -CH₂-, -CH₂CH₂-, and the like. In some embodiments, the alkyl group is a saturated alkyl group. In some embodiments, an alkyl group is an unsaturated alkyl group, also termed an alkenyl group or an alkynyl group. [0093] The term “folate moiety” means a chemical moiety based on folic acid which has been turned into a radical to enable conjugation. 70404-02 [0094] The term “mFc” means a monomer from the Fc region of an antibody, typically an IgG1 or hIgG1antibody. The Fc region is also referred to as the fragment crystallizable region. [0095] The terms “patient” and “subject” are used interchangeably and include a human patient, a laboratory animal, such as a rodent (e.g., mouse, rat, or hamster), a rabbit, a monkey, a chimpanzee, a domestic animal, such as a dog, a cat, or a rabbit, an agricultural animal, such as a cow, a horse, a pig, a sheep, or a goat, or a wild animal in captivity, such as a bear, a panda, a lion, a tiger, a leopard, an elephant, a zebra, a giraffe, a gorilla, a dolphin, or a whale. The patient to be treated is preferably a mammal, in particular a human being. [0096] The terms “protein,” “polypeptide” and “peptide” refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. [0097] Any use of section headings and subheadings is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one section heading or subheading is intended to constitute a disclosure under each and every other section heading or subheading. [0098] Various modifications and variations of the described compounds, compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims. [0099] The terms and expressions, which have been employed, are used as terms of description and not of limitation. In this regard, where certain terms are defined and are described or discussed elsewhere, the definitions and all descriptions and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. [0100] Further, all publications and patents mentioned herein are incorporated by reference in their entireties for all purposes. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls. 70404-02 ENUMERATED EMBODIMENTS [0101] Embodiment 1 relates to a monomeric, fragment crystallizable domain (mFc) fragment of human immunoglobulin G1 (IgG1) which: (i) has a molecular weight of less than 40 kD, (ii) comprises a C_H2 domain and a C_H3 domain, and (iii) binds WR^WKH^)F5Q^DQG^DW^OHDVW^RQH^)FȖ^UHFHSWRU [0102] Embodiment 2 relates to the mFc fragment of any one of Embodiment 1 wherein the at OHDVW^RQH^)FȖ^UHFHSWRU^LV^)FȖ5,^^)FȖ5,,D^^)FȖ5,,E^^DQG^RU^)FȖ5,,,^ [0103] Embodiment 3 relates to the mFc fragment of Embodiment 1, comprising an amino acid sequence of SEQ ID NO:1. [0104] Embodiment 4 relates to the mFc fragment of Embodiment 1, comprising an amino acid sequence of SEQ ID NO:2. [0105] Embodiment 5 relates to the mFc fragment of Embodiment 1, comprising an amino acid sequence of SEQ ID NO:3. [0106] Embodiment 6 relates to the mFc fragment of Embodiment 1, comprising an amino acid sequence of SEQ ID NO:4. [0107] Embodiment 7 relates to the mFc fragment of Embodiment 1, comprising an amino acid sequence of SEQ ID NO:5. [0108] Embodiment 8 relates to the mFc fragment of Embodiment 1, comprising an amino acid sequence of SEQ ID NO:6. [0109] Embodiment 9 relates to the mFc fragment of Embodiment 1, comprising at least one PXWDWLRQ^WKDW^LQFUHDVHV^DIILQLW\^WR^DW^OHDVW^RQH^)FȖ^UHFHSWRU^ [0110] Embodiment 10 relates to the mFc fragment of Embodiment 9, wherein the at least one )FȖ^UHFHSWRU^LV^)FȖ5,,,^^ [0111] Embodiment 11 relates to the mFc fragment of Embodiment 9, wherein one mutation is at least one of S239D, A330L, and I332E. [0112] Embodiment 12 relates to the mFc fragment of Embodiment 1, comprising at least one mutation that increases affinity to the FcRn receptor comparative to wild-type. [0113] Embodiment 13 relates to the mFc fragment of Embodiments 9-12, wherein the at least one mutation that increases affinity to the FcRn receptor is selected from the group consisting of H433K/N434F/Y436H and H433K/N434F. 70404-02 [0114] Embodiment 14 relates to the mFc fragment of Embodiment 1, comprising at least one mutation that increases in vivo stability of the fragment. [0115] Embodiment 15 relates to the mFc fragment of Embodiment 14, wherein the at least one mutation is at least one of L242C, K334C, P343C, and A431C. [0116] Embodiment 16 relates to the mFc fragment of Embodiment 1, which comprises at least one mutation that decreases or eliminates aggregation of the fragment. [0117] Embodiment 17 relates to the mFc fragment of Embodiment 16, wherein the at least one mutation is at least one of L351S, T366R, L368H, and P395K. [0118] Embodiment 18 relates to the mFc fragment of Embodiment 1, which comprises at least one site for conjugation. [0119] Embodiment 19 relates to the mFc fragment of Embodiment 18, wherein the at least one site for conjugation is at least one of a cysteine, a serine, a lysine, a tyrosine, and an unnatural amino acid. [0120] Embodiment 20 relates to a conjugate of Formula I: mFc-L-G (Formula I) or a pharmaceutically acceptable salt thereof, wherein: mFc is a monomeric Fc fragment of human immunoglobulin G1 (IgG1 or hIgG1; G is a folate moiety; and L comprises a linker that is covalently bound to G and mFc. [0121] Embodiment 21 relates to the conjugate of Embodiment 20, where the folate moiety comprises a methotrexate residue or:

70404-02

[0122] Embodiment 22 relates to the conjugate of Embodiment 20 or claim 21 wherein mFc comprises one or more of a cysteine residue, a lysine residue, or a tyrosine residue. [0123] Embodiment 23 relates to the conjugate of Embodiment 22, wherein one or more one or more of a cysteine residue, a lysine residue, or a tyrosine residue is covalently bound to L. [0124] Embodiment 24 relates to the conjugate of any one of Embodiments 20-23, wherein L comprises one or more unnatural amino acids. [0125] Embodiment 25 relates to the conjugate of Embodiment 24, wherein one unnatural amino acid is covalently bound to mFc. [0126] Embodiment 26 relates to the conjugate of any one of Embodiments 20-25, wherein L further comprises a spacer. [0127] Embodiment 27 relates to the conjugate of Embodiment 26, wherein the spacer is a hydrophilic spacer. [0128] Embodiment 28 relates to the conjugate of any one of Embodiments 20-27, wherein L comprises one or more of an amino acid or a polyethylene glycol (PEG) moiety. [0129] Embodiment 29 relates to the conjugate of any one of Embodiments 20-28, wherein L comprises an oligomer of peptidoglycans residues or glycan residues or a combination thereof. [0130] Embodiment 30 relates to the conjugate of any one of Embodiments 20-29, wherein the mFc is an mFc of any one of Embodiments 1-19. [0131] Embodiment 31 relates to a composition comprising a conjugate of any one of Embodiments 20-30 and a pharmaceutically acceptable carrier. [0132] Embodiment 32 relates to a method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate of any one of Embodiments 20-30 or a composition comprising the conjugate and a pharmaceutically acceptable carrier. 70404-02 SEQUENCE LISTINGS [0133] mFc-WT-G1-C152S (SEQ ID NO:1) EPKSGDKTHTSPPGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTSLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [0134] mFc-WT-G1-C210S (SEQ ID NO:2) EPKSGDKTHTSPPGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSSSVMHEALHNHYTQKSLSLSPGK [0135] mFc-WT-G2-C152S (SEQ ID NO:3) EPKSGDKTHTSPPGPAPELLGGPSVFCFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEPKCKVSNKALPAPI ECTISKAKGQCREPQVYTSPPSRDELTKNQVSLRSHVKGFYPSDIAVEWESNGQPENNY KTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHECLHNHYTQKSLSLSPGK. [0136] mFc-WT-G2-C210S (SEQ ID NO:4) EPKSGDKTHTSPPGPAPELLGGPSVFCFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEPKCKVSNKALPAPI ECTISKAKGQCREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNY KTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSSSVMHECLHNHYTQKSLSLSPGK. [0137] mFc-DLE-G2-C152S (SEQ ID NO:5) EPKSGDKTHTSPPGPAPELLGGPDVFCFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLP EECTISKAKGQCREPQVYTSPPSRDELTKNQVSLRSHVKGFYPSDIAVEWESNGQPENNY KTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHECLHNHYTQKSLSLSPGK [0138] mFc-DLE-G2-C210S (SEQ ID NO:6) EPKSGDKTHTSPPGPAPELLGGPDVFCFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLP EECTISKAKGQCREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENN YKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSSSVMHECLHNHYTQKSLSLSPGK [0139] SEQ ID NO:1 is an amino acid sequence for mFc-WT-G1-C152S, a monomeric Fc fragment payload with C152S mutation to enable site-specific conjugation with drug to monomeric Fc fragment ratio 1:1. 70404-02 [0140] SEQ ID NO:2 is an amino acid sequence for is an amino acid sequence for mFc-WT-G1- C210S, a monomeric Fc fragment payload with C210S mutation to enable site-specific conjugation with drug to monomeric Fc fragment ratio 1:1. [0141] SEQ ID NO:3 is an amino acid sequence for is an amino acid sequence for mFc-WT-G2- C152S, a monomeric Fc fragment payload with C152S mutation to enable site-specific conjugation with drug to monomeric Fc fragment ratio 1:1. [0142] SEQ ID NO:4 is an amino acid sequence for is an amino acid sequence for mFc-WT-G2- C210S, a monomeric Fc fragment payload with C210S mutation to enable site-specific conjugation with drug to monomeric Fc fragment ratio 1:1. [0143] SEQ ID NO:5 is an amino acid sequence for is an amino acid sequence for mFc-DLE-G2- C152S, a monomeric Fc fragment payload with C152S mutation to enable site-specific conjugation with drug to monomeric Fc fragment ratio 1:1. [0144] SEQ ID NO:6 is an amino acid sequence for is an amino acid sequence for mFc-DLE-G2- C210S, a monomeric Fc fragment payload with C210S mutation to enable site-specific conjugation with drug to monomeric Fc fragment ratio 1:1. EXAMPLES [0145] The following examples serve to illustrate the present disclosure and are not intended to limit the scope of the claimed invention in any way. LIST OF ABBREVIATIONS

70404-02

c^o N B ² H ₀ N -₄ ^{3 0} 6 _{1 4} O ⁰ O N₉ ² ₇ ⁷ ₉ H₅ ^. ₇ 8 ⁴ ₈ O H N ^C ₃ : : _t O _a ^h H N l _{g u} ⁱ 44 e H ⁰ _. H m^{r W} o ^r 6 ^{C: 6} a^l ₁:_t

N_{c E} o _B ^P _I D N A ^{O E} _{S T} M D O S O₅ N _a O N 1 O 8 N_{4 e} O O F₄ ^{5 l} N_{8 p} H ^{2 8} ₄ H . ⁶ _{2 1} ^. O m H₂ 3 ₈ ³ O ^C ₁ ² ₄ ⁶ a _{2 5} O F ^: _a ^l :_t ) ₂ x O N H ^C : : _t ^h _u ^h _g ^{i 7} ( E _a O ^l _{g u} ⁱ m e F F^r _{o e} ^F m^r _o ^{W F r l W} _T r ^{B Fl} _a ^l F _a ^c _{i a} ^l _{C u a u} S c_i ^c _e 3 l ^O ₃ m^c _e ^l N H m^e _o 2 _a ^e N _{h o} C M h M ^H C _N N N 6 O O N ¹ O₁ N H ₉ N₂ H N N ^{1 9} _{8 4} ^. ₇ 2) H O H^{3 4} _{7 ( 3} : H _{t S} O ^{C: h} _g H t ^r _a ^{l i N} _{, u e ,} ^O m ^W C _S O N H ^{r r} C M _o ^F _a ^l D D O ^l _{u a} ^{c c} _{i e} ^l _o 6 H m^e M O _h O C 6 N H H O O N₇ 9 ⁰ ₄ H₁ ^. ₁ ⁴ N N O N H ⁹ _{1 4} O ^C : : _{t a} ^{h l} _g ⁱ _e O N u m^r _o ^W N r H H ^l _a ^l N F _u H₂ N _a ^c _i ^c _e 1 l m_o d e_h M ⁱ _c ^a N N C ^c _i ^t _e ^c N _a O ^o _r N ^o H _u N ^lf 2 _i ^r H _T 70404-02 Experimental procedures for compound 8: [0146] All starting materials, reagents and solvents listed in the documents are procured from various commercial sources. [0147] Synthesis of Compound 3: To a solution of commercially available compound 1 (0.50 g, 1.133 mmol) and N-hydroxysuccinimide (commercially available 2, 0.143 g, 1.246 mmol, 1.10 equiv.) in GLPHWK\O^VXOIR[LGH^^^^^P/^^ZDV^DGGHG^1^^1ƍ-Dicyclohexylcarbodiimide (0.281 g, 1.359 mmol, 1.2 equiv.) at r.t under argon and the heterogeneous solution was stirred for 12 hours. Progress of the reaction was monitored by LC/MS. After completion of the reaction was confirmed by LC-MS by disappearance of compound 1, the reaction mixture was precipitated in acetone (15x) and the product was isolated by centrifugation. Further, the crude product was washed with acetone (2 x 50 mL) and EtOAc (1x30 mL), dried under vacuum, and crude compound 3 was used for the next step without further purification. LC-MS [M+H] = 539.48. [0148] Synthesis of Compound 5: To a solution of 3 (0.05 g, 0.093 mmol) in dry DMSO (1.0 mL) was added BocNH-PEG₆-NH₂, (4, procured from BroadPharma, USA, 0.032 g, 0.093 mmol, 1.05 equiv.), followed by N,N-diisopropylethyl amine (DIPEA, 0.081 mL, 0.46 mmol, 5.0 equiv.) at room temperature under argon and stirred for 12 hours. Progress of the reaction was monitored by LC/MS. After completion of the reaction was confirmed by LC/MS, the reaction mixture was precipitated by adding diethyl ether and centrifuged. The yellow precipitate was collected and washed with diethyl ether (2 x 15 mL) and crude mass purified by prep-HPLC on a C18 column (5-95% B over 60 minutes, flow 7 mL/min; B: acetonitrile; A: 20 mM NH₄OAc, pH 7 buffer, UV @ 280 nm) to afford Compound 5. LC-MS [M+H] = 848.95 [0149] Synthesis of Compound 6: To the compound 5, was added trifluoroacetic acid (1.5 mL). The solution was stirred at r.t until the reaction was completed as demonstrated by LC-MS. Reaction completed approximately in 30 min. The trifluoroacetic acid was evaporated by rotary evaporation under reduced pressure and treated with diethyl ether (3x2 mL) and dried under vacuum to give, 6 as a gummy solid. LC-MS [M+H] = 748.91 [0150] Synthesis of Compound 8: To a solution of 6 (4 mg, 5.35 μmol) in dimethylsulfoxide (200 μL) were added Sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzene- sulfonate, CBTF (7, procured from Sigma-Aldrich, USA, 2.36 mg, 5.62 μmol, 1.05 equiv.), followed by triethylamine (7.29 μL, 53.5 μmol, 10.0 equiv.) at room temperature under argon and stirred for 10-15 min. Progress of the reaction was monitored by TLC and LC/MS. After 70404-02 completion of the reaction, crude mass purified by prep-HPLC on a C18 column (5-95% B over 60 minutes, flow 7 mL/min; B: acetonitrile; A: 20 mM NH₄OAc, pH 7 buffer, UV @ 280 nm) to afford Compound 8. LC-MS [M+H] = 901.94 LCMS method conditions: [0151] Column: XBridge BEH C18 Column, 130Å, 3.5 μm, 3 mm X 100 mm [0152] Mobile phase: A: 20 mM ammonium bicarbonate buffer, pH 7; B; Acetonitrile (HPLC Grade) [0153] Method run: 5-95% B, 7 min., 0.75 mL/min. Example 2 Construction and expression of the monomeric Fc fragments for site specific cysteine mediated conjugations [0154] ExpiCHO-S™ cell line obtained from ThermoScientific was cultured as prescribed in ExpiCHO™ Expression Medium on an orbital shaker in 37 °C, 8% CO₂ in scales of 25 mL and 100 mL. For transfection with the required Fc pDNA, cells were seeded at the prescribed density and viability while counting using a trypan blue based staining method. After initial optimization, “High Titer Protocol” was followed for transfection with addition of ExpiFectamine™ CHO Enhancer feed on Day 1 post-transfection accompanied with a shifting the cells to 32°C, 5% CO₂ on that day. Day of harvesting the protein from culture was optimized to be day 10 post- transfection, based on maximum yield of protein obtained. On day 10, cultures were transferred to centrifuge tubes and centrifuged at 4000 – 5000 g for 30 minutes in a refrigerated centrifuge. Before being used for protein purification, the culture supernatant was clarified by filtering through a 0.22-μm filter. [0155] GE AKTA, placed in a 4 °C environment, was employed for purification of the Fc proteins from the clarified supernatants using HiTrap MabSelect PrismA protein A column (Cytiva). The pH of the clarified culture supernatant was adjusted to 7.2-7.4 before being passed through a washed and pre-equilibriated column. After two successive rounds of binding using the collected flow-through supernatant, unbound protein was washed using >10 CV (Column Volume) of wash buffer (20 mM NaPi pH 7.4, 0.25 M NaCl). [0156] 0.1 M sodium citrate, pH 3.0 was used for elution of purified protein in fractions of 1 mL each where the pH of each fraction was neutralized immediately after collection using 1 M Tris- HCl pH 9.0. Observing absorbance pattern on the FPLC instrument collection was stopped at 20- 70404-02 25 fractions followed by washing, CIP and equilibration for next use/storage in ethanol. Protein content in the fractions was determined using spectrophotometer (A280) and the main fractions were run on SDS-PAGE for confirmation of purity and molecular mass (26 kDa). Peak fractions were pooled and dialyzed against PBS to obtain final stocks in PBS that were stored in -20 °C by adding 20% glycerol as cryopreservant. MALDI-MS was performed to further characterize the proteins. [0157] Following expression, the molecular weight of mFc-WT-G1-C152S (Seq. ID NO:1) and mFc-WT-G1-C210S (SEQ ID NO:2) were confirmed by MALDI-MS analysis. See FIG. 3. mFc- WT-G1-C152S (or SEQ. ID No.1) had a molecular weight of 28177 Da and mFc-WT-G1-C210S (SEQ ID NO:2) had a molecular wight of 27266 Da. Example 3 [0158] Synthetic scheme for protein-drug conjugation (cysteine specific drug conjugation), Fol- mFc-WT-G1-C152S (or Fol- Seq. ID NO:1) and Fol- mFc-WT-G1-C210S (Fol- SEQ ID NO:2).

Cysteine specific drug conjugation Cysteine based site specific conjugation for Fol-mFc-WT-G1-C152S (or Seq. ID NO:1) [0159] To the purified mFc protein (mFc-WT-G1-C152S or Seq. ID NO:1) (125 μg) in PBS solution (150 μL), pH 7.2 was added 1% hydrogen peroxide solution in methanol (5 μL), followed by compound 8 (2.0 equiv.) in dimethyl sulfoxide (15 μL). Reaction kept under rotation a speed at 25 RPM at 4⁰C for 2 h and room temperature for 2 h and again 4⁰C for 16 h and continued until completion of reaction. The progress of the reaction was monitored by Elman’s test (for free cysteine) and MALDI mass analysis. After completion of reaction, the conjugated crude product was purified using molecular weight cut off (MWCO; 10kDa, Vivaspin 500, Catalog # GE28- 70404-02 9322-25) filters and centrifugation at 4⁰C, 15000/RPM, 10 min to remove all unreacted linker as well as low molecular weight impurities. The process is repeated 3-5 times (at 5 mg/mL concentration, yield: 60-70%). The purity and molecular weight of Fol-mFc-WT-G1-C152S (or Fol- Seq. ID NO:1) conjugate was confirmed by Western blot analysis, and Elman’s test (no free cysteine). The molecular weight of the product was found to be ~29 kDa, whereas molecular weight for mFc-WT-G1-C152S (or Seq. ID NO:1) alone was 28 kDa. Western blot analysis using anti—folate antibody confirmed the presence of folate moiety in the mFc protein. [0160] Followed same experimental protocol as above for other mFc-WT-G1-C210S (or Seq. ID NO:2) and confirmed the product Fol-mFc-WT-G1-C210S (or Fol- SEQ ID NO:2) conjugate by Elman’s test and Western blot analysis. Example 4 Characterization of the Folate conjugates of Fc-WT-G1-C152S (or Fol- Seq. ID NO:1) and Fc-WT- G1-C210S (or Fol- SEQ ID NO:2) [0161] To characterize the Fol-mFc conjugates, Ellman’s test was carried out, which is commonly used for detection of free thiol groups. See, e.g., Bulaj et al. (1998) Biochemistry 37, 8965-8972. As the conjugation reaction was carried out under oxidizing conditions, there should be no active thiols groups remaining in the reaction mixture if the reaction reached completion. The determination of thiol concentration was carried out as follows: Briefly, 5,5'-Dithio-bis(2- nitrobenzoic acid) or DNTB (commercially available), also called Ellman’s reagent was used to generate a calibration curve using known concentrations of acetyl cysteine. Next, 10 PL of mFc wt and mutant proteins of known concentration were incubated for 5 min with 90 PL of DNTB working solution (containing Tris base). After this, absorbance at 412 nm was read (plate reader) and % conjugation was calculated based on the consumption of free thiol groups which would be inversely related to the difference in absorbance of the unconjugated vs. conjugated protein solutions of identical concentration. As depicted in FIG. 4, in presence of the folate linker under oxidizing conditions, the reactions reach completion leaving no free thiols available to react with Ellman’s reagent. [0162] Fol-mFc conjugation was confirmed as follows. Equal amounts of pure mFc proteins and the respective conjugates were run on SDS-PAGE gel (15% acrylamide) along with prestained protein molecular weight marker (Precision Plus Protein Dual Color Standards, BioRad catalog no.1610374). BSA-folate (Molecular Depot, Catalog no. P2010026) was run as a positive control 70404-02 in one well of the gel. After the gel was run, the proteins were transferred to a PVDF membrane using wet transfer in cold condition. The resulting blot was stained with Ponceau to obtain the confirmation of equal protein loading. Ponceau stain was then washed off using TBST and blot was blocked for 1 h at room temperature using 5% BSA in TBST. See FIG. 5A. Anti-Folic acid antibody, Mouse monoclonal (Catalog number SAB4200793) from Sigma was used for probing the blot (4 °C, overnight) followed by NIR dye labeled anti-mouse secondary antibody (30 min, rt). NIR based image scanner (700 nm) was used to obtain the image depicting presence/absence of folate on the blot (shown in green). The prestained ladder was visible in the red channel (800 nm) of the scanner and combined image was recorded. See FIG. 5B. Example 5 Efficacy in vivo studies of Fol-mFc-WT-G1-C152S (Fol- Seq. ID NO:1) and Fol-mFc-WT-G1- C210S (Fol- SEQ ID NO:2) in murine lung cancer model [0163] 6-8-week-old female Balb/c mice (n = 5 per cohort) were implanted with 2-3 x 10⁶ cells of M109 murine lung cancer cell line subcutaneously (day 0). At day 11 post implantation, treatment was started with the following test articles: (a) 100 μL/mouse PBS only; (b) 50 μg G1- Fol-mFc(WT)-C152S (Fol- Seq. ID NO:1) ; (c) 50 μg G1- Fol-mFc(WT)-C210S (Fol- SEQ ID NO:2). Groups (b) and (c) received the drugs on alternate days via intraperitoneal route. Tumor size was monitored using digital vernier calipers and volume calculated using the approximation of tumor as an oblate spheroid. Example 6 Efficacy in vivo studies of Fol-mFc-WT-G1-C152S ( Fol- Seq. ID NO:1) and Fol-mFc-WT-G1- C210S (Fol- SEQ ID NO:2) in murine colorectal cancer model [0164] 6-8-week-old female Balb/c mice (n = 5 per cohort) were implanted with 2-3 x 10⁶ cells of CT-26 murine colorectal cancer cell line subcutaneously (day 0). At day 11 post implantation, treatment was started with the following : (a) 100 μL/mouse PBS only; (b) 50 μg G1- Fol- mFc(WT)-C152S ( Fol- Seq. ID NO:1); (c) 50 μg G1- Fol-mFc(WT)-C210S (Fol- SEQ ID NO:2); Groups (b) and (c) received the drugs on alternate days via intraperitoneal route. Tumor size was monitored using digital vernier calipers and volume calculated using the approximation of tumor as an oblate spheroid.

Claims

70404-02 CLAIMS 1. A monomeric, fragment crystallizable domain (mFc) fragment of human immunoglobulin G1 (IgG1) which: (iv) has a molecular weight of less than 40 kD, (v) comprises a C_H2 domain and a C_H3 domain, and (vi) binds WR^WKH^)F5Q^DQG^DW^OHDVW^RQH^)FȖ^UHFHSWRU 2. The mFc fragment of any one of claim ^^ZKHUHLQ^WKH^DW^OHDVW^RQH^)FȖ^UHFHSWRU^LV^ )FȖ5,^^)FȖ5,,D^^)FȖ5,,E^^DQG^RU^)FȖ5,,,^ 3. The mFc fragment of claim 1, comprising an amino acid sequence of SEQ ID NO:1. 4. The mFc fragment of claim 1, comprising an amino acid sequence of SEQ ID NO:2. 5. The mFc fragment of claim 1, comprising an amino acid sequence of SEQ ID NO:3. 6. The mFc fragment of claim 1, comprising an amino acid sequence of SEQ ID NO:4. 7. The mFc fragment of claim 1, comprising an amino acid sequence of SEQ ID NO:5. 8. The mFc fragment of claim 1, comprising an amino acid sequence of SEQ ID NO:6. 9. The mFc fragment of claim 1, comprising at least one mutation that increases DIILQLW\^WR^DW^OHDVW^RQH^)FȖ^UHFHSWRU. 10. The mFc fragment of claim 9, wherein the at least one )FȖ^UHFHSWRU is )FȖ5,,,^^ 70404-02 11. The mFc fragment of claim 9, wherein one mutation is at least one of S239D, A330L, and I332E. 12. The mFc fragment of claim 1, comprising at least one mutation that increases affinity to the FcRn receptor comparative to wild-type. 13. The mFc fragment of claims 9-12, wherein the at least one mutation that increases affinity to the FcRn receptor is selected from the group consisting of H433K/N434F/Y436H and H433K/N434F. 14. The mFc fragment of claim 1, comprising at least one mutation that increases in vivo stability of the fragment. 15. The mFc fragment of claim 14, wherein the at least one mutation is at least one of L242C, K334C, P343C, and A431C. 16. The mFc fragment of claim 1, which comprises at least one mutation that decreases or eliminates aggregation of the fragment. 17. The mFc fragment of claim 16, wherein the at least one mutation is at least one of L351S, T366R, L368H, and P395K. 18. The mFc fragment of claim 1, which comprises at least one site for conjugation. 19. The mFc fragment of claim 18, wherein the at least one site for conjugation is at least one of a cysteine, a serine, a lysine, a tyrosine, and an unnatural amino acid. 20. A conjugate of Formula I: mFc-L-G (Formula I) 70404-02 or a pharmaceutically acceptable salt thereof, wherein: mFc is a monomeric Fc fragment of human immunoglobulin G1 (IgG1 or hIgG1; G is a folate moiety; and L comprises a linker that is covalently bound to G and mFc. 21. The conjugate of claim 20, where the folate moiety comprises a methotrexate residue or:

_. 22. The conjugate of claim 20 or claim 21 wherein mFc comprises one or more of a cysteine residue, a lysine residue, or a tyrosine residue. 23. The conjugate of claim 22, wherein one or more one or more of a cysteine residue, a lysine residue, or a tyrosine residue is covalently bound to L. 24. The conjugate of any one of claims 20-23, wherein L comprises one or more unnatural amino acids. 25. The conjugate of claim 24, wherein one unnatural amino acid is covalently bound to mFc. 70404-02 26. The conjugate of any one of claims 20-25, wherein L further comprises a spacer. 27. The conjugate of claim 26, wherein the spacer is a hydrophilic spacer. 28. The conjugate of any one of claims 20-27, wherein L comprises one or more of an amino acid or a polyethylene glycol (PEG) moiety. 29. The conjugate of any one of claims 20-28, wherein L comprises an oligomer of peptidoglycans residues or glycan residues or a combination thereof. 30. The conjugate of any one of claims 20-29, wherein the mFc is an mFc of any one of claims 1-19. 31. A composition comprising a conjugate of any one of claims 20-30 and a pharmaceutically acceptable carrier. 32. A method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a conjugate of any one of claims 20-30 or a composition comprising the conjugate and a pharmaceutically acceptable carrier.