WO2019152827A1

WO2019152827A1 - Novel antibody-drug conjugates (adc), methods of making, and methods of use thereof

Info

Publication number: WO2019152827A1
Application number: PCT/US2019/016325
Authority: WO
Inventors: Yuefeng Lu
Original assignee: AskGene Pharma, Inc.
Priority date: 2018-02-02
Filing date: 2019-02-01
Publication date: 2019-08-08

Abstract

Described herein are novel antibody-drug conjugates (ADC), including antibody-albumin-drug conjugates (AADC). Further included in the present application are methods of using the novel antibody-drug conjugates and antibody-albumin-drug conjugates. The antibody fusion molecule can include an antigen binding moiety having an antibody heavy chain or fragment, and an antibody light chain or fragment. The antigen binding moiety is fused to a drug conjugation moiety, which includes a heterologous peptide having a cysteine and, optionally, an albumin or an albumin fragment. A payload, such as a drug molecule, can be conjugated to the cysteine residue on the heterologous peptide.

Description

NOVEL ANTIBODY-DRUG CONJUGATES (ADC), METHODS OF MAKING, AND

METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority benefit to U.S. Provisional Patent Application No. 62/625,709, entitled“NOVEL ANTIBODY-DRUG CONJUGATES (ADC) AND

METHODS OF USE THEREOF,” filed February 2, 2018, the entire contents of which are incorporated herein by reference.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

[0002] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 737252000l40SEQLIST.TXT, date recorded: February 1, 2019, size: 80 KB).

FIELD OF THE INVENTION

[0003] The present application relates to novel antibody-drug conjugates (ADC), including antibody-albumin-drug conjugates (AADC). Further included in the present application are methods of using the novel antibody-drug conjugates and antibody-albumin-drug conjugates

BACKGROUND

[0004] Antibody-drug conjugates (ADC) allow for targeted delivery of a therapeutic agent, particularly to cancer cells, which can reduce side-effects commonly associated with chemotherapy. The antibody portion of the ADC can also allow for biological modulation that further enhances the efficacy of the conjugated drug. The drug molecule is generally conjugated through a cleavable or non-cleavable linker that attaches to a cysteine or lysine on the antibody molecule. The drug to antibody ration (DAR) of the ADC is limited, in part, by the number of moieties within the antibody that the drug can be linked to and the

hydrophobicity of the drug. Most ADCs having an average DAR between 0 and 8. Higher DAR of the complex allows for more potent ADC for use as therapeutics.

[0005] Additionally, most ADC molecules in clinical trials have half-lives of about 7 days or shorter (Deslandes, Comparative clinical pharmacokinetics of antibody-drug conjugates in first-in-human Phase 1 studies , MAbs, vol. 6, no. 4, pp. 859-870 (2014)), which is significantly shorter than most non-conjugated antibodies. Hydrophobicity of the ADC molecules due to the conjugated drugs is believed to be a significant contribution to the diminished stability, particularly in vivo , Lyon et al., Reducing hydrophobicity of

homogenous antibody-drug conjugates improvise pharmacokinetics and therapeutic index , Nature Biotechnology, vol. 33, pp. 733-7355 (2015).

[0006] The disclosures of all publications, patents, and patent applications referred to herein are each hereby incorporated herein by reference in their entireties.

SUMMARY OF THE INVENTION

[0007] Described herein are antibody fusion molecules, including antibody fusion molecules conjugated to a payload molecule, such as a drug molecule (e.g., a cytotoxic drug molecule). Also described are methods of making such antibody fusion molecules and methods of treating a disease in a subject comprising administering an effective amount of the antibody fusion molecule to the subject.

[0008] The antibody fusion molecule comprises an antigen-binding moiety comprising an antibody heavy chain and an antibody light chain, or a fragment thereof, and a drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof wherein the drug conjugation moiety comprises a heterologous peptide (Pl) less than about 50 amino acids in length, and wherein Pl comprises a cysteine residue. In some embodiments, the heterologous peptide (Pl) is about 5 to about 20 amino acids in length.

[0009] In some embodiments, the antigen-binding moiety binds to one or more antigen on a surface of a cancer cell. In some embodiments, the cancer cell has upregulated

macropinocytosis. In some embodiments, the cancer cell comprises one or more mutant RAS family genes.

[0010] In some embodiments, the heterologous peptide (Pl) is unstructured or is helical.

[0011] In some embodiments, the drug conjugation moiety is fused to the N-terminus or the C-terminus of the antibody heavy chain, or the fragment thereof. In some embodiments, the drug conjugation moiety is fused to the N-terminus or the C-terminus of the antibody light chain, or the fragment thereof.

[0012] In some embodiments, the antigen-binding moiety comprises a full length antibody. In some embodiments, the antigen-binding moiety comprises an antibody fragment.

[0013] In some embodiments, the drug conjugation moiety comprises an albumin, or an albumin fragment, fused to Pl. In some embodiments, the cysteine residue of Pl is about 30 amino acid residues or less away from a first residue a last residue of the albumin or the albumin fragment. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or antibody light chain or fragment thereof via the albumin or the albumin fragment. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or antibody light chain or fragment thereof via Pl. In some

embodiments, the albumin or the albumin fragment has a point mutation at Cys34. In some embodiments, the point mutation is Cys34Ser. In some embodiments, the drug conjugation moiety comprises a full length albumin. In some embodiments, the drug conjugation moiety comprises the albumin fragment. In some embodiments, the albumin has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0014] In some embodiments, the drug conjugation moiety comprises a second heterologous peptide (P2), which is fused to the albumin or albumin fragment. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or antibody light chain or fragment thereof via the second heterologous peptide (P2). In some embodiments, the second heterologous peptide (P2) comprises a cysteine residue. In some embodiments, the second heterologous peptide (P2) does not comprise a cysteine residue. In some embodiments, the second heterologous peptide (P2) is helical or unstructured. In some embodiments, the second heterologous peptide (P2) is about 5 to about 20 amino acid residues in length. In some embodiments, the second heterologous peptide (P2) comprises one or more glycine residues or one or more serine residues. In some embodiments, the second heterologous peptide (P2) comprises an amino acid sequence of (GGGGS)_q (SEQ ID NO: 34), wherein q is 1 or more.

In some embodiments, the second heterologous peptide (P2) comprises an amino acid sequence of (GGGGS)_q (SEQ ID NO: 34), wherein q is 1, 2, 3, or 4.

[0015] In some embodiments, the drug conjugation moiety comprises two or more albumins or albumin fragments. In some embodiments, the drug conjugation moiety comprises three or more heterologous peptides. In some embodiments, each of the heterologous peptides comprises a cysteine residue.

[0016] In some embodiments, the antibody fusion molecules comprising a polypeptide configured, from N-terminus to C-terminus, according to:

HL-P1;

HL-P1-A

HL-P1-A-P3;

HL-P1-A-P3-A;

HL-P 1 - A-P3 - A-P4 ; HL-P 1 - A-P3 - A-P4- A;

HL-P 1 - A-P3 - A-P4- A-P5 ;

HL-P2-A-P1;

HL-P2-A-P1-A;

HL-P2- A-P 1 - A-P3 ;

HL-P2- A-P 1 - A-P3 - A; or

HL-P2- A-P 1 - A-P3 - A-P4 ;

wherein HL is the antibody heavy chain or the antibody light chain, or the fragment thereof; wherein Pl, P3, P4, and P5 are heterologous peptides, wherein at least one of Pl, P3, P4, and P5 comprises a cysteine residue; wherein P2 is a heterologous peptide; and wherein A is an albumin or a fragment thereof.

[0017] In some embodiments, each of Pl, P3, P4, and P5 is about 5 to about 20 amino acid residues in length, and Pl, P3, P4, and P5 are of the same length or of different lengths. In some embodiments, at least one of Pl, P3, P4, and P5 comprises at least one charged amino acid. In some embodiments, at least one of Pl, P3, P4, and P5 comprises one or more positively charged amino acid residues. In some embodiments, the one or more positively charged amino acid residues are selected from the group consisting of lysine, histidine, arginine, and combinations thereof. In some embodiments, at least one of Pl, P3, P4, and P5 comprises one or more negatively charged amino acid residues. In some embodiments, the one or more negatively charged amino acid residues are selected from the group consisting of aspartic acid, glutamic acid, and combinations thereof.

[0018] In some embodiments, at least one of Pl, P3, P4, and P5 comprises a sequence according to:

(GGGGS)_nCXaa₁Xaa₂Xaa3Xaa₄Xaa₅Xaa6(GGGGS)_m (SEQ ID NO: 35);

(GGGGS)_nXaa₁CXaa₂Xaa3Xaa₄Xaa₅Xaa₆(GGGGS)_m (SEQ ID NO: 36);

(GGGGS)_nXaa₁Xaa₂CXaa₃Xaa₄Xaa₅Xaa₆(GGGGS)_m (SEQ ID NO: 37);

(GGGGS)„XaaiXaa₂Xaa₃CXaa₄Xaa5Xaa₆(GGGGS)_m (SEQ ID NO: 38);

(GGGGS)„XaaiXaa₂Xaa3Xaa4CXaa₅Xaa6(GGGGS)_m (SEQ ID NO: 39);

(GGGGS)_nXaa₁Xaa₂Xaa₃Xaa₄Xaa₅CXaa₆(GGGGS)_m (SEQ ID NO: 40); or

(GGGGS)_nXaa₁Xaa₂Xaa₃Xaa₄Xaa₅Xaa₆C(GGGGS)_m (SEQ ID NO: 41);

wherein Xaai, Xaa₂, Xaa3, Xaa₄, Xaas, and Xaa₆ are each either (1) a single amino acid, or none; wherein Xaai, Xaa₂, Xaa3, Xaa4, Xaas, and Xaa₆ has a net positive charge or a net negative charge; wherein n = 0, 1, 2, 3, or 4; wherein m = 0, 1, 2, 3, or 4; and wherein the sequence of Pl, P3, P4, and P5 may be the same or different. In some embodiments Xaai, Xaa₂, Xaa₃, Xaa₄, Xaa₃, and Xaa₆ comprise at least one positively charged amino acid residue and no negatively charged amino acid residues; or at least one negatively charged amino acid residue and no positively charged amino acid residues. In some embodiments, Xaai, Xaa₂, Xaa₃, Xaa4, Xaa₃, and Xaa₆ are each either (1) selected from the group consisting of histidine, lysine, arginine, aspartic acid, glutamic acid, glycine, serine, and threonine; or (2) none.

[0019] In some embodiments, the antibody fusion molecule comprises a full length antibody heavy chain or a full length antibody light chain.

[0020] In some embodiments, the antibody fusion molecule comprises a first drug

conjugation moiety fused to the antibody light chain, and a second drug conjugation moiety fused to the antibody heavy chain.

[0021] In some embodiments, the antibody fusion molecule comprises a first drug

conjugation moiety fused to a first antibody heavy chain, and a second drug conjugation moiety fused to a second antibody heavy chain, and wherein the first drug conjugation moiety and the second drug conjugation moiety are the same or different.

[0022] In some embodiments, the antibody fusion molecule comprises a first drug

conjugation moiety fused to a first antibody light chain, and a second drug conjugation moiety fused to a second antibody light chain, and wherein the first drug conjugation moiety and the second drug conjugation moiety are the same or different.

[0023] In some embodiments, the antibody fusion molecule further comprises a payload molecule covalently bound to the cysteine residue in any one or more of Pl, P3, P4, and P5.

In some embodiments, the payload molecule is covalently bound to the cysteine residue in Pl, P3, P4, or P5 through a conjugation linker.

[0024] In some embodiments, the payload molecule is a drug molecule. In some

embodiments, the drug molecule is a cytotoxic drug molecule. In some embodiments, the drug molecule is an anti- viral agent or an anti-bacterial agent. In some embodiments, the payload molecule is an imaging agent.

[0025] In some embodiments, the antibody fusion molecule comprises 2-30 payload molecules.

[0026] In some embodiments, the conjugation linker binds to the cysteine in Pl, P3, P4, or P5 through a maleimide moiety. In some embodiments, the maleimide moiety is a N-alkyl maleimide moiety.

[0027] In some embodiments, the conjugation linker is bound to two or more payload molecules. [0028] In some embodiments, the drug molecule is a microtubule disrupting agent, a DNA modifying agents, an RNA polymerase inhibitors, or a topoisomerase I inhibitor.

[0029] In some embodiments, the conjugation linker is not cleavable. In some embodiments, the conjugation linker is cleavable.

[0030] In some embodiments, the conjugation linker comprises amino acids valine-alanine or valine-citrulline. In some embodiments, the conjugation linker and drug has a structure of:

wherein z is 4-24; and wherein the maleimide group is covalently attached to the cysteine in any one of Pl, P3, P4, or P5 through the maleimide group.

[0031] In some embodiments, binding of the conjugation linker to the cysteine residue in Pl, P3, P4, or P5 results in a ring-opened counterpart of a hydrolyzed succinimide thioether formed by maleimide-thiol conjugation.

[0032] Also provided herein is a pharmaceutical composition comprising any of the antibody fusion molecules described above, and a pharmaceutically acceptable excipient. Also provided herein is a method of treating a cancer in a subject, comprising administering to the subject with cancer an effective amount of the pharmaceutical composition, wherein the payload molecule is a cytotoxic drug. In some embodiments, the cancer expresses an antigen targeted by the antibody or fragment thereof, and comprises a mutant RAS gene.

[0033] Also provided herein is a method of making an antibody fusion molecule, comprising conjugating a payload molecule to the antibody fusion molecule as described above. In some embodiments, the payload molecule is conjugated to cysteine residue in Pl, P3, P4, or P5 through a conjugation linker. In some embodiments, the conjugation linker is conjugated to the payload molecule prior to conjugating the conjugation linker to the cysteine residue in Pl, P3, P4, or P5. In some embodiments, the conjugation linker is conjugated to the antibody fusion molecule prior to conjugating the conjugation linker to the drug molecule. In some embodiments, two or more drug molecules are conjugated to the conjugation linker. In some embodiments, the conjugation linker comprises a maleimide group. In some embodiments, the method comprises mixing the antibody fusion molecule with a reducing agent prior to conjugating the payload molecule to the fusion molecule. In some embodiments, the method comprises removing the reducing agent prior to conjugating the payload molecule to the fusion molecule. In some embodiments, the method comprises mixing the antibody fusion molecule with an oxidizing agent after mixing the antibody fusion molecule with the reducing agent, and prior to conjugating the payload molecule to the fusion molecule. In some embodiments, the method comprises removing the oxidizing agent prior to conjugating the payload molecule to the fusion molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 illustrates the chemical structure of an exemplary drug with conjugating linker, namely MC-vc-PAB-MMAE, which can be conjugated to an antibody fusion molecule.

[0035] FIG. 2 shows a hydrophobic interaction chromatography, high performance liquid chromatography (HIC-HPLC) chromatogram for an expressed and purified antibody- albumin fusion protein (top panel) and the purified antibody- albumin fusion protein conjugated to MC -vc-PAB-MMAE under storage buffer conditions (2 mM Histidine, 4% mannitol, 2% sucrose, pH 6.7) (bottom panel).

[0036] FIG. 3 shows a HIC-HPLC chromatogram for an expressed and purified antibody- albumin fusion protein (top panel) and the purified antibody- albumin fusion protein conjugated to MC-vc-PAB-MMAE under conjugation buffer conditions (50 mM sodium phosphate, 4 mM EDTA, pH 7.0) (bottom panel).

[0037] FIG. 4 shows a HIC-HPLC chromatogram for an expressed and purified antibody- albumin fusion protein (top panel) and the purified antibody- albumin fusion protein conjugated to MC-vc-PAB-MMAE under conjugation buffer conditions (50 mM sodium phosphate, 4 mM EDTA, pH 7.0 after the antibody- albumin fusion protein was reduced with tris(2-carboxyethyl)phosphine (TCEP) at a 2.5x molar ratio (TCEP to protein) (bottom panel). Peaks were observed for antibody- albumin fusion protein conjugated to 0, 1, 2, 3, 4, and 5 molecules of MC-vc-PAB-MMAE, with an average DAR of 2.3.

[0038] FIG. 5 shows a HIC-HPLC chromatogram for an expressed and purified antibody- albumin fusion protein (top panel) and the purified antibody- albumin fusion protein conjugated to MC-vc-PAB-MMAE under conjugation buffer conditions (50 mM sodium phosphate, 4 mM EDTA, pH 7.0) after the antibody- albumin fusion protein was reduced with tris(2-carboxyethyl)phosphine (TCEP) at a 20x molar ratio (TCEP to protein) (bottom panel). Peaks were observed for antibody- albumin fusion protein conjugated to 0, 1, 2, 3, 4, and 5 molecules of MC-vc-PAB-MMAE, with an average DAR of 3.3. [0039] FIG. 6 shows a HIC-HPLC chromatogram for an expressed and purified antibody- albumin fusion protein (top panel) and the purified antibody- albumin fusion protein conjugated to MC-vc-PAB-MMAE under conjugation buffer conditions (50 mM sodium phosphate, 4 mM EDTA, pH 7.0) after the antibody- albumin fusion protein was reduced with tris(2-carboxyethyl)phosphine (TCEP) at a 20x molar ratio (TCEP to protein), and then oxidized using ascorbic acid (bottom panel). Peaks were observed for antibody- albumin fusion protein conjugated to 0, 1, 2, 3, 4, and 5 molecules of MC-vc-PAB-MMAE, with an average DAR of 4.5.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention relates to compositions for targeted delivery of drug molecules. An antibody fusion molecule includes an antigen-binding moiety, which may include, for example, an antibody heavy chain and an antibody light chain, which binds to an antigen on a cell surface. Fused to the antigen-binding moiety or a polypeptide component of the antigen binding moiety, there is a drug conjugation moiety. The drug conjugation moiety includes a heterologous peptide that includes a cysteine residue, which can be conjugated to the drug. The antigen-binding moiety targets the drug to the cell by binding the antigen, which internalizes the antibody fusion molecule. Through this mechanism, the antibody fusion molecule can target the drug to the cell.

[0041] Optionally, the heterologous peptide of the drug conjugation moiety is fused to an albumin or an albumin fragment. The albumin or albumin fragment may be fused to the C-terminal end or the N-terminal end of the heterologous peptide. The albumin or the albumin fragment can bind the drug linked to the heterologous polypeptide, which enhances solubility of the drug. Accordingly, the antibody fusion molecule described herein is particularly beneficial for targeted delivery of hydrophobic payload molecules, such as drug molecules.

[0042] Some embodiments of the present invention include a drug conjugation moiety with a plurality of fused heterologous polypeptides, which allow the drug to antibody ratio to be increased by extending the number of heterologous polypeptides. Additionally, the drug conjugation moiety can optionally include a plurality of albumins or albumin fragments, which can enhance the solubility of the drugs molecules attached to the heterologous polypeptides. For example, an albumin or an albumin fragment can be included adjacent to each heterologous polypeptide to support the solubility of the drug molecule attached to that heterologous polypeptide. Although antibody-drug conjugates (ADCs) are known, the drug molecules are generally conjugated directly to the antibody molecule. Such configuration limits the number of drug molecules that can be bound to the complex (that is, the drug to antibody ratio) due to the limited number of drug attachment sites on the antibody molecule. Additionally, attaching hydrophobic drugs to an antibody molecule can result in decreased solubility and stability of the complex.

[0043] Described herein is an antibody fusion molecule, comprising an antigen-binding moiety, and a drug conjugation moiety fused to the antigen-binding domain, wherein the drug conjugation moiety comprises a heterologous peptide (Pl), and wherein Pl comprises a cysteine residue. In some embodiments, Pl is less than about 50 amino acids in length. In some embodiments, the antigen-binding moiety includes an antibody heavy chain (or a fragment thereof) and an antibody light chain (or a fragment thereof), and the drug conjugation moiety is fused to the antibody heavy chain (or fragment thereof) or the antibody light chain (or fragment thereof). Also described are methods of making the antibody fusion molecule. The drug conjugation moiety can be fused to the N-terminus or the C-terminus of the antibody heavy chain or the antibody light chain. Optionally, the drug conjugation moiety includes an albumin or an albumin fragment, which is fused to Pl. In some embodiments, the drug conjugation moiety includes more than one heterologous peptides, which may be the same or different, or more than one albumins or albumin fragments.

[0044] The heterologous peptide includes a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or shorter. In some embodiments, a payload molecule, such as a cytotoxic drug molecule or any other drug molecule, is conjugated to the heterologous peptide, for example through the cysteine residue. In some embodiments, the antibody fusion molecule comprises 2 or more payload molecules. The payload molecule can be conjugated to the heterologous peptide through a cleavable or a non- cleavable linker.

[0045] Also described herein are methods of treating cancer in a subject by administering an effective amount of the antibody fusion molecule to the subject. The antibody fusion molecule can target an antigen on the surface of the cancer cells, thereby delivering the drug to the cancer. In certain embodiments, the cancer cell has upregulated micropinocytosis compared to a non-cancerous cell. For example, in some embodiments, the cancer has a RAS mutation, which has been linked to upregulated micropinocytosis.

[0046] It is understood that aspects and variations of the invention described herein include “consisting” and/or“consisting essentially of’ aspects and variations. Definitions

[0047] As used herein and in the appended claims, the singular forms“a,”“or,” and“the” include plural referents unless the context clearly dictates otherwise.

[0048] Reference to“about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of“X.” Additionally, use of“about” preceding any series of numbers includes“about” each of the recited numbers in that series. For example, description referring to“about X, Y, or Z” is intended to describe“about X, about Y, or about Z.”

[0049] The term“antigen-binding moiety” refers to a polypeptide or a set of interacting polypeptides that specifically bind to an antigen, and includes, but is not limited to, an antibody or antibody fragment, such as a monoclonal antibody, polyclonal, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')₂, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope-binding fragment thereof, bifunctional hybrid antibodies, a single chain antibody, and a Fc-containing polypeptide, such as an immunoadhesion. In some embodiments, the antibody may be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD). In some embodiments, the antibody may be of any light chain isotype (e.g., kappa or gamma). The antibody may be non-human (e.g., from mouse, goat, or any other animal), fully human, humanized, or chimeric. In some embodiments, the antibody is a derivatized antibody.

[0050] The term“effective amount” used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition, or disease, such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In reference to a disease such as a cancer, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in the cancer. In some embodiments, the effective amount is an amount sufficient to delay development of a cancer. In some embodiments, the effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. In the case of a cancer, the effective amount of the drug or composition may: (i) reduce the number of epithelioid cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop the cancer cells infiltration into peripheral organs; (iv) inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0051] The term“fused” or“fusion” in reference to two or more polypeptide sequences (such as an antibody heavy chain, antibody light chain, an antibody heavy chain fragment, an antibody light chain fragment, a drug conjugation moiety, a heterologous peptide, an albumin, or an albumin fragment) refers to joining of the polypeptide sequences through a backbone peptide bond.

[0052] The term“pharmaceutically acceptable” when used to refer to a compound or composition means that the compound or composition is suitable for administration to a subject, including a human subject, to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0053] The term“subject” refers to a mammal and includes, but is not limited to, human, bovine, horse, feline, canine, rodent, or primate.

[0054] As used herein,“treatment” or“treating” is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of a disease, stabilizing a disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of a disease, preventing or delaying the recurrence of a disease, delaying or slowing the progression of a disease, ameliorating a disease state, providing remission (partial or total) of a disease, decreasing the dose of one or more other medications required to treat a disease, delaying the progression of a disease, increasing the quality of life, and/or prolonging survival. Also encompassed by“treatment” is a reduction of a pathological consequence of a disease (such as cancer). The methods of the invention contemplate any one or more of these aspects of treatment.

[0055] It is to be understood that one, some or all of the properties of the various

embodiments described herein may be combined to form other embodiments of the present invention.

[0056] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Antibody Fusion Molecule

[0057] The antibody fusion molecule includes an antigen-binding moiety fused to a drug conjugation moiety. In some embodiments, the antigen-binding moiety includes an antibody heavy chain or a fragment thereof, and an antibody light chain or a fragment thereof. The drug conjugation moiety cam be fused to the antibody heavy chain or fragment thereof or the antibody light chain or fragment thereof. The drug conjugation moiety includes a

heterologous peptide (Pl), which includes a cysteine residue.

[0058] The antigen-binding moiety can be an antibody or a fragment thereof. For example, in some embodiments, the antigen binding moiety is a full length antibody, a Fab fragment, a Fab' fragment, a F(ab')₂ fragment, a Fv fragment, a disulfide linked Fv fragment, or a single chain variable fragment (scFv) fragment. In some embodiments, the antigen-binding moiety includes a full-length antibody heavy chain or a full-length antibody light chain. In some embodiments, the antigen-binding moiety includes an antibody heavy chain fragment or an antibody light chain fragment.

[0059] The antigen-binding moiety can bind an antigen on the surface of a cell, such as a cancer cell. In some embodiments, the antigen-binding moiety is a bispecific antigen-binging moiety, can bind to two different antigens or two different epitopes on the same antigen. In some embodiments, the antigen-binding moiety binds to Guanyl cyclase C (GCC), carbohydrate antigen 19-9 (CA19-9), glycoprotein A33 (gpA33), mucin 1 (MUC1), carcinoembryonic antigen (CEA), insulin-like growth factor 1 receptor (IGF1-R), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGFR1) 1, vascular endothelial growth factor receptor 2 (VEGFR2), Nectin-4, Liv-l, glycoprotein NMB (GPNMB), pro state- specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), Thomsen-Friedenrech antigen (TF), sodium-dependent phosphate transport protein 2B (NaPi2b), six transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor alpha (FR-a), SLIT and NTRK- like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide

pyrophosphatase/phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD123, CD138, CD352, programmed death ligand 1 (PD-L1), Claudin 18.2, Claudin 6, or deacetylated b-1-6 polymer of N-acetyl glucosamine (dPNAG). In some embodiments, the antigen-binding moiety binds to an epidermal growth factor (EGF)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to a Delta/S errate/Lag2 (DSL)-like domain of DLL3. In some embodiments, the antigen-binding moiety binds to an epitope located after the 374th amino acid of GPC3. In some

embodiments, the antigen-binding moiety binds to a heparin sulfate glycan of GPC3. In some embodiments, the antigen-binding moiety binds to Claudin 18.2 and does not bind to Claudin 18.1. In some embodiments, the antigen-binding moiety binds to Claudin 18.1 with at least 10 times weaker binding affinity than to Claudin 18.2

[0060] Exemplary antigen-binding moieties include trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, and fragments thereof. In some embodiments, the antigen-binding moiety that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to trastuzumab, rituximab, brentuximab, cetuximab, or panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen-binding moiety has an antibody heavy chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibody heavy chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. In some embodiments, the antigen binding moiety has an antibody light chain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the antibody light chain of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33 (or a humanized version thereof), anti-EGFR antibody mAb806 (or a humanized version thereof), anti-dPNAG antibody F598, or a fragment thereof. The antigen-binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a drug molecule is conjugated to the cysteine residue.

[0061] In some embodiments, the antigen-binding moiety comprises the six complementarity determining regions (CDRs) of trastuzumab, rituximab, brentuximab, cetuximab, panitumumab, GC33, anti-EGFR antibody mAb806, or anti-dPNAG antibody F598, A number of CDR delineations are known in the art and are encompassed herein. A person of skill in the art can readily determine a CDR for a given delineation based on the sequence of the heavy or light chain variable region. The“Rabat” Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). “Chothia” CDRs refer to the location of the structural loops (Chothia & Lesk, Canonical structures for the hypervariable regions of immunoglobulins, J. Mol. Biol., vol. 196, pp. 901-917 (1987)). The“AbM” CDRs represent a compromise between the Rabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software. The“Contact” CDRs are based on an analysis of the available complex crystal structures. The residues from each of these CDRs are noted below in Table 1, in reference to common antibody numbering schemes. Unless otherwise specified herein, amino acid number of antibodies refers to the Rabat numbering scheme as described in Rabat et al., supra, including when CDR delineations are made in reference to Rabat, Chothia, AbM, or Contact schemes. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a framework region (FR) or CDR of the variable domain. For example, a heavy-chain variable domain may include a single amino acid insert (residue 52a according to Rabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Rabat) after heavy-chain FR residue 82. The Rabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a“standard” Rabat numbered sequence.

Table 1: CDR Delineations According to Various Schemes

[0062] In some embodiments, the CDRs are“extended CDRs,” and encompass a region that begins or terminates according to a different scheme. For example, an extended CDR can be as follows: F24— F36, F26— F34, or F26— F36 (VF-CDR1); F46— F52, F46— F56, or F50— F55 (VF-CDR2); F91— F97 (VF-CDR3); H47— H55, H47— H65, H50— H55, H53— H58, or H53— H65 (VH-CDR2); and/or H93— H102 (VH-CDR3). [0063] In some embodiments, the antigen-binding domain binds to HER2, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3, or a fragment thereof. In some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 2, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 3. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 2, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 3. The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule is conjugated to the cysteine residue.

[0064] In some embodiments, the antigen-binding domain binds to CD20, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5, or a fragment thereof. I n some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 4, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 5. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 4, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 5. The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue. [0065] In some embodiments, the antigen-binding domain binds to CD30, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 7, or a fragment thereof. In some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 6, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 7. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 6, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 7. . The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue.

[0066] In some embodiments, the antigen-binding domain binds to EGF, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 8, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 9, or a fragment thereof. In some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 8, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 9. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 8, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 9. The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule is conjugated to the cysteine residue. [0067] In some embodiments, the antigen-binding domain binds to EGFR, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 10 or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11 or a fragment thereof. I n some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 10, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 11. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 10, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 11. The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue.

[0068] In some embodiments, the antigen-binding domain binds to c-MET, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 12, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 13, or a fragment thereof. In some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 12, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 13. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 12, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 13. The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue. [0069] In some embodiments, the antigen-binding domain binds to GPC3, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14 or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 15 or a fragment thereof. In some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 15. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 15. The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue.

[0070] In some embodiments, the antigen-binding domain binds to Claudin 18.2, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 16, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 17, or a fragment thereof. In some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 16, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 17. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 16, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 17. The antigen-binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue.

[0071] In some embodiments, the antigen-binding domain binds to dPNAG, and comprises a light chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 18, or a fragment thereof, and a heavy chain having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 19, or a fragment thereof. In some embodiments, the antigen-binding domain comprises an antibody light chain or an antibody light chain variable region comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 18, and an antibody heavy chain or an antibody heavy chain variable region comprising CDR1, CDR2, and CDR3 of the antibody heavy chain according to SEQ ID NO: 19. In some embodiments, the antigen-binding domain comprises the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 18, and the antibody heavy chain variable region of the antibody heavy chain according to SEQ ID NO: 19. The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue.

[0072] In some embodiments, the antigen-binding domain binds to Trop-2, and comprises a light chain variable region comprising a CDR1 comprising an amino acid sequence of KASQDVSIAVA (SEQ ID NO: 20), a CDR2 comprising an amino acid sequence of SASYRYT (SEQ ID NO: 21), and a CDR3 comprising an amino acid sequence of

QQHYITPLT (SEQ ID NO: 22); and a heavy chain variable region comprising a CDR1 comprising an amino acid sequence of NYGMN (SEQ ID NO: 23), a CDR2 comprising an amino acid sequence of WINTYTGEPTYTDDFKG (SEQ ID NO: 24), and a CDR3 comprising an amino acid sequence of GGFGSSYWYFDV (SEQ ID NO: 25). The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue.

[0073] In some embodiments, the antigen-binding domain binds to mesothelin, and comprises light chain variable region comprising a CDR1 comprising an amino acid sequence of SASSSVSYMH (SEQ ID NO: 26), a CDR2 comprising an amino acid sequence of DTSKLAS (SEQ ID NO: 27), and a CDR3 comprising an amino acid sequence of

QQWSGYPLT (SEQ ID NO: 28); and a heavy chain variable region comprising a CDR1 comprising an amino acid sequence of GYTMN (SEQ ID NO: 29), a CDR2 comprising an amino acid sequence of LITP YN GAS S YN QKFRG (SEQ ID NO: 30), and a CDR3 comprising an amino acid sequence of GGYDGRGFDY (SEQ ID NO: 31). The antigen binding moiety is fused to a drug conjugation moiety comprising a heterologous peptide, wherein the heterologous peptide comprises a cysteine residue. In some embodiments, the heterologous peptide is about 50 amino acids in length or less. In some embodiments, the heterologous peptide is fused to an albumin or an albumin fragment. In some embodiments, a payload molecule, such as a drug molecule, is conjugated to the cysteine residue.

[0074] The drug conjugation moiety is a polypeptide that includes at least one heterologous peptide comprising a cysteine residue. In some embodiments, the drug conjugation moiety further includes one or more albumins or albumin fragment. The drug conjugation moiety is fused to the antibody heavy chain or the antibody light chain (or antibody heavy chain fragment or antibody light chain fragment) of the antigen fusion molecule, which may be at the N-terminus or the C-terminus of the antibody heavy chain (or fragment) or antibody light chain (or fragment).

[0075] In some embodiments, the heterologous peptide is unstructured. In some

embodiments, the heterologous peptide is helical. In some embodiments, the heterologous peptide does not have a tertiary structure in isolation. In some embodiments, the

heterologous peptide does not have a biological function. In some embodiments, the heterologous peptide is synthetically designed, and is not derived from a naturally occurring peptide. In some embodiments, the heterologous peptide is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in length. In some embodiments, the heterologous peptide comprises about 50 or fewer, about 45 or fewer, about 40 or fewer, about 35 or fewer, about 30 or fewer, about 25 or fewer, about 20 or fewer, about 15 or fewer, or about 10 or fewer amino acids in length. In some embodiments, the heterologous peptide is about 5 to about 20 amino acid residues in length, such as about 5 to about 10, about 10 to about 15, or about 15 to about 20 amino acid residues in length. If the antibody fusion molecule includes two or more heterologous peptides, the heterologous peptides can be of the same length or of different lengths.

[0076] In some embodiments, the heterologous peptide includes at least one charged amino acid, which may be a positively charged amino acid or a negatively charged amino acid (as determined at physiological pH). In some embodiments, the positively charged amino acid residue is lysine, histidine, or arginine. In some embodiments, the negatively charged amino acid residue is aspartic acid or glutamic acid. In some embodiments, the heterologous peptide has a net negative charge or a net positive charge.

[0077] The heterologous peptide can optionally include one or more (such as 1, 2, 3, or 4) GGGGS (SEQ ID NO: 42) sequences at the N-terminal end, the C-terminal end, or both the N-terminal end and the N-terminal end of the heterologous peptide. For example, in some embodiments, the heterologous peptide has the sequence:

(GGGGS)_nX_aalX_aa2X_aa3X_aa4X_aa5X_aa6X_aa7(GGGGS)_m, (SEQ ID NO: 43) wherein n is 0, 1, 2, 3, 4 or more, and wherein m is 0, 1, 2, 3, 4 or more. At least one of X^i, X_aa2. X_aa3, X_aa4, X_aa5. X_aa6, or X_aa7 is the cysteine reside included in the heterologous peptide. In some

embodiments, the X_aai, X_aa2, X_aa3, X_aa4, X_aa5, X_aa(„ and X_aa7 amino acid residues have a net positive charge or a net negative charge. In some embodiments, the X_aai, X_aa2, X_aa3, X_aa4, X_aa5, X_{aaf ,} and X_aa7 amino acid residues include at least one positively charged amino acid residue and no negatively charged amino acid residues. In some embodiments, the X^i, X_aa2, X_aa3, X_aa4, X_aa5, X_aa(„ and X_aa7 amino acid residues include at least one negatively charged amino acid residue and no positively charged amino acid residues. In some embodiments, the X_aa amino acid residues are selected from histidine, lysine, arginine, aspartic acid, glutamic acid, glycine, serine, threonine, and combinations thereof. Although this example shows seven X_aa residues, in other embodiments, the heterologous peptide can include 1,2 ,3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20 or more X_aa amino acid residues. Accordingly, in some embodiments, one or more of the X_aa amino acid residues are deleted, and in some embodiments, one or more additional X_aa amino acid residues are included between the (GGGGS)n (SEQ ID NO: 44) sequence and the (GGGGS)_m (SEQ ID NO: 45) sequence.

[0078] In some embodiments, the drug conjugation moiety includes an albumin or a fragment thereof fused to the heterologous peptide. The albumin or fragment thereof can be fused to the N-terminal end or the C-terminal end of the heterologous peptide. The drug conjugation moiety can be fused to the antibody heavy chain (or fragment) or the antibody light chain (or fragment) via the albumin or albumin fragment or via the heterologous peptide. For example, the antibody fusion molecule can include a polypeptide having the configuration HL-A-P (wherein HL indicates the antibody heavy chain (or fragment) or antibody light chain (or fragment), A indicates the albumin (or fragment), and P indicates the heterologous peptide) or the configuration HL-P-A. [0079] In some embodiments, the cysteine residue of the heterologous peptide is about 30,

25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues or less from a first residue or a last residue of the albumin or the albumin fragment.

[0080] In some embodiments, the albumin is human albumin or a variant thereof. SEQ ID NO: 1 is the sequence of human albumin. In some embodiments, the albumin or albumin fragment is about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.8% or more identical to human albumin or a fragment thereof.

[0081] In some embodiments, the drug conjugation moiety comprises a full length albumin. In some embodiments, the drug conjugation moiety comprises an albumin fragment that is about 10 or more, 20 or more, 30 or more 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, or 550 or more amino acids in length. In some embodiments, the albumin fragment is between about 10 amino acids and about 584 amino acids in length (such as between about 10 and about 20, about 20 and about 40, about 40 and about 80, about 80 and about 160, about 160 and about 250, about 250 and about 350, about 350 and about 450, or about 450 and about 550 amino acids in length). In some embodiments, the albumin fragment includes the Sudlow I domain or a fragment thereof, or the Sudlow II domain or the fragment thereof.

[0082] In some embodiments, the albumin or albumin fragment includes one or more (such as 2, 3, 4, 5 or more) point mutations, amino acid insertions, or amino acid deletions.

Exemplary albumin variants are described in U.S. Patent Publication 2011/0313133. In some embodiments, the albumin or albumin fragment includes a point mutation at one or more cysteine residues. In some embodiments, the albumin or albumin fragment includes a point mutation at one or more of Dl, A2, C75, T79, E82, E86, C91, D121, C124, D129, C168, C169, C177, S270, C316, A364, C360, C361, C369, Q397, K500, A504, E505, D549, C558, D562, C567, K573, K574, A578, A581, or L585. In some embodiments, the albumin or albumin fragment includes a point mutation, insertion, or deletion adjacent to one or more of Dl, A2, C75, T79, E82, E86, C91, D121, C124, D129, C168, C169, C177, S270, C316,

A364, C360, C361, C369, Q397, K500, A504, E505, D549, C558, D562, C567, K573, K574, A578, A581, or L585. In some embodiments, the albumin or albumin fragment includes a point mutation at Cys34. In some embodiments, the albumin or albumin fragment includes a Cys34Ser point mutation. [0083] Optionally, the drug conjugation moiety includes a second heterologous peptide in addition to one or more of the heterologous peptides described above. In some embodiments, the second heterologous peptide includes a cysteine residue. In some embodiments, the second heterologous peptide does not include a cysteine residue. The second heterologous peptide is fused to one of the other components of the drug conjugation moiety (i.e., an albumin or albumin fragment, and/or another heterologous peptide). For example, in some embodiments, the antibody fusion molecule includes a polypeptide having the configuration HL-P2-A-P1 (wherein HL indicates the antibody heavy chain (or fragment) or antibody light chain (or fragment), P2 indicates the second heterologous peptide that may or may not include a cysteine, A indicates the albumin (or fragment), and Pl indicates the heterologous peptide having a cysteine). In this configuration, the drug conjugation moiety is fused to the antibody heavy chain (or fragment) or the antibody light chain (or fragment) via the second heterologous peptide (P2), which is fused to the albumin or albumin fragment (A), which is fused to the first heterologous peptide (Pl).

[0084] In some embodiments, the second heterologous peptide (P2) is helical. In some embodiments, the second heterologous peptide (P2) is unstructured. In some embodiments, the second heterologous peptide (P2) is helical. In some embodiments, the heterologous peptide does not have a tertiary structure in isolation. In some embodiments, the

heterologous peptide does not have a biological function. In some embodiments, the heterologous peptide is synthetically designed, and is not derived from a naturally occurring peptide. In some embodiments, the second heterologous peptide (P2) is about 5, 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues in length. In some embodiments, the second heterologous peptide (P2) comprises about 50 or fewer, about 45 or fewer, about 40 or fewer, about 35 or fewer, about 30 or fewer, about 25 or fewer, about 20 or fewer, about 15 or fewer, or about 10 or fewer amino acids in length. In some

embodiments, the second heterologous peptide (P2) is about 5 to about 20 amino acid residues in length, such as about 5 to about 10, about 10 to about 15, or about 15 to about 20 amino acid residues in length.

[0085] In some embodiments, the second heterologous peptide (P2) includes one or more glycine residues or one or more serine residues. In some embodiments, the second

heterologous peptide (P2) includes an amino acid sequence of (GGGGS)_q (SEQ ID NO: 34), wherein q is 1, 2, 3, 4 or more.

[0086] The drug conjugation moiety can include 0, 1, 2, 3, 4, 5 or more albumins or albumin fragments, which may be the same or different, and 1, 2, 3, 4, 5, or more heterologous peptides comprising a cysteine residue. The components of the drug conjugation moiety (i.e., the one or more albumin or albumin fragments, if present, the one or more heterologous peptides comprising a cysteine residue, and the second heterologous peptide (P2) if present) are fused together in a single polypeptide chain, which is fused to the antibody heavy chain (or fragment) or the antibody light chain (or fragment). The components of the drug conjugation moiety can be fused in any order, but are preferably fused such that at least one heterologous peptide is included between each albumin or albumin fragment when more than one albumins or albumin fragments are present.

[0087] By way of example, the antibody fusion molecule can include a polypeptide configured, from N-terminus to C-terminus, according to any of the following configurations:

HL-P1;

HL-P1-A

HL-P1-A-P3;

HL-P1-A-P3-A;

HL-P 1 - A-P3 - A-P4 ;

HL-P 1 - A-P3 - A-P4- A;

HL-P 1 - A-P3 - A-P4- A-P5 ;

HL-P2-A-P1;

HL-P2-A-P1-A;

HL-P2- A-P 1 - A-P3 ;

HL-P2- A-P 1 - A-P3 - A; or

HL-P2- A-P 1 - A-P3 - A-P4 ;

wherein HL is the antibody heavy chain or the antibody light chain, or the fragment thereof; Pl, P2, P3, P4, and P5 are heterologous peptides; and A is an albumin or a fragment thereof. In some embodiments, at least one or more of Pl, P3, P4, and P5 include a cysteine residue. In some embodiments, each of Pl, P3, P4, and P5 include a cysteine residue. In some embodiments, the second heterologous peptide (P2) includes a cysteine residue, and in some embodiments the second heterologous peptide (P2) does not include a cysteine residue.

[0088] The antibody fusion molecule can include more than one drug conjugation moieties. For example, in some embodiments, the antigen fusion molecule includes a first drug conjugation moiety and a second drug conjugation moiety, wherein the first drug conjugation moiety is fused to the antibody heavy chain (or fragment) and the second drug conjugation moiety is fused to the antibody light chain (or fragment). The first drug conjugation moiety and the second drug conjugation moiety may be the same or different. In another example, the antibody fusion molecule can include two antibody heavy chains (or fragments) and two antibody light chains (or fragments), and a first drug conjugation moiety is fused to a first antibody heavy chain (or fragment), and a second drug conjugation moiety is fused to a second antibody heavy chain (or fragment), wherein the first drug conjugation moiety and the second drug conjugation moiety may be the same or different. In some embodiments, the antibody fusion molecule includes two antibody heavy chains (or fragments) and two antibody light chains (or fragments), and a first drug conjugation moiety is fused to a first antibody light chain (or fragment), and a second drug conjugation moiety is fused to a second antibody light chain (or fragment), wherein the first drug conjugation moiety and the second drug conjugation moiety may be the same or different. In some embodiments, the antibody fusion molecule includes two antibody heavy chains (or fragments) and two antibody light chains (or fragments), and a first drug conjugation moiety is fused to one of the antibody heavy chains (or fragments), and a second drug conjugation moiety is fused to one of the antibody light chains (or fragments), wherein the first drug conjugation moiety and the second drug conjugation moiety may be the same or different. In some embodiments, the antibody fusion molecule includes two antibody heavy chains (or fragments) and two antibody light chains (or fragments), and a first drug conjugation moiety is fused to a first antibody light chain (or fragment), a second drug conjugation moiety is fused to a second antibody light chain (or fragment), a third drug conjugation moiety is fused to a first antibody heavy chain (or fragment), and a fourth drug conjugation moiety is fused to a second antibody heavy chain (or fragment), wherein the first drug conjugation moiety and the second drug conjugation moiety may be the same or different.

[0089] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising an antibody heavy chain, or a fragment thereof, and an antibody light chain, or a fragment thereof; and drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), wherein the heterologous peptide comprises a cysteine residue. In some embodiments, Pl is charged. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or fragment thereof. In some embodiments, the drug conjugation moiety is fused to the antibody light chain or fragment thereof. In some embodiments, the antigen-binding moiety comprises a full length antibody. In some embodiments, the antigen-binding moiety comprises an antibody fragment. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0090] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising an antibody heavy chain, or a fragment thereof, and an antibody light chain, or a fragment thereof; and drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising (1) a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue, and (2) an albumin or an albumin fragment fused to Pl. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of Pl. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via Pl. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via the albumin or the albumin fragment. In some embodiments, Pl is charged. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or fragment thereof. In some embodiments, the drug conjugation moiety is fused to the antibody light chain or fragment thereof. In some embodiments, the antigen-binding moiety comprises a full length antibody. In some embodiments, the antigen binding moiety comprises an antibody fragment. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0091] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising two full-length antibody heavy chains and two full-length antibody light chain; and a drug conjugation moiety fused to the C-terminus of each antibody heavy chain, comprising (1) a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue, and (2) an albumin or an albumin fragment fused to Pl. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of Pl. In some embodiments, the drug conjugation moiety is fused to the antigen -binding moiety via Pl. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via the albumin or the albumin fragment. In some embodiments, Pl is charged. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0092] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising two full-length antibody heavy chains and two full-length antibody light chain; and a drug conjugation moiety fused to the C-terminus of each antibody light chain, comprising (1) a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue, and (2) an albumin or an albumin fragment fused to Pl. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of Pl. In some embodiments, the drug conjugation moiety is fused to the antigen -binding moiety via Pl. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via the albumin or the albumin fragment. In some embodiments, Pl is charged. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0093] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising an antibody heavy chain, or a fragment thereof, and an antibody light chain, or a fragment thereof; and drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising (1) a first heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue, (2) an albumin or an albumin fragment fused to Pl, and (3) a second heterologous peptide (P2) fused to the albumin or the albumin fragment. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of P2. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via P2. In some embodiments, P2 does not comprise a cysteine. In some embodiments, Pl is charged. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or fragment thereof. In some embodiments, the drug conjugation moiety is fused to the antibody light chain or fragment thereof. In some embodiments, the antigen-binding moiety comprises a full length antibody. In some embodiments, the antigen-binding moiety comprises an antibody fragment. In some embodiments, Pl or P2 comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0094] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising two full-length antibody heavy chains and two full-length antibody light chain; and a drug conjugation moiety fused to the C-terminus of each antibody heavy chain, comprising (1) a first heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue, (2) an albumin or an albumin fragment fused to Pl, and (3) a second heterologous peptide (P2) fused to the albumin or the albumin fragment. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of P2. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via P2. In some embodiments, P2 does not comprise a cysteine. In some embodiments, Pl is charged. In some embodiments, Pl or P2 comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0095] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising two full-length antibody heavy chains and two full-length antibody light chain; and a drug conjugation moiety fused to the C-terminus of each antibody light chain, comprising (1) a first heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue, (2) an albumin or an albumin fragment fused to Pl, and (3) a second heterologous peptide (P2) fused to the albumin or the albumin fragment. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of P2. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via P2. In some embodiments, P2 does not comprise a cysteine. In some embodiments, Pl is charged. In some embodiments, Pl or P2 comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0096] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32. In some

embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some

embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0097] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence according to SEQ ID NO: 32. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some

[0098] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antigen binding moiety comprises an antibody light chain or antibody light chain fragment, and an antibody heavy chain or antibody heavy chain fragment comprising CDR1, CDR2, and CDR3 of the sequence according to SEQ ID NO: 32, wherein the antigen binding moiety is fused to a polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32. In some embodiments, the polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32 is fused to the antibody heavy chain or antibody heavy chain fragment. In some embodiments, the polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32 is fused to the antibody light chain or antibody light chain fragment. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO:

14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0099] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antigen binding moiety comprises an antibody light chain or antibody light chain fragment, and an antibody heavy chain or antibody heavy chain fragment comprising the antibody heavy chain variable region of the sequence according to SEQ ID NO: 32, wherein the antigen binding moiety is fused to a polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32. In some embodiments, the polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32 is fused to the antibody heavy chain or antibody heavy chain fragment. In some embodiments, the polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32 is fused to the antibody light chain or antibody light chain fragment. In some

[0100] Amino acid residues 1-19 of SEQ ID NO: 32 are a signal peptide, which is optionally excluded from the antibody fusion molecule. Accordingly, in some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 20-1072 of SEQ ID NO: 32. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0101] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence according to residues 20-1072 of SEQ ID NO: 32. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0102] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33. In some

[0103] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence according to SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some

[0104] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antigen binding moiety comprises an antibody light chain or antibody light chain fragment, and an antibody heavy chain or antibody heavy chain fragment comprising CDR1, CDR2, and CDR3 of the sequence according to SEQ ID NO: 33, wherein the antigen binding moiety is fused to a polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33. In some embodiments, the polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33 is fused to the antibody heavy chain or antibody heavy chain fragment. In some embodiments, the polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33 is fused to the antibody light chain or antibody light chain fragment. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO:

[0105] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antigen binding moiety comprises an antibody light chain or antibody light chain fragment, and an antibody heavy chain or antibody heavy chain fragment comprising the antibody heavy chain variable region of the sequence according to SEQ ID NO: 33, wherein the antigen binding moiety is fused to a polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33. In some embodiments, the polypeptide comprising residues465-l075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33 is fused to the antibody heavy chain or antibody heavy chain fragment. In some embodiments, the polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO:

33 is fused to the antibody light chain or antibody light chain fragment. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some

[0106] Amino acid residues 1-19 of SEQ ID NO: 32 are a signal peptide, which is optionally excluded from the antibody fusion molecule. Accordingly, in some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 20-1075 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0107] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence according to residues 20-1075 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0108] In some embodiments, there is a polypeptide or a drug conjugation moiety, comprising (1) a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue, and (2) an albumin or an albumin fragment fused to Pl. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of Pl. In some embodiments, Pl is charged. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0109] In some embodiments, there is a polypeptide or a drug conjugation moiety, comprising (1) a first heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue, (2) an albumin or an albumin fragment fused to Pl, and (3) a second heterologous peptide (P2) fused to the albumin or the albumin fragment. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of P2. In some embodiments, P2 does not comprise a cysteine. In some embodiments, Pl is charged. In some embodiments, Pl or P2 comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0110] In some embodiments, there is a polypeptide or a drug conjugation moiety that comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 465-1072 of SEQ ID NO: 32. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 465- 1072 of SEQ ID NO: 32. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 480-1072 of SEQ ID NO: 32. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 480- 1072 of SEQ ID NO: 32. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 465-1064 of SEQ ID NO: 32. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 465- 1064 of SEQ ID NO: 32.

[0111] In some embodiments, there is a polypeptide or drug conjugation moiety that comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 465-1075 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 465- 1075 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 483-1075 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 483- 1075 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 465-1067 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 465- 1067 of SEQ ID NO: 33.

Antibody Fusion Molecule Drug Conjugates

[0112] The antibody fusion molecules described herein can further include a payload molecule, such as a drug molecule, covalently bound to the cysteine residue in any one or more of the heterologous peptides through a conjugation linker. The number of payload molecules attached to the antibody fusion molecule can be determined based on the number of heterologous peptides comprising cysteines, and the number of drug molecules that are conjugated to each cysteine.

[0113] The payload molecule can be a hydrophobic payload molecule, such as a hydrophobic drug molecule. In some embodiments, the payload molecule has a solubility of about 10 mg/mL or less, about 5 mg/mL or less, about 2.5 mg/mL or less, about 1 mg/mL or less, about 0.5 mg/mL or less, about 0.2 mg/mL or less, about 0.1 mg/mL or less, about 0.005 mg/mL or less, about 0.02 mg/mL or less, or about 0.01 mg/mL or less) in water at pH 7 at 25 °C.

[0114] In some embodiments, the payload molecule is a drug molecule. In some

embodiments, the drug molecule is a cytotoxic drug, an anti-viral drug, or an anti-bacterial drug. Exemplary drugs that can be conjugated to the antibody fusion molecule includes an antibiotic, a microtubule disrupting agent, a DNA modifying agent, an RNA polymerase inhibitor, or a topoisomerase I inhibitor. In some embodiments, the drug is azaribine, anastrozole, azacitidine, bleomycin, bortezomib, bryostatin-l, busulfan, camptothecin, 10- hydroxycamptothecin, calicheamicin, carmustine, celebrex, chlorambucil, cisplatin, irinotecan, carboplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycin glucuronide, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin, doxorubicin glucuronide, duocarmycin, duomycin, epirubicin, ethinyl estradiol, estramustine, etoposide, etoposide glucuronide, floxuridine, fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide, leucovorin, lomustine, mechlorethamine, medroxyprogesterone acetate, megestrol, melphalan, mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), paclitaxel, phenylbutyrate, prednisone, procarbazine, paclitaxel, pentostatin,

pyrrolobenzodiazepine (PBD), semustine, SN-38, streptozocin, tamoxifen, a taxane, testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinblastine, vinorelbine, or vincristine. In some embodiments, the payload molecule is an imaging agent, such as a fluorescent imaging agent or an MRI contrast agent.

[0115] In some embodiments, the antibody fusion molecule includes one or more payload molecules, such as 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 or more payload molecules. In some embodiments, the antibody fusion molecule includes 2 to 30 payload molecules, such as 2 to 4, 4 to 8, 8 to 12, 12 to 16, 16 to 20, 20 to 25, or 25 to 30 payload molecules.

[0116] The payload molecule can be conjugated to the cysteine on the heterologous peptide through any suitable conjugation linkers. In some embodiments, the payload molecule is conjugated to the cysteine through a maleimide moiety (i.e., through maleimide alkylation) or through cysteine arylation (e.g., through the use of a palladium complex). In some embodiments, the maleimide moiety is a N-alkyl maleimide moiety. Binding of the maleimide moiety to the thiol of the cysteine results in a succinimide thioether, which can be hydrolyzed into a ring-opened counterpart.

[0117] In some embodiments, the conjugation linker is not cleavable. Payload molecules, such as drug molecules, conjugated to the antibody fusion molecule through a non-cleavable linker are leased after the antibody fusion molecule is internalized by the target cell and the polypeptide components of the antibody fusion molecule are degraded by the lysosome.

[0118] In some embodiments, the conjugation linker is a cleavable linker. Exemplary cleavable linkers may include a pH- sensitive cleavable linker (such as a linker with a hydrazine moiety which is cleavable in acidic conditions), an enzymatically cleavable linker, such as a linker with a protease-cleavable moiety (such as a valine-alanine (Val-Ala) dipeptide component or a valine-citrulline dipeptide component, which may be

/; - a m i n o b e n zy 1 o x y c a r b o n y 1 (PABC)), linker cleavable under reducing conditions (such as a linker having a disulfide bond, which is cleavable in intracellular reducing conditions), or a pyrophosphate diester. Exemplary conjugation linkers for conjugating the drug to a cysteine in the heterologous peptide are discussed in Tsuchikama et ah, Antibody-drug conjugates: recent advances in conjugation and linker chemistries, Protein & Cell, vol. 9, pp. 33-46 (2018), first available online Oct. 14, 2016.

[0119] An exemplary conjugation linker that can be used to conjugate the payload to the cysteine of the heterologous peptide is shown in Formula I:

wherein z is 4-24; and the maleimide group is covalently attached to the cysteine of the heterologous peptide through the maleimide group.

[0120] In some embodiments, the conjugation linker conjugated to the cysteine of the heterologous peptide is bound to two or more payload molecules. The two or more payload molecules may optionally be independently cleavable from the conjugation linker, for example by including a cleavable bond for each payload molecule.

[0121] In some embodiments, there is an antibody fusion molecule, comprising antigen binding moiety that binds to DLL3, comprising an antibody heavy chain and an antibody light chain or fragment thereof and a drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising a heterologous peptide (Pl), wherein the heterologous peptide comprises a cysteine residue conjugated to a DNA- modifying agent. In some embodiments, the DNA-modifying agent is pyrrolobenzodiazepine (PBD), a PBD dimer, calicheamicin, duomycin, or duocarmycin. In some embodiments, the drug conjugation moiety comprises an albumin or an albumin fragment fused to the heterologous peptide, either at the N-terminal end or the C-terminal end of the heterologous peptide.

[0122] In some embodiments, there is an antibody fusion molecule, comprising antigen binding moiety that binds to Trop-2, comprising an antibody heavy chain and an antibody light chain or fragment thereof and a drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising a heterologous peptide (Pl), wherein the heterologous peptide comprises a cysteine residue conjugated to a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor is SN-38. In some embodiments, the drug conjugation moiety comprises an albumin or an albumin fragment fused to the heterologous peptide, either at the N-terminal end or the C-terminal end of the heterologous peptide.

[0123] In some embodiments, there is an antibody fusion molecule, comprising antigen binding moiety that binds to Claudin 18.2, but not to Claudin 18.1, or binds to Claudin 18.1 with at least 10 times weaker binding affinity than to Claudin 18.2, comprising an antibody heavy chain and an antibody light chain or fragment thereof and a drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising a heterologous peptide (Pl) , wherein the heterologous peptide comprises a cysteine residue conjugated to a DNA-modifying agent, a microtubule disrupting agent, or a topoisomerase inhibitor. In some embodiments, the cysteine residue is conjugated to monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), SN-38, PBD, a PBD dimer, calicheamicin, duomycin, or duocarmycin. In some embodiments, the drug conjugation moiety comprises an albumin or an albumin fragment fused to the heterologous peptide, either at the N-terminal end or the C-terminal end of the heterologous peptide.

[0124] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising an antibody heavy chain, or a fragment thereof, and an antibody light chain, or a fragment thereof; and drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), wherein the heterologous peptide comprises a cysteine residue conjugated to a payload molecule (such as a drug molecule, for example a cytotoxic drug molecule). In some embodiments, Pl is charged. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or fragment thereof. In some embodiments, the drug conjugation moiety is fused to the antibody light chain or fragment thereof. In some embodiments, the antigen-binding moiety comprises a full length antibody. In some embodiments, the antigen-binding moiety comprises an antibody fragment. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0125] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising an antibody heavy chain, or a fragment thereof, and an antibody light chain, or a fragment thereof; and drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising (1) a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue conjugated to a payload molecule, such as a drug molecule (such as a cytotoxic drug molecule), and (2) an albumin or an albumin fragment fused to Pl. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of Pl. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via Pl. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via the albumin or the albumin fragment. In some embodiments, Pl is charged. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or fragment thereof. In some embodiments, the drug conjugation moiety is fused to the antibody light chain or fragment thereof. In some embodiments, the antigen-binding moiety comprises a full length antibody. In some embodiments, the antigen binding moiety comprises an antibody fragment. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0126] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising two full-length antibody heavy chains and two full-length antibody light chain; and a drug conjugation moiety fused to the C-terminus of each antibody heavy chain, comprising (1) a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue conjugated to a payload molecule, such as a drug molecule (such as a cytotoxic drug molecule), and (2) an albumin or an albumin fragment fused to Pl. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of Pl. In some embodiments, the drug conjugation moiety is fused to the antigen -binding moiety via Pl. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via the albumin or the albumin fragment. In some embodiments, Pl is charged. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42). [0127] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising two full-length antibody heavy chains and two full-length antibody light chain; and a drug conjugation moiety fused to the C-terminus of each antibody light chain, comprising (1) a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue conjugated to a payload molecule, such as a drug molecule (such as a cytotoxic drug molecule), and (2) an albumin or an albumin fragment fused to Pl. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of Pl. In some embodiments, the drug conjugation moiety is fused to the antigen -binding moiety via Pl. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via the albumin or the albumin fragment. In some embodiments, Pl is charged. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0128] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising an antibody heavy chain, or a fragment thereof, and an antibody light chain, or a fragment thereof; and drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof comprising (1) a first heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue conjugated to a payload molecule, such as a drug molecule (such as a cytotoxic drug molecule), (2) an albumin or an albumin fragment fused to Pl, and (3) a second heterologous peptide (P2) fused to the albumin or the albumin fragment. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of P2. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via P2. In some embodiments, P2 does not comprise a cysteine.

In some embodiments, Pl is charged. In some embodiments, the drug conjugation moiety is fused to the antibody heavy chain or fragment thereof. In some embodiments, the drug conjugation moiety is fused to the antibody light chain or fragment thereof. In some embodiments, the antigen-binding moiety comprises a full length antibody. In some embodiments, the antigen-binding moiety comprises an antibody fragment. In some embodiments, Pl or P2 comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0129] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising two full-length antibody heavy chains and two full-length antibody light chain; and a drug conjugation moiety fused to the C-terminus of each antibody heavy chain, comprising (1) a first heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue conjugated to a payload molecule, such as a drug molecule (such as a cytotoxic drug molecule), (2) an albumin or an albumin fragment fused to Pl, and (3) a second heterologous peptide (P2) fused to the albumin or the albumin fragment. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of P2. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via P2. In some embodiments, P2 does not comprise a cysteine. In some embodiments, Pl is charged. In some embodiments, Pl or P2 comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0130] In some embodiments, an antibody fusion molecule comprises an antigen-binding moiety comprising two full-length antibody heavy chains and two full-length antibody light chain; and a drug conjugation moiety fused to the C-terminus of each antibody light chain, comprising (1) a first heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue conjugated to a payload molecule, such as a drug molecule (such as a cytotoxic drug molecule), (2) an albumin or an albumin fragment fused to Pl, and (3) a second heterologous peptide (P2) fused to the albumin or the albumin fragment. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of P2. In some embodiments, the drug conjugation moiety is fused to the antigen-binding moiety via P2. In some embodiments, P2 does not comprise a cysteine. In some embodiments, Pl is charged.

In some embodiments, Pl or P2 comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0131] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 32, wherein a payload molecule, such as a drug molecule, is conjugated to the polypeptide. In some embodiments, the payload molecule is conjugated to Cysl070 of SEQ ID NO: 32. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0132] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence according to SEQ ID NO: 32, wherein a payload molecule, such as a drug molecule, is conjugated to the polypeptide. In some embodiments, the payload molecule is conjugated to Cysl070 of SEQ ID NO: 32. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some

[0133] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antigen binding moiety comprises an antibody light chain or antibody light chain fragment, and an antibody heavy chain or antibody heavy chain fragment comprising CDR1, CDR2, and CDR3 of the sequence according to SEQ ID NO: 32, wherein the antigen binding moiety is fused to a polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32, and wherein a payload molecule, such as a drug molecule, is conjugated to the polypeptide. In some embodiments, the payload molecule is conjugated to Cysl070 of SEQ ID NO: 32. In some embodiments, the polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32 is fused to the antibody heavy chain or antibody heavy chain fragment. In some embodiments, the polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32 is fused to the antibody light chain or antibody light chain fragment. In some

[0134] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antigen binding moiety comprises an antibody light chain or antibody light chain fragment, and an antibody heavy chain or antibody heavy chain fragment comprising the antibody heavy chain variable region of the sequence according to SEQ ID NO: 32, wherein the antigen binding moiety is fused to a polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32, and wherein a payload molecule, such as a drug molecule, is conjugated to the polypeptide. In some embodiments, the payload molecule is conjugated to Cysl070 of SEQ ID NO: 32. In some embodiments, the polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32 is fused to the antibody heavy chain or antibody heavy chain fragment. In some embodiments, the polypeptide comprising residues 465-1072 of SEQ ID NO: 32, residues 480-1072 of SEQ ID NO: 32, or residues 465-1064 of SEQ ID NO: 32 is fused to the antibody light chain or antibody light chain fragment. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some

[0135] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 20-1072 of SEQ ID NO: 32, wherein a payload molecule, such as a drug molecule, is conjugated to the polypeptide. In some embodiments, the payload molecule is conjugated to Cysl070 of SEQ ID NO: 32. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some

[0136] In some embodiments, the antibody fusion molecule, comprising an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence according to residues 20-1072 of SEQ ID NO: 32, wherein a payload molecule, such as a drug molecule, is conjugated to the polypeptide. In some embodiments, the payload molecule is conjugated to Cysl070 of SEQ ID NO: 32. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0137] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33, wherein a payload molecule is conjugated to the polypeptide. In some embodiments, the drug molecule is conjugated to Cys477 of SEQ ID NO: 33. In some embodiments, the drug molecule is conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises a first drug molecule conjugated to Cys477 of SEQ ID NO: 33 and a second drug molecule conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO:

[0138] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence according to SEQ ID NO: 33, wherein a drug molecule is conjugated to the polypeptide. In some embodiments, the drug molecule is conjugated to Cys477 of SEQ ID NO: 33. In some embodiments, the drug molecule is conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises a first drug molecule conjugated to Cys477 of SEQ ID NO: 33 and a second drug molecule conjugated to Cysl073 of SEQ ID NO: 33. In some

[0139] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antigen binding moiety comprises an antibody light chain or antibody light chain fragment, and an antibody heavy chain or antibody heavy chain fragment comprising CDR1, CDR2, and CDR3 of the sequence according to SEQ ID NO: 33, wherein the antigen binding moiety is fused to a polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33, and wherein a payload molecule, such as a drug molecule, is conjugated to the polypeptide. In some embodiments, the drug molecule is conjugated to Cys477 of SEQ ID NO: 33. In some embodiments, the drug molecule is conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises a first drug molecule conjugated to Cys477 of SEQ ID NO: 33 and a second drug molecule conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33 is fused to the antibody heavy chain or antibody heavy chain fragment. In some embodiments, the polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33 is fused to the antibody light chain or antibody light chain fragment. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0140] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antigen binding moiety comprises an antibody light chain or antibody light chain fragment, and an antibody heavy chain or antibody heavy chain fragment comprising the antibody heavy chain variable region of the sequence according to SEQ ID NO: 33, wherein the antigen binding moiety is fused to a polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33, and wherein a payload molecule, such as a drug molecule, is conjugated to the polypeptide. In some embodiments, the drug molecule is conjugated to Cys477 of SEQ ID NO: 33. In some embodiments, the drug molecule is conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises a first drug molecule conjugated to Cys477 of SEQ ID NO: 33 and a second drug molecule conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33 is fused to the antibody heavy chain or antibody heavy chain fragment. In some embodiments, the polypeptide comprising residues 465-1075 of SEQ ID NO: 33, residues 483-1075 of SEQ ID NO: 33, or residues 465-1067 of SEQ ID NO: 33 is fused to the antibody light chain or antibody light chain fragment. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some

[0141] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 20-1075 of SEQ ID NO: 33, wherein a drug molecule is conjugated to the polypeptide. In some embodiments, the drug molecule is conjugated to Cys477 of SEQ ID NO: 33. In some embodiments, the drug molecule is conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises a first drug molecule conjugated to Cys477 of SEQ ID NO: 33 and a second drug molecule conjugated to Cysl073 of SEQ ID NO: 33. In some

[0142] In some embodiments, the antibody fusion molecule comprises an antigen binding moiety that specifically binds to GPC3, wherein the antibody fusion molecule comprises an antibody light chain and a polypeptide comprising a sequence according to residues 20-1075 of SEQ ID NO: 33, wherein a drug molecule is conjugated to the polypeptide. In some embodiments, the drug molecule is conjugated to Cys477 of SEQ ID NO: 33. In some embodiments, the drug molecule is conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises a first drug molecule conjugated to Cys477 of SEQ ID NO: 33 and a second drug molecule conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the antibody fusion molecule comprises an antibody light chain or antibody light chain fragment comprising CDR1, CDR2, and CDR3 of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain variable region comprising the antibody light chain variable region of the antibody light chain according to SEQ ID NO: 14. In some embodiments, the antibody fusion molecule comprises an antibody light chain comprising a sequence according to SEQ ID NO: 14.

[0143] In some embodiments, there is a polypeptide or drug conjugation moiety, comprising (1) a heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue conjugated to a drug molecule, and (2) an albumin or an albumin fragment fused to Pl. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin.

In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of Pl. In some embodiments, Pl is charged. In some embodiments, Pl comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0144] In some embodiments, there is a polypeptide or drug conjugation moiety, comprising (1) a first heterologous peptide (Pl) less than about 50 amino acids in length (or less than about 40, or less than about 30, or less than about 20 amino acids in length), comprising a cysteine residue conjugated to a drug molecule, (2) an albumin or an albumin fragment fused to Pl, and (3) a second heterologous peptide (P2) fused to the albumin or the albumin fragment. In some embodiments, the cysteine residue of Pl is within about 30 amino acids (such as within about 25, within about 20, within about 15, or within about 10 amino acids) of the albumin or albumin fragment. In some embodiments, the albumin is a full length albumin. In some embodiments, the albumin or albumin fragment is fused to the N-terminus of Pl. In some embodiments, the albumin or albumin fragment is fused to the C-terminus of P2. In some embodiments, P2 does not comprise a cysteine. In some embodiments, Pl is charged. In some embodiments, Pl or P2 comprises one or more contiguous linkers having the sequence GGGGS (SEQ ID NO: 42).

[0145] In some embodiments, there is a polypeptide or drug conjugation moiety comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 465-1072 of SEQ ID NO: 32, wherein the polypeptide or drug conjugation moiety is conjugated to a drug molecule. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 465-1072 of SEQ ID NO: 32, wherein the polypeptide or drug conjugation moiety is conjugated to a drug molecule. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 480- 1072 of SEQ ID NO: 32, wherein the polypeptide or drug conjugation moiety is conjugated to a drug molecule. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 480-1072 of SEQ ID NO: 32, wherein the polypeptide or drug conjugation moiety is conjugated to a drug molecule. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 465- 1064 of SEQ ID NO: 32, wherein the polypeptide or drug conjugation moiety is conjugated to a drug molecule. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 465-1064 of SEQ ID NO: 32, wherein the polypeptide or drug conjugation moiety is conjugated to a drug molecule. In some embodiments, the drug molecule is conjugated to Cysl070 of SEQ ID NO: 32.

[0146] In some embodiments, there is a polypeptide or drug conjugation moiety comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 465-1075 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 465-1075 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 483-1075 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 483-1075 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to residues 465- 1067 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a sequence according to residues 465-1067 of SEQ ID NO: 33. In some embodiments, the drug molecule is conjugated to Cys477 of SEQ ID NO: 33. In some embodiments, the drug molecule is conjugated to Cysl073 of SEQ ID NO: 33. In some embodiments, the polypeptide or drug conjugation moiety comprises a first drug molecule conjugated to Cys477 of SEQ ID NO: 33 and a second drug molecule conjugated to Cysl073 of SEQ ID NO: 33.

Pharmaceutical Compositions

[0147] Pharmaceutical compositions of the antibody fusion molecules and antibody fusion molecule drug conjugates are prepared by mixing the antibody fusion molecules or the antibody fusion molecule drug conjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (see Remington's Pharmaceutical Sciences l6th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;

hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;

cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

[0148] Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

[0149] Preservatives are added to retard microbial growth, and are typically present in a range from 0.2% - 1.0% (w/v). Suitable preservatives for use with the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;

benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0150] Tonicity agents, sometimes known as“stabilizers” are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed“stabilizers” because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter- and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1% to 25% by weight, or more preferably between 1% to 5% by weight, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0151] Non-ionic surfactants or detergents (also known as“wetting agents”) are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

[0152] Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0153] The choice of pharmaceutical carrier, excipient or diluent may be selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s).

[0154] There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, pharmaceutical compositions useful in the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be administered by a number of routes.

[0155] In some embodiments, an antibody formulation is a lyophilized antibody formulation. In another embodiment, an antibody formulation is an aqueous antibody formulation.

Methods of Treatment

[0156] The antibody fusion molecule conjugated to a drug can be used to treat a disease, depending on the antigen bound by the antigen-binding domain and the drug conjugated to the heterologous peptide. In some embodiments, the antibody fusion molecule drug conjugate is used to treat cancer, for example when the drug molecule is a cytotoxic agent. In some embodiments, the antibody fusion molecule drug conjugate is used to treat an infection, for example when the drug molecule is an antibacterial agent or an antiviral agent. Other diseases that may be treated include metabolic diseases (such as diabetes), bone diseases (such as osteoporosis), obesity, heart disease, or liver disease.

[0157] In some embodiments, a method of treating a disease (such as cancer, a viral infection, or a bacterial infection) in a subject comprises administering to the subject an effective amount of an antibody fusion molecule conjugated to a cytotoxic drug through a cysteine of the heterologous peptide.

[0158] In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer. Exemplary cancers that may be treated include, but are not limited to, leukemia, lymphoma, kidney cancer, bladder cancer, urinary tract cancer, cervical cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, esophageal cancer, liver cancer, colorectal cancer, stomach cancer, squamous cell carcinoma, prostate cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer. In some embodiments, the cancer has one or more mutant RAS family genes. In some embodiments, the cancer cell has upregulated macropinocytosis compared to a non- cancerous cell.

[0159] In some embodiments, the antibody fusion molecule conjugated to a drug is used to treat a bacterial infection. In some embodiments, the bacteria causing the bacterial infection is a drug-resistant bacteria. In some embodiments, the antigen-binding moiety binds to a bacterial antigen. In some embodiments, the drug conjugated to the antibody fusion molecule is an antibacterial agent.

[0160] In some embodiments, the antibody fusion molecule conjugated to a drug is used to treat a viral infection. In some embodiments, the virus causing the viral infection is hepatitis C (HCV), hepatitis B (HBV), human immunodeficiency virus (HIV), a human papilloma virus (HPV). In some embodiments, the antigen-binding moiety binds to a viral antigen. In some embodiments, the drug conjugated to the antibody fusion molecule is an anti-viral agent.

[0161] Generally, dosages and routes of administration of the nanoparticle composition are determined according to the size and condition of the subject, according to standard pharmaceutical practice. In some embodiments, the antibody fusion molecule is administered to a subject through any route, including orally, transdermally, by inhalation, intravenously, intra-arterially, intramuscularly, direct application to a wound site, application to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transcutaneously, by nebulization, intrapleurally, intraventricularly, intra-articularly, intraocularly, or intraspinally. In some embodiments, the composition is administered to a subject intravenously.

[0162] In some embodiments, the dosage of the antibody fusion molecule conjugated to the drug is a single dose or a repeated dose. In some embodiments, the doses are given to a subject once per day, twice per day, three times per day, or four or more times per day. In some embodiments, about 1 or more (such as about 2, 3, 4, 5, 6, or 7 or more) doses are given in a week. In some embodiments, the antibody fusion molecule conjugated to the drug is administered weekly, once every 2 weeks, once every 3 weeks, once every 4 weeks, weekly for two weeks out of 3 weeks, or weekly for 3 weeks out of 4 weeks. In some embodiments, multiple doses are given over the course of days, weeks, months, or years. In some embodiments, a course of treatment is about 1 or more doses (such as about 2, 2, 3, 4, 5, 7,

10, 15, or 20 or more doses).

Methods of Making the Antibody Fusion Molecule

[0163] Polypeptides that include an antibody heavy chain (or fragment) or an antibody light chain (or fragment) fused to a drug conjugation moiety can be produced using recombinant DNA methods. Nucleic acid molecules encoding the fusion polypeptide can be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid molecules may be readily isolated and sequenced using conventional methods. The antibody light chain or fragment or the variable heavy chain or fragment, wherein the antibody heavy chain or the antibody light chain is fused to the drug conjugation moiety, may be co-expressed in the same host cell. Suitable host cells for cloning or expression of fusion polypeptide vectors include prokaryotic cells or eukaryotic cells. Exemplary host cells include Chinese Hamster Ovary (CHO) cells or human embryonic kidney cells (e.g.,

HEK293).

[0164] Expression host cells express the antibody fusion molecule. After an expression period, the host cells can by lysed and the antibody fusion molecule purified. Exemplary purification methods include liquid chromatography, such as ion exchange chromatography, affinity chromatography (such as Protein A affinity chromatography), or size exclusion chromatography.

[0165] Drug molecules can be attached to a conjugation linker, for example during synthesis of the drug molecule or through a conjugation chemistry. The linker can include, for example, a maleimide moiety, which can react with a thiol group. The drug attached to the conjugation linker can be combined with the purified antibody fusion molecule, such that the drug molecule conjugates to a cysteine of one of the heterologous peptides comprising a cysteine. The maleimide moiety reacts with the cysteine residue in the heterologous peptide to form a succinimide thioether. The ring in the succinimide thioether can open after hydrolysis, thereby forming a stable conjugation between the heterologous peptide and the drug.

[0166] In some embodiments, the conjugation linker is conjugated to the antibody fusion molecule prior to attaching the conjugation linker to the drug.

[0167] The drug can be conjugated to the antibody fusion molecule by mixing the antibody fusion molecule and the drug in solution. The drug is generally mixed with the antibody fusion molecule in molar excess (that is, a molar ratio of drug to antibody fusion molecule of about 1 or more). In some embodiments, the molar ratio of drug to antibody fusion molecule is about 1 to about 1 or more, about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, about 7 or more, or about 8 or more. In some embodiments, the molar ratio of drug to antibody fusion molecule is about 1 to about 20, about 2 to about 15, about 4 to about 10, or about 6 to about 8.

[0168] The antibody fusion molecule can be conjugated to the drug with or without mixing the antibody fusion molecule with a reducing agent. Exemplary reducing agents that may be used include tris (2-carboxyethyl)phosphine (TCEP), b-mercaptoethanol (B-ME) and dithiothreitol (DTT). The reducing agent can be mixed with the antibody fusion molecule to reduce thiolates on the protein prior to conjugating the drug to the antibody fusion molecule. The reducing agent may be mixed with the antibody fusion molecule at a molar ratio

(reducing agent to antibody fusion molecule) of about 1 or more, about 1.5 or more, about 2 or more, about 2.5 or more, about 3 or more, about 5 or more, about 10 or more, about 15 or more, or about 20 or more. In some embodiments, the reducing agent is mixed with the antibody fusion molecule at a molar ratio (reducing agent to antibody fusion molecule) of about 1 to about 100, about 2 to about 75, about 5 to about 50, about 10 to about 40, or about 20 to about 30. In some embodiments, the reducing agent is removed from the composition containing the antibody fusion molecule prior to conjugating the drug to the antibody fusion molecule. After treating the antibody fusion molecule with the reducing agent, the antibody fusion molecule is optionally treated with an oxidizing agent prior to conjugating the antibody fusion molecule with the drug. Exemplary oxidizing agents can include ascorbic acid, cysteine, oxidized glutathione, and copper ions.

[0169] Once the antibody fusion molecule is conjugated to a drug molecule, the antibody fusion molecule can be further purified, for example to isolate the antibody fusion molecule conjugated to the drug from unreacted polypeptides, unreacted drug molecules, or antibody fusion molecules that are over- or under- conjugated to the drug molecules. Exemplary purification methods include liquid chromatography, such as ion exchange chromatography, affinity chromatography (such as Protein A affinity chromatography), or size exclusion chromatography. The purification methods can be used to isolate antibody drug conjugate molecules with a uniform drug-antibody ratio (DAR).

EXAMPLES

[0170] The following non-limiting examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments now contemplated. These examples are intended to be a mere illustration only and not to constitute a limitation on the scope of the invention. Thus, these examples should not be construed to limit any of the embodiments described in the present specification.

Example 1: Expression and Purification of Antibody- Albumin Fusion Protein

[0171] Expression plasmid constructs containing DNA sequences encoding the genes of the GPC3 antibody light chain (SEQ ID NO: 14) and antibody heavy chain-albumin fusion protein (SEQ ID NO: 33) were transfected into HEK-293 cells using polyethylenimine (PEI) for transient expression. The transfected cells were treated with valproic acid (VPA) 24 hours post transfection to enhance protein expression. The supernatants were harvested on day 6 and the antibodies were purified.

[0172] The antibody- albumin fusion protein was first captured by a Protein A affinity column, and eluted using a 0.5 M arginine buffer, pH 3.8. The pH of the eluted antibody- albumin fusion protein was adjusted to ~5.6, and the pH-adjusted elute was filtered. The antibody fusion protein was further purified by an anion exchange chromatography using Capto Q Impres resin. The purified antibody- albumin fusion protein was the formulated in a formulation containing 12 mM Histidine, 4% mannitol, 2% sucrose, pH 6.7. It was stored at 2-8 °C or -80 °C until use. Example 2: Drug Conjugation to Antibody- Albumin Fusion Protein

[0173] The antibody- albumin fusion protein expressed and purified as described in Example 1 was conjugated to MC-vc-PAB-MMAE (FIG. 1) according to the methods described in Reactions A-E, below.

[0174] Reaction A. The antibody- albumin fusion protein expressed and purified as described in Example 1 was conjugated to MC-vc-PAB-MMAE in the fusion protein storage buffer (2 mM Histidine, 4% mannitol, 2% sucrose, pH 6.7). The resulting antibody solution was adjusted to a concentration of >3 mg/mL. The presence of free, accessible thiols for cysteine- maleimide conjugation was measured using a standard Ellman’s reagent assay (ThermoFisher Scientific, Catalog # 22582). Briefly, Ellman’s reagent stock solution was prepared in DMSO at 0.1M before 1: 100 dilution into conjugation buffer. An equal volume of antibody aliquot and 1 mM Ellman’s reagent solution were mixed and incubated at room temperature for 5 min. The resulting absorbance was measured at 412 nm on a NanoDrop or Denovix instrument relative to buffer or suitable negative control. The ratio of free thiol-to-antibody (SH/Ab) was determined using Ellman’s reagent product extinction coefficient at 412 nm (e = 14,150 M^cnT¹). Immediately prior to conjugation, but before drug-linker addition, propylene glycol (PG) was added to the antibody solution to give a final concentration of 30% (v/v). Solid MC-vc-PAB-MMAE drug-linker reagent (Levena Biopharma U.S.) was first dissolved in DMSO at 5 mM concentration before being added in 4-8-fold molar excess to the PG-containing antibody solution. After thorough mixing, the resulting conjugation reaction was allowed to incubate at room temperature for a minimum of 1.5 h.

[0175] Reaction progress was monitored via hydrophobic interaction chromatography (HIC) on an Agilent 1100 HPLC system using a MAbPac™ HIC- 10 column (5 pm particles, 1000 A pore size, 4.6 x 100 mm column, ThermoFisher Scientific, Catalog # 088480). The column temperature was set to 30 °C and a flow rate was set at 1 mL/min. The column was loaded with approximately 50-100 pg protein for analysis, and the column elute was monitored with by UV at wavelengths of 280 nm and 252 nm. The column was run with a gradient between Buffer A (1.5 M ammonium sulfate) and Buffer B (25 mM sodium phosphate, pH 7.0, 25% isopropanol) according to the following: 0 min, 0% Buffer B, to 24 min: 80% Buffer B; then 95% Buffer B to 30 min; 0% Buffer B. FIG. 2 shows the HIC-HPLC chromatograph of the protein-drug conjugate formed by Reaction A.

[0176] The A280 peaks indicating conjugate formation were measured relative to unmodified antibody and drug-linker control injections. Average DAR (drug-to-antibody ratio) and % unmodified antibody-albumin fusion protein were determined via A280 peak area integration. The drug-antibody ratio (DAR) was calculated using the following equitation:

Fusion Protein

A -252 _

252 A 280 Fusion Protein

'280

[MMAE] MMAE

DAR = '252

[Fusion Protein] MMAE

f^c280

A₂80 A 252 MMAE

f^c252

Fusion Protein

f^c280

wherein the extinction coefficients (e) are defined in Table 2:

Table 2: Extension Coefficients for Determining Drug-Antibody Ratio (DAR)

[0177] Reaction B. The antibody- albumin fusion protein expressed and purified as described in Example 1 was buffer exchanged into conjugation buffer (50 mM sodium phosphate, 4 mM EDTA, pH 7.0) using multiple rounds of centrifugal ultrafiltration (using Amicon Ultra- 4 10 KDa centrifugal filter units (Millipore Sigma)). The antibody- albumin fusion protein was conjugated to MC-vc-PAB-MMAE in the conjugation buffer as otherwise described for Reaction A, and the product analyzed by HIC-HPLC as described for Reaction A. FIG. 3 shows the HIC-HPLC chromatograph of the protein-drug conjugate formed by Reaction B.

[0178] Reaction C. The antibody- albumin fusion protein expressed and purified as described in Example 1 was buffer exchanged into conjugation buffer (50 mM sodium phosphate, 4 mM EDTA, pH 7.0) using multiple rounds of centrifugal ultrafiltration (using Amicon Ultra- 4 10 KDa centrifugal filter units (Millipore Sigma)) before being partially reduced using 2.5x molar ratio of the reducing reagent tris(2-carboxyethyl)phosphine (TCEP). The conjugation reaction was conducted using 8x of MC-vc-PAB-MMAE at room temperature for

approximately 1 hour, as otherwise described for Reaction A, and the product analyzed by HIC-HPLC as described for Reaction A. FIG. 4 shows the HIC-HPLC chromatograph of the protein-drug conjugate formed by Reaction C.

[0179] Reaction D. The antibody- albumin fusion protein expressed and purified as described in Example 1 was buffer exchanged into conjugation buffer (50 mM sodium phosphate, 4 mM EDTA, pH 7.0) using multiple rounds of centrifugal ultrafiltration (using Amicon Ultra- 4 10 KDa centrifugal filter units (Millipore Sigma)) before being reduced using 20x molar ratio of the reducing reagent TCEP. The antibody-albumin fusion protein was then incubated with TCEP for 48 hours at 2-8 °C to fully reduce the antibody- albumin fusion protein. The TECP was removed and free thiol content was measured. The conjugation reaction was conducted using 4x of MC-vc-PAB-MMAE at room temperature for approximately 1 hour, as otherwise described for Reaction A, and the product analyzed by HIC-HPLC as described for Reaction A. FIG. 5 shows the HIC-HPLC chromatograph of the protein-drug conjugate formed by Reaction D.

[0180] Reaction E. Reaction D. The antibody- albumin fusion protein expressed and purified as described in Example 1 was buffer exchanged into conjugation buffer (50 mM sodium phosphate, 4 mM EDTA, pH 7.0) using multiple rounds of centrifugal ultrafiltration (using Amicon Ultra-4 10 KDa centrifugal filter units (Millipore Sigma)) before being reduced using 20x molar ratio of the reducing reagent TCEP. The antibody- albumin fusion protein was then incubated with TCEP for 48 hours at 2-8 °C to fully reduce the antibody- albumin fusion protein. The antibody-albumin fusion protein was then partially re-oxidized using ascorbic acid at room temperature for 1 hour. The conjugation reaction was conducted using 8x of MC-vc-PAB-MMAE at room temperature for approximately 1 hour, as otherwise described for Reaction A, and the product analyzed by HIC-HPLC as described for Reaction A. FIG. 6 shows the HIC-HPLC chromatograph of the protein-drug conjugate formed by Reaction E.

[0181] The reaction conditions and results for Reactions A-E are summarized in Table 3.

Table 3: Antibody-Albumin-Drug Conjugation Results Summary

[0182] While reactivity demonstrated with different equivalents of reducing agent (TCEP), highest DAR was achieved with the partial re-oxidation of fully reduced antibody- albumin fusion protein sample (Reaction E).

[0183] It is understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciated that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is not intended to be limited to a particular compound, composition, article, or method, unless expressly stated as such. In addition, those of ordinary skill in the art will recognize that certain changes, modification, permutations, alterations, additions, subtractions, and sub-combinations thereof can be made in accordance with the teachings herein without departing from the spirit of the present specification.

Claims

CLAIMS What is claimed is:

1. An antibody fusion molecule, comprising:

an antigen-binding moiety comprising an antibody heavy chain and an antibody light chain, or a fragment thereof; and

a drug conjugation moiety fused to the antibody heavy chain or the antibody light chain or fragment thereof, wherein the drug conjugation moiety comprises a heterologous peptide (Pl) less than about 50 amino acids in length, and wherein Pl comprises a cysteine residue.

2. The antibody fusion molecule of claim 1, wherein the heterologous peptide (Pl) is unstructured or helical.

3. The antibody fusion molecule of claim 1 or 2, wherein the heterologous peptide (Pl) is about 5 to about 20 amino acids in length.

4. The antibody fusion molecule of any one of claims 1-3, wherein the drug conjugation moiety is fused to the N-terminus or the C-terminus of the antibody heavy chain, or the fragment thereof.

5. The antibody fusion molecule of any one of claims 1-3, wherein the drug conjugation moiety is fused to the N-terminus or the C-terminus of the antibody light chain, or the fragment thereof.

6. The antibody fusion molecule of any one of claims 1-5, wherein the antigen-binding moiety comprises a full length antibody or an antibody fragment.

7. The antibody fusion molecule of any one of claims 1-6, wherein the drug conjugation moiety comprises an albumin or an albumin fragment fused to Pl.

8. The antibody fusion molecule of claim 7, wherein the cysteine residue of Pl is about 30 amino acid residues or less away from a first residue or a last residue of the albumin or the albumin fragment.

9. The antibody fusion molecule of claim 7 or 8, wherein the drug conjugation moiety is fused to the antibody heavy chain or antibody light chain or fragment thereof via the albumin or the albumin fragment.

10. The antibody fusion molecule of claim 7 or 8, wherein the drug conjugation moiety is fused to the antibody heavy chain or antibody light chain or fragment thereof via Pl.

11. The antibody fusion molecule of any one of claims 7-10, wherein the albumin or the albumin fragment has a point mutation at Cys34.

12. The antibody fusion molecule of claim 11, wherein the point mutation is Cys34Ser.

13. The antibody fusion molecule of any one of claims 7-12, wherein the drug conjugation moiety comprises a full length albumin.

14. The antibody fusion molecule of any one of claims 7-12, wherein the drug conjugation moiety comprises an albumin fragment.

15. The antibody fusion molecule of any one of claims 7-14, wherein the drug conjugation moiety comprises a second heterologous peptide (P2), which is fused to the albumin or albumin fragment.

16. The antibody fusion molecule of claim 15, wherein the drug conjugation moiety is fused to the antibody heavy chain or antibody light chain or fragment thereof via the second heterologous peptide (P2).

17. The antibody fusion molecule of claim 15 or 16, wherein the second heterologous peptide (P2) comprises a cysteine residue.

18. The antibody fusion molecule of claim 15 or 16, wherein the second heterologous peptide (P2) does not comprise a cysteine residue.

19. The antibody fusion molecule of any one of claims 15-18, wherein the second heterologous peptide (P2) is helical or unstructured.

20. The antibody fusion molecule of anyone of claims 7-19, wherein the drug conjugation moiety comprises two or more albumins or albumin fragments.

21. The antibody fusion molecule of claim 20, wherein the drug conjugation moiety comprises three or more heterologous peptides.

22. The antibody fusion molecule of claim 21, wherein each of the heterologous peptides comprises a cysteine residue.

23. The antibody fusion molecule of any one of claims 1-22, comprising a polypeptide configured, from N-terminus to C-terminus, according to:

HL-P1;

HL-P1-A

HL-P1-A-P3;

HL-P1-A-P3-A;

HL-P 1 - A-P3 - A-P4 ;

HL-P 1 - A-P3 - A-P4- A;

HL-P 1 - A-P3 - A-P4- A-P5 ;

HL-P2-A-P1;

HL-P2-A-P1-A;

HL-P2- A-P 1 - A-P3 ;

HL-P2- A-P 1 - A-P3 - A; or

HL-P2- A-P 1 - A-P3 - A-P4 ;

wherein HL is the antibody heavy chain or the antibody light chain, or the fragment thereof;

wherein Pl, P3, P4, and P5 are heterologous peptides, wherein at least one of Pl, P3, P4, and P5 comprises a cysteine residue;

wherein P2 is a heterologous peptide; and

wherein A is an albumin or a fragment thereof.

24. The antibody fusion molecule of any one of claims 15-23, wherein the second heterologous peptide (P2) is about 5 to about 20 amino acid residues in length.

25. The antibody fusion molecule of any one of claims 15-24, wherein the second heterologous peptide (P2) comprises one or more glycine residues or one or more serine residues.

26. The antibody fusion molecule of any one of claims 15-25, wherein the second heterologous peptide (P2) comprises an amino acid sequence of (GGGGS)_q (SEQ ID NO: 34), wherein q is 1, 2, 3, 4, or more.

27. The antibody fusion molecule of any one of claims 23-26, wherein each of Pl, P3, P4, and P5 is about 5 to about 20 amino acid residues in length, and wherein Pl, P3, P4, and P5 are of the same length or of different lengths.

28. The antibody fusion molecule of any one of claims 1-27, wherein at least one of Pl, P3, P4, and P5 comprises at least one charged amino acid.

29. The antibody fusion molecule of any one of claims 1-28, wherein at least one of Pl, P3, P4, and P5 comprises one or more positively charged amino acid residues.

30. The antibody fusion molecule of claim 29, wherein the one or more positively charged amino acid residues are selected from the group consisting of lysine, histidine, arginine, and combinations thereof.

31. The antibody fusion molecule of any one of claims 1-28, wherein at least one of Pl, P3, P4, and P5 comprises one or more negatively charged amino acid residues.

32. The antibody fusion molecule of claim 31, wherein the one or more negatively charged amino acid residues are selected from the group consisting of aspartic acid, glutamic acid, and combinations thereof.

33. The antibody fusion molecule of any one of claims 1-32, wherein at least one of Pl, P3,

P4, and P5 comprises a sequence according to: (GGGGS)_nCX_aalX_aa2X_aa3X_aa4X_aa5X_aa6(GGGGS)_m (SEQ ID NO: 35);

(GGGGS)_nX_aalCX_aa2X_aa3X_aa4X_aa5X_aa6(GGGGS)_m (SEQ ID NO: 36);

(GGGGS)_nX_aaiX_aa2CX_aa3X_aa4X_aa5X_aa6(GGGGS)_m (SEQ ID NO: 37);

(GGGGS)_nX_aaiX_aa2X_aa3CX_aa4X_aa5X_aa6(GGGGS)_m (SEQ ID NO: 38);

(GGGGS)_nX_aalX_aa2X_aa3X_aa4CX_aa5X_aa6(GGGGS)_m (SEQ ID NO: 39);

(GGGGS)„X_aalX_aa2X_aa3X_aa4X_aa5CX_aa6(GGGGS)_m (SEQ ID NO: 40); or

(GGGGS)_nX_aaiX_aa2X_aa3X_aa4X_aa5X_aa6C(GGGGS)_m (SEQ ID NO: 41);

wherein X_aai, X_aa2, X_aa3, X_aa3, X _3, and X_aa6 are each either a single amino acid, or none;

wherein X_aai, X_aa2, X_aa3, X_aa4, X_aa3, and X_aa6 has a net positive charge or a net negative charge;

wherein n = 0, 1, 2, 3, or 4;

wherein m = 0, 1, 2, 3, or 4; and

wherein the sequence of Pl, P3, P4, and P5 may be the same or different.

34. The antibody fusion molecule of claim 33, wherein X^i, X_aa2, X_aa3, X_aa4, X_aa5, and X_aa6 comprise:

at least one positively charged amino acid residue and no negatively charged amino acid residues; or

at least one negatively charged amino acid residue and no positively charged amino acid residues.

35. The antibody fusion molecule of claim 33 or 34, wherein X^i, X_aa2, X_aa3, X_aa4, X_aa5, and X_aa6 are each either:

an amino acid selected from the group consisting of histidine, lysine, arginine, aspartic acid, glutamic acid, glycine, serine, and threonine; or

none.

36. The antibody fusion molecule of any one of claims 1-35, wherein the antibody fusion molecule comprises a full length antibody heavy chain or a full length antibody light chain.

37. The antibody fusion molecule of any one of claims 1-36, wherein the albumin has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1.

38. The antibody fusion molecule of any one of claims 1-37, wherein the antibody fusion molecule comprises a first drug conjugation moiety fused to the antibody light chain, and a second drug conjugation moiety fused to the antibody heavy chain.

39. The antibody fusion molecule of any one of claims 1-38, further comprising a payload molecule covalently bound to the cysteine residue in any one or more of Pl, P3, P4, and P5.

40. The antibody fusion molecule of claim 39, wherein the payload molecule comprises a drug molecule or an imaging agent.

41. The antibody fusion molecule of claim 40, wherein the drug molecule comprises a cytotoxic drug molecule, an anti- viral agent, or an anti-bacterial agent.

42. The antibody fusion molecule of claim 40 or 41, wherein the antibody fusion molecule comprises 2-30 payload molecules.

43. The antibody fusion molecule of any one of claims 40-42, wherein the payload molecule is covalently bound to the cysteine residue in Pl, P3, P4, or P5 through a conjugation linker.

44. The antibody fusion molecule of claim 43, wherein the conjugation linker is bound to two or more payload molecules.

45. The antibody fusion molecule of claim 43 or 44, wherein the conjugation linker is not cleavable.

46. The antibody fusion molecule of claim 43 or 44, wherein the conjugation linker is cleavable.

47. The antibody fusion molecule of claim 39, wherein the payload molecule is MC-vc- PAB-MMAE or a conjugation product thereof.

48. The antibody fusion molecule of claim 47, wherein the antigen binding moiety specifically binds to GPC3.

49. The antibody fusion molecule of claim 48, wherein the antigen binding moiety comprises an antibody light chain with at least 95% identity to SEQ ID NO: 14, and an antibody heavy chain with at least 95% identity to SEQ ID NO: 32 or SEQ ID NO: 33.

50. The antibody fusion molecule of any one of claims 1-49, wherein the antigen-binding moiety binds to one or more antigens on the surface of a cancer cell.

51. The antibody fusion molecule of claim 50, wherein the cancer cell has upregulated macropinocytosis.

52. The antibody fusion molecule of claim 50 or 51, wherein the cancer cell comprises one or more mutant RAS family genes.

53. A pharmaceutical composition comprising an antibody fusion molecule of any one of claims 39-52, and a pharmaceutically acceptable excipient.

54. A method of treating a cancer in a subject, comprising administering to the subject with cancer an effective amount of the pharmaceutical composition according to claim 53, wherein the payload molecule is a cytotoxic drug.

55. The method of claim 54, wherein the cancer expresses an antigen targeted by the antibody or fragment thereof, and comprises a mutant RAS gene.

56. A method of making an antibody fusion molecule, comprising conjugating a payload molecule to the antibody fusion molecule according to any one of claims 1-55.

57. The method of claim 56, wherein the drug molecule is conjugated to a cysteine residue in Pl, P3, P4, or P5 through a conjugation linker.

58. The method of claim 57, wherein the conjugation linker is attaching to the payload molecule prior to attaching the conjugation linker to the cysteine residue in Pl, P3, P4, or P5.

59. The method of claim 57, wherein the conjugation linker is attached to the antibody fusion molecule prior to attaching the conjugation linker to the payload molecule.

60. The method of any one of claims 56-59, comprising mixing the antibody fusion molecule with a reducing agent prior to conjugating the payload molecule to the fusion molecule.

61. The method of claim 60, comprising mixing the antibody fusion molecule with an oxidizing agent after mixing the antibody fusion molecule with the reducing agent, and prior to conjugating the payload molecule to the fusion molecule.