Chimeric proteins comprising masked il-2

Abstract

Provided herein are IL-2 prodrugs, as well as methods of using the same to modulate the immune system in a subject.

Description

CHIMERIC PROTEINS COMPRISING MASKED IL-2 CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority from PCT / US2023 / 082385, filedDecember 4, 2023, and U.S. Provisional Application No.63 / 722,770, filed on November 20, 2024, the contents of which are incorporated herein by reference in their entirety. BACKGROUND OF THE INVENTION

[0002] Interleukin-2 (IL-2) plays a central role in lymphocyte generation, survival, andhomeostasis. It has 133 amino acids and consists of four antiparallel, amphipathic alpha- helices that form a quaternary structure essential for its function (Smith, Science (1988) 240:1169-76; Bazan, Science (1992) 257:410-13).

[0003] IL-2 exerts its activities by binding to IL-2 receptors (IL-2R), which consist of upto three individual subunits. Association of the α (CD25 or Tac antigen), β (CD122), and γ (γc, common γ chain, or CD132) subunits results in a trimeric, high-affinity receptor for IL-2 (KD ~ 0.01 nM). Dimeric IL-2 receptor consisting of the β and γ subunits is termed intermediate-affinity IL-2R (KD ~ 1 nM). The α subunit alone forms the monomeric low affinity IL-2 receptor (KD ~ 10 nM). See, e.g., Kim et al., Cytokine Growth Factor Rev. (2006) 17:349-66. Although the dimeric intermediate-affinity IL-2 receptor binds IL-2 with approximately 100-fold lower affinity than the trimeric high-affinity receptor, both the dimeric and trimeric IL-2 receptors can transmit signal upon IL-2 binding (Minami et al., Annu Rev Immunol. (1993) 11:245-68). Thus, it appears that the α subunit, while conferring high-affinity binding of the receptor to IL-2, is not essential for IL-2 signaling. However, the β and γ subunits are essential for IL-2 signaling (Krieg et al., Proc Natl Acad Sci. (2010) 107:11906-11). The trimeric IL-2 receptor is expressed by CD4+FoxP3+ regulatory T (Treg) cells. Treg cells consistently express the highest level of IL-2Rα (CD25) in vivo (Fontenot et al., Nature Immunol. (2005) 6:1142-51). The trimeric IL-2 receptor is also transiently induced on conventional activated T cells, whereas in the resting state these cells express only the dimeric IL-2 receptor.

[0004] Mutated versions of IL-2 have been developed to optimize treatment of cancer andautoimmune diseases. IL-2 prodrugs have also been in development, which in generaldepends on protease cleavage for activation. There is a need to develop IL-2 prodrugs which does not need protease cleavage for activation to further increase efficacy of the prodrugs. SUMMARY OF THE INVENTION

[0005] The present disclosure provides a prodrug comprising an IL-2 cytokine moiety, amasking moiety, and a carrier moiety, wherein the IL-2 cytokine moiety comprises SEQ ID NO:2, the masking moiety comprises an scFv specific for the IL-2 cytokine moiety, and the carrier moiety is an antibody against human PD-1, wherein the anti-PD-1 antibody comprises a heavy chain variable domain comprising amino acid residues 1-113 of SEQ ID NO:13 and a light chain variable domain comprising amino acid residues 1-107 of SEQ ID NO:15.

[0006] In some embodiments, the scFv comprises a light chain variable domaincomprising amino acid residues 452-560 of SEQ ID NO:13 and a heavy chain variable domain comprising amino acid residues 584-703 of SEEQ ID NO:13.

[0007] In some embodiments, the prodrug comprises a fusion polypeptide in which thescFv is fused to the C-terminus of a first heavy chain of the anti-PD-1 antibody through a first peptide linker comprising SEQ ID NO:16 and the IL-2 cytokine moiety is fused to the C- terminus of the scFv through a second peptide linker comprising SEQ ID NO:16. In further embodiments, the fusion polypeptide comprises SEQ ID NO:13 or an amino acid sequence at least 95% identical thereto, the second heavy chain of the anti-PD-1 antibody comprises SED ID NO:14 or an amino acid sequence at least 95% identical thereto, and the light chains of the anti-PD-1 antibody each comprise SEQ ID NO:15 or an amino acid sequence at least 95% identical thereto. In certain embodiments, the fusion polypeptide comprises SEQ ID NO:13, the second heavy chain of the anti-PD-1 antibody comprises SED ID NO:14, and the light chains of the anti-PD-1 antibody each comprise SEQ ID NO:15.

[0008] Provided herein also is a chimeric protein comprising a first heavy chainpolypeptide chain, a second heavy chain polypeptide chain, and two identical light chains, wherein the first heavy chain polypeptide chain comprises SEQ ID NO:13 or an amino acid sequence at least 95% identical thereto, the second heavy chain polypeptide chain comprises SEQ ID NO:14 or an amino acid sequence at least 95% identical thereto, and two identical light chains each comprise SEQ ID NO:15 or an amino acid sequence at least 95% identical thereto.

[0009] In another aspect, the present disclosure provides pharmaceutical compositioncomprising the prodrug or chimeric protein herein and a pharmaceutically acceptable excipient.

[0010] In another aspect, the present disclosure provides polynucleotides encoding thepresent prodrug or chimeric protein, expression vectors comprising the polynucleotides, and host cells comprising the expression vector(s). The present disclosure also provides a method of making a prodrug or chimeric protein, comprising culturing the present host cell under conditions that allow expression of the prodrug or chimeric protein, and isolating the expressed prodrug or chimeric protein from the culture.

[0011] In another aspect, the present disclosure provides a method of treating a cancer oran infectious disease or modulating the immune system in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the present prodrug, chimeric protein, or pharmaceutical composition. Also provided are prodrugs, chimeric proteins and pharmaceutical compositions for use in treating a cancer or an infectious disease or modulating the immune system in a patient in need thereof, and use of these prodrugs, chimeric proteins, and pharmaceutical compositions for the manufacture of a medicament for treating a cancer or an infectious disease or modulating the immune system in a patient in need thereof. In some embodiments, the patient has HIV infection; has a cancer selected from the group consisting of 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 such as non- small cell lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer, and medullary thyroid cancer; or an inflammatory or an autoimmune disease, optionally selected from asthma, Type I diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, organ graft rejection, and graft-versus-host disease.

[0012] Other features, objects, and advantages of the invention are apparent in thedetailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0013] FIGs. 1A-1E show schematic illustrations of the structures of antibody-cytokinefusion molecules and the sequences of the polypeptide chains, which form the fusion molecules.

[0014] FIG. 2 shows the results of the in vitro cell based activity assay (HEK BlueReporter Assay).

[0015] FIGs. 3A and 3B show the in vivo efficacy and safety results of the two fusionmolecules (812mN-mut4 and 812mW5-mut4) in comparison with the reference molecule Ref3, and an anti-mouse PD-1 antibody.

[0016] FIG. 4 shows the results of the cell-based activity assay of IL-2 prodrugsASKG812 EC DS and ASKG812N (also termed 812NW5-Mut4 or NW5-Mut4 herein) as compared to un-conjugated human IL-2.

[0017] FIG. 5 shows the activity of ASKG812N in an HEK blue reporter assay from thecynomolgus monkey PK study. In this study, cynomolgus monkey was dosed intravenously with test article ASKG812N at 5 mg / kg at Day 1 and Day 15, and 10 mg / kg at Day 40. Blood was sampled at pre-dose, 0, and 5 min, 2h, 8h, 24h, 48h, 72h, 120h, 168h, 240h, and 336h post-dose. The concentrations of ASKG812N in the serum samples were assayed for test article by ELISA.

[0018] FIG. 6 shows the serum concentrations of ASKG812N and PK parameters incynomolgus monkey.

[0019] FIG. 7 shows the expansion of immune cells induced by ASKG812N.DETAILED DESCRIPTION OF THE INVENTION

[0020] As used herein and in the appended claims, the singular forms “a,” “or,” and “the”include plural referents unless the context clearly dictates otherwise.

[0021] 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.”

[0022] The term “antigen-binding moiety” refers to a polypeptide or a set of interactingpolypeptides that specifically bind to an antigen, and includes, but is not limited to, an antibody (e.g., a monoclonal antibody, polyclonal antibody, a multi-specific antibody, a dualspecific or bispecific antibody, an anti-idiotypic antibody, or a bifunctional hybrid antibody) or an antigen-binding fragment thereof (e.g., a Fab, a Fab’, a F(ab’)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), or a diabody), a single chain antibody, and an Fc-containing polypeptide such as an immunoadhesin or an scFv-Fc. In some embodiments, the antibody may be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibody may be of any light chain isotype (e.g., kappa or lambda). The antibody may be human, non-human (e.g., from mouse, rat, rabbit, goat, or another non-human animal), chimeric (e.g., with a non-human variable region and a human constant region), or humanized (e.g., with non-human CDRs and human framework and constant regions). In some embodiments, the antibody is a derivatized antibody.

[0023] The term “cytokine agonist polypeptide” refers to a wildtype cytokine, or ananalog thereof. An analog of a wildtype cytokine has the same biological specificity (e.g., binding to the same receptor(s) and activating the same target cells) as the wildtype cytokine, although the activity level of the analog may be different from that of the wildtype cytokine. The analog may be, for example, a mutein (i.e., mutated polypeptide) of the wildtype cytokine, and may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten mutations relative to the wildtype cytokine.

[0024] The term “prodrug” herein refers to a cytokine fusion protein that comprises acytokine moiety bound by a masking moiety and has not yet been activated to remove the mask from the cytokine moiety. Once the cytokine moiety is unbound, the fusion molecule becomes “activated.”

[0025] The terms “cytokine antagonist,” “cytokine mask,” and “masking moiety” refer toa moiety (e.g., a polypeptide) that binds to a cytokine and thereby inhibits the cytokine from binding to its receptor on the surface of a target cell and / or exerting its biological functions while being bound by the antagonist or mask. Examples of a cytokine antagonist or mask include, without limitations, a polypeptide derived from an extracellular domain of the cytokine’s natural receptor that makes contact with the cytokine and an antibody that binds to the cytokine or an antigen-binding fragment thereof (e.g., scFv).

[0026] The term “effective amount” or “therapeutically effective amount” refers to anamount 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 cancer, an effective amount may be an amount sufficient todelay cancer development or progression (e.g., decrease tumor growth rate, and / or delay or prevent tumor angiogenesis, metastasis, or infiltration of cancer cells into peripheral organs), reduce the number of epithelioid cells, cause cancer regression (e.g., shrink or eradicate a tumor), and / or prevent or delay cancer occurrence or recurrence. An effective amount can be administered in one or more administrations.

[0027] The term “functional analog” refers to a molecule that has the same biologicalspecificity (e.g., binding to the same ligand) and / or activity (e.g., activating or inhibiting a target cell) as a reference molecule.

[0028] The term “fused” or “fusion” in reference to two polypeptide sequences refers tothe joining of the two polypeptide sequences through a backbone peptide bond. Two polypeptides may be fused directly or through a peptide linker that is one or more amino acids long. A fusion polypeptide may be made by recombinant technology from a coding sequence containing the respective coding sequences for the two fusion partners, with or without a coding sequence for a peptide linker in between. In some embodiments, fusion encompasses chemical conjugation.

[0029] The term “pharmaceutically acceptable excipient” when used to refer to aningredient in a composition means that the excipient is suitable for administration to a treatment subject, including a human subject, without undue deleterious side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).

[0030] The term “subject” refers to a mammal and includes, but is not limited to, ahuman, a pet (e.g., a canine or a feline), a farm animal (e.g., cattle or horse), a rodent, or a primate.

[0031] As used herein, “treatment” or “treating” is an approach for obtaining beneficialor desired clinical results. 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, ameliorating a disease state, stabilizing a disease (e.g., preventing or delaying the worsening or progression of the disease), preventing or delaying the spread (e.g., metastasis) of a disease, preventing or delaying the recurrence of a disease, providing partial or total remission of a disease, decreasing the dose of one or more other medications required to treat a disease, increasing the patient’s quality of life, and / or prolonging survival. The methods of the present disclosure contemplate any one or more of these aspects of treatment.

[0032] It is to be understood that one, some, or all of the properties of the variousembodiments described herein may be combined to form other embodiments of the present invention. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described thereunder. I. Mutant IL-2 Prodrugs

[0033] The present disclosure further provides cytokine prodrugs that are metabolized invivo at a target site to become active cytokine therapeutics. The cytokine prodrugs have fewer side effects (including less immunogenicity risk from the cytokine as compared to the wildtype cytokine), better in vivo PK profiles (e.g., longer half-life) and better target specificity, and are more efficacious as compared to prior cytokine therapeutics. The present prodrugs comprise a cytokine moiety comprising a cytokine agonist polypeptide linked to a carrier moiety and masked (bound) by a cytokine antagonist (masking moiety). The cytokine antagonist may be, for example, an extracellular domain of a receptor for the cytokine, and is linked to the cytokine moiety or to the carrier moiety through a peptide linker (e.g., a cleavable or non-cleavable peptide linker). The mask inhibits the cytokine moiety’s biological functions while the mask is binding to it. The prodrugs may be activated at a target site (e.g., at a tumor site or the surrounding environment) in the patient by cleavage of the linker and the consequent release of the cytokine mask from the prodrug, exposing the previously masked cytokine moiety and allowing the cytokine moiety to bind to its receptor on a target cell and exert its biological functions on the target cell. In other embodiments, when the masking moiety is linked to the carrier moiety through a non-cleavable peptide linker, the prodrugs disclosed herein can engage the target cell via “cis-binding” of a cytokine receptor and an antigen expressed on the cell surface, leading to increased activity of the prodrug without cleavage and removal of the masking moiety. The cytokine moiety of the prodrugs may increase in activity at a target site (e.g., at a tumor site or the surrounding environment), where both the antigen targeted by the carrier and a receptor of the cytokine are expressed on the same cell.

[0034] In some embodiments, the carriers for the prodrugs are antigen-binding moieties,such as antibodies or antigen-binding fragments thereof, that bind an antigen at the target site.

[0035] In some embodiments, the present prodrugs are pro-inflammatory cytokineprodrugs that are metabolized to become pro-inflammatory cytokines at a target site in the body targeted by the carrier moiety. In further embodiments, the carrier moiety in the prodrug is an antibody or antigen-binding fragment thereof targeting a tumor antigen suchthat the prodrug is delivered to a tumor site in a patient and is metabolized locally (e.g., inside or in the vicinity of the tumor microenvironment) through cleavage of the linker linking the cytokine mask to the carrier moiety or the cytokine moiety, making the pro-inflammatory cytokine moiety available to interact with its receptor on a target cell and stimulating the target immune cells locally.

[0036] An IL-2 prodrug may comprise a cytokine moiety comprising an IL-2 agonistpolypeptide, a carrier moiety, and a masking moiety (an IL-2 antagonist), wherein the cytokine moiety is fused to the carrier moiety directly or through a linker (e.g., cleavable or non-cleavable peptide linker), and the IL-2 antagonist is linked to the IL-2 agonist polypeptide or to the carrier moiety through a cleavable peptide linker. In the present IL-2 prodrugs, the IL-2 agonist polypeptide may be an IL-2 mutein such as an IL-2 mutein described herein derived from a human IL-2. The IL-2 mutein may have significantly reduced affinity for CD25 or the trimeric high-affinity IL-2R, as compared to wildtype IL-2. In some embodiments, the IL-2 mutein has binding affinity for the high-affinity IL-2R that is 100 times, 300 times, 500 times, 1,000 times, or 10,000 times lower as compared to wildtype IL-2. Unless otherwise indicated, all residue numbers in IL-2 and IL-2 muteins described herein are in accordance with the numbering in SEQ ID NO:1. In some embodiments, the IL- 2 agonist is an IL-2 mutein comprising SEQ ID NO:2.

[0037] The cytokine antagonist, i.e., the masking moiety, in the present immunoconjugatemay comprise a peptide, an antibody, or antibody fragment that binds to the cytokine moiety in the prodrug, thereby masking the cytokine moiety and inhibiting its biological functions. In some embodiments, the prodrug comprises a masking moiety, wherein the masking moiety binds to a mutant IL-2 polypeptide disclosed herein and inhibits a biological activity of the mutant IL-2 polypeptide.

[0038] By way of example, IL-2 antagonists may comprise peptides and antibodies thatbind IL-2 and interfere with the binding of the IL-2 moiety to its receptors, leading to the reduced biological activities of the IL-2 moiety while masked. In some embodiments, the IL- 2 antagonist comprises an IL-2Rβ or IL-2Rγ extracellular domain or its functional analog such as one derived from human IL-2Rβ or IL-2Rγ. In some embodiments, the IL-2 antagonist comprises a peptide identified from the screening of a peptide library. In some embodiments, the masking moiety comprises an antibody or an antigen-binding fragment thereof. In particular embodiments, the IL-2 antagonist comprises an antibody or fragment (e.g., scFv) thereof that blocks the binding of IL-2 or IL-2 muteins to an IL-2 receptor. In some embodiments, the scFv comprises a light chain variable domain comprising amino acidresidues 452-560 of SEQ ID NO:13 and a heavy chain variable domain comprising amino acid residues 584-703 of SEEQ ID NO:13.

[0039] In some other embodiments, the prodrug further comprises a peptide linker,wherein the peptide linker links the masking moiety to the carrier. In some other embodiments, the peptide linker links the mutant IL-2 polypeptide to the carrier moiety.

[0040] The carrier moiety of the present prodrugs may be an antigen-binding moiety, or amoiety that does not bind an antigen. The carrier moiety may improve the PK profiles such as serum half-life of the cytokine agonist polypeptide, and may also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site.

[0041] The carrier moiety may be an antibody or an antigen-binding fragment thereof, oran immunoadhesin. In some embodiments, the antigen-binding moiety is a full-length antibody with two heavy chains and two light chains, a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment, a Fv fragment, a disulfide linked Fv fragment, a single domain antibody, a nanobody, or a single-chain variable fragment (scFv). In some embodiments, the antigen- binding moiety is a bispecific antigen-binding moiety and can bind to two different antigens or two different epitopes on the same antigen. The antigen-binding moiety may provide additional and potentially synergetic therapeutic efficacy to the cytokine agonist polypeptide.

[0042] The cytokine agonist polypeptide and its mask may be fused to the N-terminus orC-terminus of the light chains and / or heavy chains of the antigen-binding moiety. By way of example, the cytokine agonist polypeptide and its mask may be fused to a heavy chain of an antibody or an antigen-binding fragment thereof. In some embodiments, the cytokine agonist polypeptide and its mask may be fused to the light chain of an antibody or an antigen-binding fragment thereof. In some embodiments, the cytokine agonist polypeptide is fused to the C- terminus of one or both of the heavy chains of an antibody, and the cytokine’s mask (masking moiety) is fused to the C-terminus of the cytokine agonist polypeptide through a cleavable or non-cleavable peptide linker. In some embodiments, the cytokine agonist polypeptide is fused to the C-terminus of one of the heavy chains of an antibody, and the cytokine’s mask is fused to the C-terminus of the other heavy chain of the antibody through a cleavable or non- cleavable peptide linker, wherein the two heavy chains contain mutations that allow their specific pairing.

[0043] In some embodiments, the PD-1-binding moiety includes an antibody or fragmentthereof known in the art that binds to PD-1 and disrupts the interaction between the PD-1 and its ligand (PD-L1) to stimulate an anti-tumor immune response. In some embodiments, the antibody or antigen-binding portion thereof binds specifically to PD-1. In someembodiments, the antigen-binding moiety is an anti-PD1 antibody. In some embodiments, the anti-PD-1 antibody is pembrolizumab or nivolumab.

[0044] The IL-2 agonist polypeptide may be fused to the carrier moiety with or without apeptide linker. The peptide linker may be non-cleavable. In particular embodiments, the peptide linker are G / S-rich linkers (i.e., 50% or more amino acids of the linker are glycine and / or serine (e.g., linkers comprising 1, 2, 3, 4, or 5 repeats of a motif having four consecutive glycine residues following by a serine). In some embodiments, the peptide linker comprises a motif of G4S (SEQ ID NO:16). In further embodiments, the peptide linker comprises SEQ ID NO:17.

[0045] The prodrugs of the present disclosure may be made by well-known recombinanttechnology. For example, one or more expression vectors comprising the coding sequences for the polypeptide chains of the prodrugs may be transfected into mammalian host cells (e.g., CHO cells), and cells are cultured under conditions that allow the expression of the coding sequences and the assembly of the expressed polypeptides into the prodrug complex. II. Pharmaceutical Compositions

[0046] Pharmaceutical compositions comprising the prodrugs and muteins (i.e., the activepharmaceutical ingredient or API) of the present disclosure may be prepared by mixing the API having the desired degree of purity with one or more optional pharmaceutically acceptable excipients (see, e.g., Remington's Pharmaceutical Sciences, 16th Edition., Osol, A. Ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable excipients (or carriers) are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers containing, for example, phosphate, citrate, succinate, histidine, acetate, or another inorganic or organic acid or salt thereof; 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 sucrose, 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).

[0047] Buffers are used to control the pH in a range which optimizes the therapeuticeffectiveness, 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, and acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

[0048] Preservatives are added to retard microbial growth, and are typically present in arange 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.

[0049] Tonicity agents, sometimes known as “stabilizers” are present to adjust ormaintain 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, considering 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.

[0050] Non-ionic surfactants or detergents (also known as “wetting agents”) are presentto 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.

[0051] 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 beused include sodium lauryl sulfate, dioctyle sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0052] The choice of pharmaceutical carrier, excipient or diluent may be selected withregard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may additionally comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s).

[0053] There may be different composition / formulation requirements dependent on thedifferent 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.

[0054] In some embodiments, the pharmaceutical composition of the present disclosure isa lyophilized protein formulation. In other embodiments, the pharmaceutical composition may be an aqueous liquid formulation. III. Methods of Treatment

[0055] The prodrugs of the present invention can be used to treat a disease. In someembodiments, the prodrugs are used to treat cancer. In some embodiments, the prodrugs are used to treat an infection, for example, when the drug molecule is an antibacterial agent or an antiviral agent.

[0056] In some embodiments, the method of treating a disease (such as cancer, a viralinfection, or a bacterial infection) in a subject comprises administering to the subject an effective amount of the prodrugs disclosed herein.

[0057] In some embodiments, the cancer is a solid cancer. In some embodiments, thecancer is a blood cancer or a solid tumor. 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 such as non-small cell lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer, and medullary thyroid cancer.

[0058] In some embodiments, the prodrugs are used to treat a bacterial infection such assepsis. In some embodiments, the bacteria causing the bacterial infection are drug-resistantbacteria. In some embodiments, the antigen-binding moiety (carrier moiety) disclosed herein binds to a bacterial antigen.

[0059] In some embodiments, the prodrugs are used to treat a viral infection. In someembodiments, 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 disclosed herein binds to a viral antigen.

[0060] Generally, dosages and routes of administration of the present pharmaceuticalcompositions are determined according to the weight and conditions of the subject, according to standard pharmaceutical practice. In some embodiments, the pharmaceutical composition 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, intracranially, or intraspinally. In some embodiments, the composition is administered to a subject intravenously.

[0061] In some embodiments, the dosage of the pharmaceutical composition is a singledose 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 pharmaceutical composition 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, 3, 4, 5, 7, 10, 15, or 20 or more doses).

[0062] Unless otherwise defined herein, scientific and technical terms used in connectionwith the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group ofintegers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context.

[0063] According to the present disclosure, back-references in the dependent claims aremeant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0064] In order that this invention may be better understood, the following examples areset forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner. EXAMPLES Example 1: In Vivo Efficacy Study with Colon 26 Syngeneic Tumor Model

[0065] An in vivo efficacy study was carried out with the Colon 26 syngeneic tumormodel. Briefly, female 7 to 8 week old Balb / C mice were implanted with 5 x 105Colon 26 tumor cells delivered subcutaneously over the abdomen. When mean tumor volume reached approximately 82 mm3, mice were randomized into treatment groups such that each group had approximately the same mean tumor volume. Mice were treated with test article or vehicle by intraperitoneal injection in 100 µL of PBS given 3 days apart for a total of two treatments. Tumor volume was determined by measuring the longest (a) and shortest (b) diameter and calculating the volume using the formula = ab2π / 6.

[0066] Fusion molecule 812mN-mut4 comprises two identical light chains of an anti-mouse PD-1 antibody, and two identical heavy chain polypeptide chains; wherein the heavy chain polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain of the anti-mouse PD-1 antibody, a mask which binds to IL-2, and an IL-2 mutein. The structure of this molecule is illustrated in FIG.1A, and its sequence information is shown in FIG.1D.

[0067] Fusion molecule 812mW5-mut4 has the structure illustrated in FIG. 1B. It issimilar to 812mN-mut4 except that it comprises only one mask and one IL-2 mutein at the C- terminus of the heavy chain of the anti-mouse PD-1 antibody.

[0068] Ref3 is an anti-PD-1 antibody-IL-2 mutein fusion molecule, which has a structureas illustrated in FIG.1C. It is a mouse analog of eciskafusp alfa which is currently in clinical development.

[0069] All three molecules have the same anti-PD-1 antibody as that of mPD-1 molecule.812mN-mut4 comprises two copies of the masked IL-2 mutein. The human version (i.e., with anti-human PD-1 antibody, 812KN-mut4) of the fusion molecule 812mN-mut4 showed stronger in vitro cell-based biological activity than that of the human version (812KW5- mut4) of 812mW5-mut4, which comprises only one copy of the same masked IL-2 mutein (FIG.2). The IL-2 mutein in Ref3 is not masked. It has the strongest in vitro cell-based IL-2 activity among the three molecules (FIG.2). Hence, 812KW5-mut4 has weaker activity than 812KN-mut4 and the reference molecule Ref3. The IL-2 moieties are the same between the human versions and the mouse versions.

[0070] The in vivo efficacy and safety results of the two chimeric molecules, Ref3, andthe anti-mouse PD-1 antibody are shown in FIG.3A and FIG.3B. Surprisingly, 812mW5- mut4 has similar anti-tumor efficacy as that of Ref3 (FIG.3A), yet 812mW5-mut4 is safer than Ref3 as there was no body weight drop for 812mW5-mut4, but significant body weight loss was observed with Ref3 (FIG.3B). It was also surprising that 812mW5-mut4 was observed to have stronger in vivo efficacy than that of 812mN-mut4, as the latter was expected to have stronger in vitro cell-based activity. Example 2: Cell-Based Bio-Assay

[0071] HEK-Blue™ IL-2 cells were generated by stable transfection of HEK 293 cellswith the human CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ) genes, along with the human JAK3 and STAT5 genes. STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene was also introduced. The HEK Blue IL-2 reporter cell line was maintained at sub-confluent density in assay medium containing 1:250 dilution of HEK-Blue CLR selection and 1 µg / mL puromycin. Cells were harvested when reaching adherence, using Cellstripper™ (Corning) to detach. Serial dilutions of test articles were performed in 96-well tissue culture plates in 50 µL / well assay medium. Cells were counted and adjusted to a concentration to 600,000 cells / mL. 50 µL / well (30,000 cells / well) were dispensed into assay plates containing sample dilutions. The assay plates were incubatedovernight at 37oC in a CO2incubator. 20 µL of supernatant was transferred then from the assay plates to ELISA plates. 180 µL QUANTI-Blue™ reagent (InvivoGen) was added to the plates and the plates were incubated at 37oC for 1 hour. OD650 was measured using a microplate spectrophotometer. Absorbance data were analyzed using GraphPad Prism software, perform non-linear regression analysis (4-parameter) to determine EC50.

[0072] FIG. 4 shows the results of the cell-based activity assay. The data show thatASKG812N (i.e., NW5-Mut4) was active but had reduced activity compared to the wild type IL-2 and ASKG812 EC. ASKG812N comprises a first heavy chain polypeptide chain of SEQ ID NO:13, a second heavy chain polypeptide chain of SEQ ID NO:14, and two identical light chains of SEQ ID NO:15. ASKG812 EC comprises a first heavy chain polypeptide chain of SEQ ID NO:18, a second heavy chain polypeptide chain of SEQ ID NO:19, and two identical light chains of SEQ ID NO:20. Example 3: Cynomolgus Monkey PK and PD Study

[0073] One male cynomolgus monkey was dosed intravenously with test articleASKG812N at 5 mg / kg at Day 1 and Day 15, and 10 mg / kg at Day 40. Blood was sampled at pre-dose, 0, 5 min, 2 h, 8 h, 24 h, 48 h, 72 h, 120 h, 168 h, 240 h, and 336 h post-dose. No body weight loss and no overt toxicity was observed, suggesting that ASKG812N was well tolerated at up to 10 mg / kg in monkey.

[0074] For PK analysis, serum samples were assayed for test article by ELISA. Briefly,ELISA plates were coated with 100 µL / well PD -1 (ACRO, Cat. #PD1-H5221) at 1 µg / mL in PBS. Plates were blocked with 100 µl / well of 3% BSA. After 2 hours of incubation and subsequent wash (four times with PBST), 100 µL of serum samples diluted in 1% BSA or standard was added to each well (1:100). After incubation of 1 hour and wash (3 times with PBST), 100 µL of anti-human IgG HRP (Jackson ImmunoResearch, Cat. #109-036- 006,1:10000) in1% BSA was added to each well. After incubation (1 hour) and wash (3 times with PBST). The color reaction was started by adding 100 µL of the TMB substrate to each well. The reaction was stopped with the addition of 100 µL / well of 1 N H2SO4solution. OD450 was then measured.

[0075] For PD analysis, the population of CD4+ T cells, CD8+ T cells, Treg cells, NKcells, and NKT cells were measured. For Immune cells expansion and proliferation analysis, whole blood samples were assayed for test article by FACS. Briefly, 100 μL whole blood sample was incubated with 5 μL Fc receptor blocker (BD, Cat. # 564220) for 5 min at RT.Then the following antibodies or reagent were added: Live / Dead stain (Invitrogen, Cat. # L34957, 1:1000 dilution), PerCP-Cy5.5 Anti-CD3 (BD, Cat. # 552852, 5 μL / sample), FITC Anti-CD4 (BD, Cat. # 550628, 5 μL / sample), APC / Cy7 anti-CD8 (BD, Cat. # 557760 , 1.5 μL / sample), BV421 anti-CD56 (Biolegend, Cat. # 318328 , 1.5 μL / sample), BV605 anti- CD25 (Biolegend, Cat. # 356142 , 2 μL / sample). Samples were incubated at 4℃ for 25 min in dark and then lysed by red blood cells lysis buffer (BD, Cat. # 555899). Samples were washed by PBS + 1% FBS buffer twice and then fixated and permeabilized by Fixation / Permeabilization kit (Invitrogen, Cat. # 00-5523-00). After two washes with permeabilization buffer, APC anti-Foxp3 (Invitrogen, Cat. # 17-4776-42, 1 μL / sample) and PE anti-Ki67 (Invitrogen, Cat. # 12-5698-82, 0.025 μL / sample) were added. Samples were incubated at 4℃ for 25 min in dark and then washed twice by permeabilization buffer, detected by CytoFlex FACS machine and analysed by CytExpert software. The CD4+ T, CD8+ T, Treg, NK and NKT cell numbers were calculated by Volume-Method.

[0076] FIG. 5 shows the PK of ASKG812N in cynomolgus monkey, while FIG. 6 showsthe PK parameters and serum concentrations of ASKG812N in cynomolgus monkey. The results show that ASKG812N had a half-life of approximately 40 hours after the first dose in cynomolgus monkey. The fusion protein exhibited good serum concentration after the second and third doses, suggesting minimum or low risk of immunogenicity in monkey.

[0077] FIG. 7 shows that ASKG812N significantly expanded CD4+ and CD8+ T cells,and Treg cells in cynomolgus monkey. It also expanded NK cells, though to a lesser extent. Those results demonstrate that ASKG812N was active in stimulating the immune system and preferentially expanded the T cells over NK cells.SEQUENCES

[0078] Sequences described in the present disclosure are shown below.SEQ ID NO:1 – human IL-2 APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT SEQ ID NO:2 – human IL-2 Mut4 (T3A / L36I / R38S / C125A) APASSSTKKT QLQLEHLLLD LQMILNGINN YKNPKITSML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFAQSIIS TLT SEQ ID NO:3 - mPD1-mIgG2-L234A / L2356A / P329G CX11_158_3 EVQLVESGGG LVQPGGSLKL SCAASGFTFS NSGLAWVRQA PEKGLEWVAT ITYNGTSTYY RDSVKGRFTI SRDNAKNTLY LQMSSLRSED TATYYCARWV PGSGNFDYWG QGTLVTVSSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRG PTIKPCPPCK CPAPNAAGGP SVFIFPPKIK DVLMISLSPI VTCVVVDVSE DDPDVQISWF VNNVEVHTAQ TQTHREDYNS TLRVVSALPI QHQDWMSGKE FKCKVNNKDL GAPIERTISK PKGSVRAPQV YVLPPPEEEM TKKQVTLTCM VTDFMPEDIY VEWTNNGKTE LNYKNTEPVL DSDGSYFMYS KLRVEKKNWV ERNSYSCSVV HEGLHNHHTT KSFSRTPGK SEQ ID NO:4 - mDX400-LC CX11_154_1 DIVLTQSPAS LAVSLGQRAT ISCRASQSVT ISRYTLMHWY QQKPGQPPKL LIYRASNLAS GIPARFSGSG SGTDFTLNIH PVEEDDAATY YCQQSRESPW TFGGGTKLEI KRADAAPTVS IFPPSSEQLT SGGASVVCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS STLTLTKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC SEQ ID NO:5 - mPD1-mIgG2-L234A / L2356A / P329G, K409E / K439D IL-2vR ASKG812_CX15_154_2 EVQLVESGGG LVQPGGSLKL SCAASGFTFS NSGLAWVRQA PEKGLEWVAT ITYNGTSTYY RDSVKGRFTI SRDNAKNTLY LQMSSLRSED TATYYCARWV PGSGNFDYWG QGTLVTVSSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRG PTIKPCPPCK CPAPNAAGGP SVFIFPPKIK DVLMISLSPI VTCVVVDVSE DDPDVQISWF VNNVEVHTAQ TQTHREDYNS TLRVVSALPI QHQDWMSGKE FKCKVNNKDL GAPIERTISK PKGSVRAPQV YVLPPPEEEM TKKQVTLTCM VTDFMPEDIY VEWTNNGKTE LNYKNTEPVL DSDGSYFMYS ELRVEKKNWV ERNSYSCSVV HEGLHNHHTT DSFSRTPGGG GGSGGGGSGG GGSAPASSST KKTQLQLEHL LLDLQMILNG INNYKNPKLT RMLTAKFAMP KKATELKHLQ CLEEELKPLE EVLNGAQSKN FHLRPRDLIS NINVIVLELK GSETTFMCEY ADETATIVEF LNRWITFAQS IISTLT SEQ ID NO:6 - mPD1-mIgG2-L234A / L2356A / P329G, E356K / D399KCX11_158_2 EVQLVESGGG LVQPGGSLKL SCAASGFTFS NSGLAWVRQA PEKGLEWVAT ITYNGTSTYY RDSVKGRFTI SRDNAKNTLY LQMSSLRSED TATYYCARWV PGSGNFDYWG QGTLVTVSSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRG PTIKPCPPCK CPAPNAAGGP SVFIFPPKIK DVLMISLSPI VTCVVVDVSE DDPDVQISWF VNNVEVHTAQ TQTHREDYNS TLRVVSALPI QHQDWMSGKE FKCKVNNKDL GAPIERTISK PKGSVRAPQV YVLPPPEKEM TKKQVTLTCM VTDFMPEDIY VEWTNNGKTE LNYKNTEPVL KSDGSYFMYS KLRVEKKNWV ERNSYSCSVV HEGLHNHHTT KSFSRTP SEQ ID NO:7 - CX21_87_4 EVQLVESGGG LVQPGGSLKL SCAASGFTFS NSGLAWVRQA PEKGLEWVAT ITYNGTSTYY RDSVKGRFTI SRDNAKNTLY LQMSSLRSED TATYYCARWV PGSGNFDYWG QGTLVTVSSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRG PTIKPCPPCK CPAPNAAGGP SVFIFPPKIK DVLMISLSPI VTCVVVDVSE DDPDVQISWF VNNVEVHTAQ TQTHREDYNS TLRVVSALPI QHQDWMSGKE FKCKVNNKDL GAPIERTISK PKGSVRAPQV YVLPPPEEEM TKKQVTLTCM VTDFMPEDIY VEWTNNGKTE LNYKNTEPVL DSDGSYFMYS KLRVEKKNWV ERNSYSCSVV HEGLHNHHTT KSFSRTPGGG GGSGGGGSEI VLTQSPGTLS LSPGERATLS CRASQSVSSS YLAWYQQKPG QAPRLLIYDA SSRATGIPDR FSGSGSGTDF TLTISRLEPE DFAVYYCQQY GSSFPWTFGQ GTKVEIKSGG GGSGGGGSAA GGGGSGGGGS QVQLVESGGG VVQPGRSLRL SCAASGFTFS TYGMHWVRQA PGKGLEWVAV ISHEGNNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDW FYGRARFDPW GQGTLVTVSS GGGGSGGGGS AAGGGGSGGG GSAPASSSTK KTQLQLEHLL LDLQMILNGI NNYKNPKITS MLTFKFYMPK KATELKHLQC LEEELKPLEE VLNLAQSKNF HLRPRDLISN INVIVLELKG SETTFMCEYA DETATIVEFL NRWITFAQSI ISTLT SEQ ID NO:8 - mPD1-mIgG2-L234A / L2356A / P329G, K409E / K439D-F7- IL-2v- mut4 ASKG812_CX21_132_2 EVQLVESGGG LVQPGGSLKL SCAASGFTFS NSGLAWVRQA PEKGLEWVAT ITYNGTSTYY RDSVKGRFTI SRDNAKNTLY LQMSSLRSED TATYYCARWV PGSGNFDYWG QGTLVTVSSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRG PTIKPCPPCK CPAPNAAGGP SVFIFPPKIK DVLMISLSPI VTCVVVDVSE DDPDVQISWF VNNVEVHTAQ TQTHREDYNS TLRVVSALPI QHQDWMSGKE FKCKVNNKDL GAPIERTISK PKGSVRAPQV YVLPPPEEEM TKKQVTLTCM VTDFMPEDIY VEWTNNGKTE LNYKNTEPVL DSDGSYFMYS ELRVEKKNWV ERNSYSCSVV HEGLHNHHTT DSFSRTPGGG GSGGGGSEIV LTQSPGTLSL SPGERATLSC RASQSVSSSY LAWYQQKPGQ APRLLIYDAS SRATGIPDRF SGSGSGTDFT LTISRLEPED FAVYYCQQYG SSFPWTFGQG TKVEIKSGGG GSGGGGSAAG GGGSGGGGSQ VQLVESGGGV VQPGRSLRLS CAASGFTFST YGMHWVRQAP GKGLEWVAVI SHEGNNKYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARDWF YGRARFDPWG QGTLVTVSSG GGGSGGGGSA AGGGGSGGGG SAPASSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKITSM LTFKFYMPKK ATELKHLQCL EEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN RWITFAQSII STLT SEQ ID NO:9 - mPD1-mIgG2-L234A / L2356A / P329G, E356K / D399K CX11_158_2EVQLVESGGG LVQPGGSLKL SCAASGFTFS NSGLAWVRQA PEKGLEWVAT ITYNGTSTYY RDSVKGRFTI SRDNAKNTLY LQMSSLRSED TATYYCARWV PGSGNFDYWG QGTLVTVSSA KTTAPSVYPL APVCGDTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRG PTIKPCPPCK CPAPNAAGGP SVFIFPPKIK DVLMISLSPI VTCVVVDVSE DDPDVQISWF VNNVEVHTAQ TQTHREDYNS TLRVVSALPI QHQDWMSGKE FKCKVNNKDL GAPIERTISK PKGSVRAPQV YVLPPPEKEM TKKQVTLTCM VTDFMPEDIY VEWTNNGKTE LNYKNTEPVL KSDGSYFMYS KLRVEKKNWV ERNSYSCSVV HEGLHNHHTT KSFSRTP SEQ ID NO:10 - ASKG812K_CX21_134_4 QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPCREE MTKNQVSLWC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGG GGSGGGGSEI VLTQSPGTLS LSPGERATLS CRASQSVSSS YLAWYQQKPG QAPRLLIYDA SSRATGIPDR FSGSGSGTDF TLTISRLEPE DFAVYYCQQY GSSFPWTFGQ GTKVEIKSGG GGSGGGGSAA GGGGSGGGGS QVQLVESGGG VVQPGRSLRL SCAASGFTFS TYGMHWVRQA PGKGLEWVAV ISHEGNNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDW FYGRARFDPW GQGTLVTVSS GGGGSGGGGS AAGGGGSGGG GSAPASSSTK KTQLQLEHLL LDLQMILNGI NNYKNPKITS MLTFKFYMPK KATELKHLQC LEEELKPLEE VLNLAQSKNF HLRPRDLISN INVIVLELKG SETTFMCEYA DETATIVEFL NRWITFAQSI ISTLT SEQ ID NO:11 - 812K-HC L234A,L235A,G237A, hole ASKG812K_CX13_152_5 QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW GQGTTVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPEAAGA PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VCTLPPSREE MTKNQVSLSC AVKGFYPSDI AVEWESNGQP ENNYKTTPPVLDSDGSFFLV SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK SEQ ID NO:12 - ASKG812K_CX13_157_4 EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC* SEQ ID NO:13 - ASKG812N-HC knob CX22_31_1 QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSSVVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEA AGAPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPC REEMTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGGGGSGGGG SEIVLTQSPG TLSLSPGERA TLSCRASQSV SSSYLAWYQQ KPGQAPRLLI YDASSRATGI PDRFSGSGSG TDFTLTISRL EPEDFAVYYC QQYGSSFPWT FGQGTKVEIK SGGGGSGGGG SAAGGGGSGG GGSQVQLVES GGGVVQPGRS LRLSCAASGF TFSTYGMHWV RQAPGKGLEW VAVISHEGNN KYYADSVKGR FTISRDNSKN TLYLQMNSLR AEDTAVYYCA RDWFYGRARF DPWGQGTLVT VSSGGGGSGG GGSAAGGGGS GGGGSAPASS STKKTQLQLE HLLLDLQMIL NGINNYKNPK ITSMLTFKFY MPKKATELKH LQCLEEELKP LEEVLNLAQS KNFHLRPRDL ISNINVIVLE LKGSETTFMC EYADETATIV EFLNRWITFA QSIISTLT SEQ ID NO:14 - 812N-HC L234A,L235A,G237A, hole CX22_31_2 QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEA AGAPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVCTLPPS REEMTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK SEQ ID NO:15 – PD1-Nivolumab-LC CX3.73.3 EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC SEQ ID NO:16 – Peptide linker GGGGS SEQ ID NO:17 – Peptide Linker (GGGGS)n, where n= 1, 2, 3, 4, or 5 SEQ ID NO:18812E-PD1 L234A,L235A,G237A -IL2v / L36I knob-2xG4S ASKG812E_CX15_19_1 QVQLVQSGAE VKKPGSSVKV SCKASGFTFT TYYISWVRQA PGQGLEYLGY INMGSGGTNY NEKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCAIIG YFDYWGQGTM VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAP EAAGAPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PCREEMTKNQ VSLWCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPGGGGSGG GGSAPASSST KKTQLQLEHL LLDLQMILNG INNYKNPKIT SMLTAKFAMPKKATELKHLQ CLEEALKPLE EVLNLAQSKN FHLRPRDLIS NINVIVLELK GSETTFMCEY ADETATIVEF LNRWITFAQS IISTLT SEQ ID NO:19 – 812E-HC L234A,L235A,G237A, hole ASKG812E_CX15_1_6 QVQLVQSGAE VKKPGSSVKV SCKASGFTFT TYYISWVRQA PGQGLEYLGY INMGSGGTNY NEKFKGRVTI TADKSTSTAY MELSSLRSED TAVYYCAIIG YFDYWGQGTM VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE PVTVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDK THTCPPCPAP EAAGAPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVCTLP PSREEMTKNQ VSLSCAVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK SEQ ID NO:20 – ASKG812E_CX15_5_1 DVVMTQSPLS LPVTLGQPAS ISCRSSQSLL DSDGGTYLYW FQQRPGQSPR RLIYLVSTLG SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCMQLTHWP YTFGQGTKLE IKRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ SGNSQESVTE QDSKDSTYSL SSTLTLSKAD YEKHKVYACE VTHQGLSSPV TKSFNRGEC

Claims

CLAIMS1. A prodrug comprising an IL-2 cytokine moiety, a masking moiety, and a carriermoiety, wherein the IL-2 cytokine moiety comprises SEQ ID NO:2, the masking moiety comprises an scFv specific for the IL-2 cytokine moiety, and the carrier moiety is an antibody against human PD-1, wherein the anti-PD-1 antibody comprises a heavy chain variable domain comprising amino acid residues 1-113 of SEQ ID NO:13 and a light chain variable domain comprising amino acid residues 1-107 of SEQ ID NO:15.

2. The prodrug of claim 1, wherein the scFv comprises a light chain variable domaincomprising amino acid residues 452-560 of SEQ ID NO:13 and a heavy chain variable domain comprising amino acid residues 584-703 of SEEQ ID NO:13.

3. The prodrug of claim 1 or 2, comprising a fusion polypeptide in which the scFv isfused to the C-terminus of a first heavy chain of the anti-PD-1 antibody through a first peptide linker comprising SEQ ID NO:16 and the IL-2 cytokine moiety is fused to the C- terminus of the scFv through a second peptide linker comprising SEQ ID NO:16.

4. The prodrug of claim 3, whereinthe fusion polypeptide comprises SEQ ID NO:13 or an amino acid sequence at least 95% identical thereto, the second heavy chain of the anti-PD-1 antibody comprises SED ID NO:14 or an amino acid sequence at least 95% identical thereto, and the light chains of the anti-PD-1 antibody each comprise SEQ ID NO:15 or an amino acid sequence at least 95% identical thereto.

5. The prodrug of claim 4, whereinthe fusion polypeptide comprises SEQ ID NO:13, the second heavy chain of the anti-PD-1 antibody comprises SED ID NO:14, and the light chains of the anti-PD-1 antibody each comprise SEQ ID NO:15.

6. A chimeric protein comprising a first heavy chain polypeptide chain, a second heavychain polypeptide chain, and two identical light chains, wherein: the first heavy chain polypeptide chain comprises SEQ ID NO:13 or an amino acid sequence at least 95% identical thereto, the second heavy chain polypeptide chain comprises SEQ ID NO:14 or an amino acid sequence at least 95% identical thereto, and two identical light chains each comprise SEQ ID NO:15 or an amino acid sequence at least 95% identical thereto.

7. A pharmaceutical composition comprising the prodrug or chimeric protein of any oneof claims 1-6 and a pharmaceutically acceptable excipient.

8. One or more polynucleotides encoding the prodrug or chimeric protein of any one ofclaims 1-6.

9. One or more expression vectors comprising the polynucleotide(s) of claim 8.

10. A host cell comprising the expression vector(s) of claim 9.

11. A method of making a prodrug or chimeric protein, comprising:culturing the host cell of claim 10 under conditions that allow expression of the prodrug or chimeric protein, and isolating the expressed prodrug or chimeric protein from the culture.

12. A method of treating a cancer or an infectious disease or modulating the immunesystem in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the prodrug or chimeric protein of any one of claims 1-6, or the pharmaceutical composition of claim 7.

13. The prodrug or chimeric protein of any one of claims 1-6, or the pharmaceuticalcomposition of claim 7 for use in treating a cancer or an infectious disease or modulating the immune system in a patient in need thereof.

14. Use of the prodrug or chimeric protein of any one of claims 1-6, or the pharmaceuticalcomposition of claim 7 for the manufacture of a medicament for treating a cancer or an infectious disease or modulating the immune system in a patient in need thereof.

15. The method of claim 12, the prodrug, chimeric protein, or pharmaceuticalcomposition of claim 13, or the use of claim 14, wherein the patient has HIV infection, has a cancer selected from the group consisting of 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 such as non-small cell lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer, and medullary thyroid cancer, or an inflammatory or an autoimmune disease, optionally selected from asthma, Type I diabetes, rheumatoid arthritis, allergy, systemic lupus erythematosus, organ graft rejection, and graft-versus-host disease.

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