Modified IL-12 and IL-23 polypeptides and their use

Abstract

To provide compositions and methods for modulating signal transduction mediated by IL-12 and IL-23. [Solution] This disclosure generally relates to compositions and methods for modulating interleukin 12 and interleukin 23-mediated signaling. In particular, this disclosure provides novel variants of interleukin 12 subunit p40 having reduced binding affinity to IL-12Rβ1. Also provided are compositions and methods useful for producing such IL-12p40 polypeptide variants, as well as methods for modulating IL-12p40-mediated signaling and / or methods for treating conditions associated with disruption of IL-12p40-mediated signaling.

Description

[Technical Field]

[0001] Statement on federally supported research and development This invention was made with government support under contracts AI051321 and CA177684, awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 011,742, filed April 17, 2020, and U.S. Provisional Patent Application No. 63 / 150,451, filed February 17, 2021. The disclosures of the applications referenced above, including any drawings, are expressly incorporated herein by reference in their entirety.

[0003] Importing sequence lists The attached sequence listing document is incorporated into this application by reference. The accompanying sequence listing text file, named 078430-517001WO-Sequence Listing.txt, was created on April 12, 2021, and is 76.5KB in size.

[0004] field This disclosure relates in general to compositions and methods for modulating IL-12 and IL-23-mediated signaling. In particular, this disclosure provides novel IL-12p40 polypeptide variants having reduced binding affinity to IL-12Rβ1. Also provided are compositions and methods useful for producing such IL-12p40 polypeptide variants, as well as methods for modulating IL-12p40-mediated signaling and / or for treating conditions associated with disruption of IL-12p40-mediated signaling. [Background technology]

[0005] background The use of biopharmaceuticals or pharmaceutical formulations containing therapeutic proteins for the treatment of health conditions and diseases is a central strategy for many pharmaceutical and biotechnology companies. For example, several members of the cytokine family have been reported to be effective in treating cancer and are playing a major role in the development of cancer immunotherapy. Therefore, cytokine families are the focus of much clinical research and effort to improve their administration and bioanabolism.

[0006] Interleukin-12 (IL-12) and interleukin-23 (IL-23), cytokines of the IL-12 family, have become among the most promising targets in cancer immunotherapy and autoimmune diseases, respectively. While the IL-12 and IL-23 complexes share the IL-12p40 cytokine subunit and the cell surface receptor IL-12 receptor beta-1 (IL-12Rβ1), they induce different downstream signaling pathways. Specifically, IL-12 signals via the IL-12Rβ1 and IL-12Rβ2 receptor complex to induce STAT4 phosphorylation in both NK cells and activated T cells. STAT4 signaling leads to interferon-gamma (IFNγ) expression and enhanced tumor cell killing. In contrast, IL-23 signals via the IL-12Rβ1 and IL-23R receptor complex to promote STAT3 phosphorylation and IL-17 expression. IL-23 plays a crucial role in immunity against extracellular pathogens, but abnormal IL-23 signaling is associated with the development of several autoimmune diseases.

[0007] The clinical success of existing therapeutic approaches involving cytokines is limited due to off-target toxicity and pleiotropy, primarily because cytokines have receptors on both desirable and undesirable response cells, which can cancel each other out and lead to undesirable side effects. For example, in the case of IL-12, systemic administration of IL-12 results in toxicity due to NK cell-mediated IFNγ production.

[0008] In recent years, cytokine modification has emerged as a promising strategy for preparing recombinant cytokines with desirable activity and low toxicity. Therefore, additional approaches are needed to improve the properties of IL-12 and IL-23 for therapeutic use. In particular, there is a need for variants of IL-12 and IL-23 that can selectively activate specific downstream functions and actions more than others, retaining many of the beneficial properties of IL-12 and IL-23, but without their known toxic side effects, resulting in improved applications as antitumor or immunomodulatory agents. [Overview of the Initiative]

[0009] overview This disclosure relates generally to the field of immunology and includes compositions and methods for modulating interleukin-12 (IL-12) and / or interleukin-23 (IL-23) mediated signaling pathways. More specifically, in some embodiments, this disclosure provides various recombinant interleukin-12 subunit p40 (IL-12p40) polypeptides in which the binding affinity to its innate receptor, interleukin-12 receptor subunit beta-1 (IL-12Rβ1), is altered. As described in more detail below, IL-12p40 can be modulated to achieve different levels of STAT3-mediated and / or STAT4-mediated signaling. Some embodiments of this disclosure provide IL-12p40 partial agonists that can result in cell-type biased IL-12p40 signaling. Some embodiments provide IL-12p40 partial agonists that can confer cell-type biased IL-12 signaling, for example, reduced IL-12 signaling in natural killer (NK) cells, while substantially preserving IL-12 signaling in CD8+ T cells. Also provided are compositions and methods useful for producing such IL-12p40 polypeptide variants, methods for modulating IL-12p40-mediated signaling in subjects, and methods for treating conditions associated with disruptions in downstream IL-12p40 signaling, such as IL-12 signaling and / or IL-23 signaling.

[0010] In one embodiment, a recombinant polypeptide comprising the following is provided herein: (a) an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with the IL-12p40 polypeptide having the amino acid sequence of SEQ ID NO: 1; and further, one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 1.

[0011] Non-limiting exemplary embodiments of the disclosed recombinant polypeptides may include one or more of the following features: In some embodiments, one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO: 1. In some embodiments, one or more amino acid substitutions are independently selected from the group consisting of alanine (A) substitutions, arginine (R) substitutions, asparagine (N) substitutions, aspartic acid (D) substitutions, leucine (L) substitutions, lysine (K) substitutions, phenylalanine (F) substitutions, lysine substitutions, glutamine (Q) substitutions, glutamic acid (E) substitutions, serine (S) substitutions, and threonine (T) substitutions, and any combination thereof. In some embodiments, one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 in SEQ ID NO: 1.

[0012] In some embodiments, the recombinant polypeptides of the present disclosure comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 1, and further comprise amino acid substitutions corresponding to the following: (a) W37A; (b) P39A; (c) D40A; (d) E81A; (e) F82A; (f) K106A; (g) D109A; (h) K217A; (i) K219A; (j) E81A / F82A; (k) W37A / E81A / F82A; (l) E81A / F82A / K106A; (m )E81A / F82A / K106A / K219A, (n)E81A / F82A / K106A / K217A, (o)81A / F82A / K 106A / E108A / D115A, (p)E81F / F82A, (q)E81K / F82A, (r)E81L / F82A, (s)E8 1H / F82A, (t)E81S / F82A, (u)E81A / F82A / K106N, (v)E81A / F82A / K106Q, (w )E81A / F82A / K106T, (x)E81A / F82A / K106R, or (y)P39A / D40A / E81A / F82A. In some embodiments, the recombinant polypeptides of this disclosure comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-8 and 13-16.

[0013] In one embodiment, several embodiments of the present disclosure relate to a polypeptide comprising: (a) an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with the IL-12p40 polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) and further, one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 2.

[0014] In some embodiments, one or more amino acid substitutions are at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO: 2. In some embodiments, one or more amino acid substitutions are independently selected from the group consisting of alanine (A) substitution, arginine (R) substitution, asparagine (N) substitution, aspartic acid (D) substitution, leucine (L) substitution, lysine (K) substitution, phenylalanine (F) substitution, lysine substitution, glutamine (Q) substitution, glutamic acid (E) substitution, serine (S) substitution, and threonine (T) substitution. In some embodiments, one or more amino acid substitutions are at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and E219 of SEQ ID NO: 2. In some embodiments, one or more amino acid substitutions are at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, E81, F82, K106, K217, and E219 of SEQ ID NO: 2.

[0015] In some embodiments, the recombinant polypeptides of the present disclosure comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 2, and further comprise amino acid substitutions corresponding to the following: (a) W37A; (b) P39A; (c) D40A; (d) E81A; (e) F82A; (f) K106A; (g) D109A; (h) K217A; (i) E219A; (j) E81A / F82A; (k) W37A / E81A / F82A, (l)E81A / F82A / K106A, (m)E81A / F82A / K106A / K217A, (n)E81A / F82A, (o)E81K / F82A, (p)E81L / F82A, (q)E81H / F82A, (r)E81S / F82A, (s)E81A / F82A / K106N, (t)E81A / F82A / K106Q, (u)E81A / F82A / K106T, (v)E81A / F82A / K106R, or (w)P39A / D40A / E81A / F82A. In some embodiments, the recombinant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs. 9-11 and 17-25.

[0016] In some embodiments, the recombinant polypeptides of this disclosure have a modified binding affinity to interleukin-12 receptor beta-1 (IL-12Rβ1) compared to the binding affinity of a reference polypeptide lacking one or more amino acid substitutions. In some embodiments, the recombinant polypeptides have a reduced binding affinity to IL-12Rβ1 compared to the binding affinity of a reference polypeptide lacking one or more amino acid substitutions. In some embodiments, the recombinant polypeptides, as determined by surface plasmon resonance (SPR), have a binding affinity to IL-12Rβ1 that is reduced by about 10% to about 100% compared to the binding affinity of a reference polypeptide lacking one or more amino acid substitutions. In some embodiments, when combined with the interleukin-12 subunit p35 (IL-12p35) polypeptide, the recombinant polypeptides of this disclosure have a reduced ability to stimulate STAT4 signaling compared to a reference polypeptide lacking one or more amino acid substitutions. In some embodiments, recombinant polypeptides, when combined with interleukin 23 subunit p19 (IL-23p19) polypeptides, exhibit reduced ability to stimulate STAT3 signaling compared to reference polypeptides lacking one or more amino acid substitutions. In some embodiments, STAT3 and / or STAT4 signaling are determined by assays selected from the group consisting of gene expression assays, phosphoflow signaling assays, and enzyme-linked immunosorbent assays (ELISAs).

[0017] In some embodiments, one or more amino acid substitutions in the disclosed recombinant polypeptide result in cell type-biased signaling of downstream signaling mediated by interleukin-12 (IL-12) and / or interleukin-23 (IL-23), as compared to a reference polypeptide lacking the one or more amino acid substitutions. In some embodiments, cell type-biased signaling includes a decrease in the ability of the recombinant polypeptide to stimulate IL-12-mediated signaling in NK cells. In some embodiments, cell type-biased signaling includes substantially unmodified ability of the recombinant polypeptide to stimulate IL-12 signaling in CD8+ T cells. In some embodiments, the one or more amino acid substitutions result in a decrease in the ability of the recombinant polypeptide to stimulate IL-12 signaling in NK cells, but substantially retain its ability to stimulate IL-12 signaling in CD8+ T cells.

[0018] In another aspect, recombinant nucleic acids are provided herein, wherein the nucleic acid comprises a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of the polypeptides of the present disclosure.

[0019] Non-limiting exemplary embodiments of the disclosed nucleic acid molecules can include one or more of the following features. In some embodiments, the nucleic acid sequence is operably linked to a heterologous nucleic acid sequence. In some embodiments, the nucleic acid molecule is further defined as an expression cassette or an expression vector.

[0020] In one aspect, some embodiments of the present disclosure relate to recombinant cells, wherein the recombinant cells comprise (a) a recombinant polypeptide of the present disclosure; and (b) one or more of the recombinant nucleic acids of the present disclosure. In some embodiments, the recombinant cells are eukaryotic cells. In some embodiments, the eukaryotic cells are mammalian cells. In a related aspect, some embodiments of the present disclosure relate to a cell culture comprising at least one recombinant cell of the present disclosure and a medium.

[0021] In another embodiment, some embodiments of the present disclosure relate to a method for producing polypeptides, wherein the method comprises (a) providing one or more recombinant cells of the present disclosure, and (b) culturing one or more recombinant cells in a culture medium so that the cells produce polypeptides encoded by recombinant nucleic acid molecules.

[0022] In some embodiments, a method for producing the polypeptide of the Disclosure further comprises isolating and / or purifying the produced polypeptide. In some embodiments, a method for producing the polypeptide of the Disclosure further comprises structurally modifying the produced polypeptide to extend its half-life. In some embodiments, the modification includes one or more modifications selected from the group consisting of fusion to a human Fc antibody fragment, fusion to albumin, and PEGylation. Accordingly, in relevant embodiments, recombinant polypeptides produced by the method of the Disclosure are also provided herein.

[0023] In one embodiment, several embodiments of the present disclosure relate to pharmaceutical compositions, wherein a pharmaceutical composition comprises one or more of the following: (a) recombinant polypeptides of the present disclosure; (b) recombinant nucleic acids of the present disclosure; (c) recombinant cells of the present disclosure; and (d) pharmaceutically acceptable carriers.

[0024] Non-limiting exemplary embodiments of the disclosed pharmaceutical compositions may include one or more of the following features: In some embodiments, the composition comprises the recombinant polypeptide of the Disclosure and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises the recombinant cells of the Disclosure and a pharmaceutically acceptable carrier. In some embodiments, the recombinant cells express the recombinant polypeptide of the Disclosure. Examples of recombinant cells genetically modified to express and secrete therapeutic polypeptides have already been described, for example, in Steidler L. et al., Nature Biotechnology, Vol. 21, No. 7, July 2003 and Oh JH et al., mSphere, Vol. 5, Issue 3, May / June 2020. In some embodiments, the composition comprises the recombinant nucleic acid of the Disclosure and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises the recombinant cells of the Disclosure and a pharmaceutically acceptable carrier.

[0025] In one embodiment, several embodiments of the present disclosure relate to a method for modulating IL-12p40-mediated signaling in a subject, the method comprising administering to the subject a composition comprising one or more of the following: (a) recombinant IL-12p40 polypeptide of the present disclosure; (b) recombinant nucleic acid of the present disclosure; (c) recombinant cell of the present disclosure; and (d) pharmaceutical composition of the present disclosure. In some embodiments, IL-12p40-mediated signaling comprises IL-12-mediated signaling. In some embodiments, IL-12p40 signaling comprises IL-23-mediated signaling.

[0026] Accordingly, some embodiments of the present disclosure relate to methods for modulating IL-12-mediated signaling in a subject, wherein the method comprises administering to a subject a composition comprising one or more of the following: (a) recombinant IL-12p40 polypeptide of the present disclosure; (b) recombinant nucleic acid of the present disclosure; (c) recombinant cell of the present disclosure; and (d) pharmaceutical composition of the present disclosure. In some embodiments, the method further comprises administering to a subject an IL-12p35 polypeptide, or a nucleic acid encoding the IL-12p35 polypeptide.

[0027] In some embodiments, methods are provided for modulating IL-23-mediated signaling in a subject, the method comprising administering to the subject a composition comprising one or more of the following: (a) recombinant IL-12p40 polypeptide of the Disclosure; (b) recombinant nucleic acid of the Disclosure; (c) recombinant cell of the Disclosure; and (d) pharmaceutical composition of the Disclosure. In some embodiments, the method further comprises administering to the subject an IL-23p19(p19) polypeptide or nucleic acid encoding the IL-23p19 polypeptide.

[0028] In another embodiment, various methods for treating a condition in a subject are provided herein, each method comprising administering to the subject a composition comprising one or more of the following: (a) recombinant IL-12p40 polypeptide of the Disclosure; (b) recombinant nucleic acid of the Disclosure; (c) recombinant cells of the Disclosure; and (d) pharmaceutical compositions of the Disclosure. In some embodiments, the method further comprises administering to the subject a composition comprising one or more of the following: (a) IL-12p35(p35) polypeptide; (b) IL-23p19 polypeptide; and (c) nucleic acid encoding (a) or (b).

[0029] Non-limiting exemplary embodiments of the disclosed methods for modulating IL-12p40-mediated signaling in subjects and / or for treating conditions in subjects requiring such modulation may include one or more of the following features: In some embodiments, the recombinant polypeptide has a modified binding affinity to interleukin-12 receptor subunit beta 1 (IL-12Rβ1) compared to the binding affinity of a reference polypeptide lacking one or more amino acid substitutions. In some embodiments, the recombinant polypeptide has a reduced binding affinity to IL-12Rβ1 compared to the binding affinity of a reference polypeptide lacking one or more amino acid substitutions. In some embodiments, the recombinant polypeptide has a binding affinity to IL-12Rβ1 that is reduced by about 10% to about 100% compared to the binding affinity of a reference polypeptide lacking one or more amino acid substitutions, as determined by surface plasmon resonance (SPR). In some embodiments, the reduced binding affinity of the recombinant polypeptide to the IL-12Rβ1 receptor results in a reduction of STAT4-mediated signaling compared to a reference polypeptide lacking one or more amino acid substitutions. In some embodiments, reduced binding affinity of recombinant polypeptides to the IL-12Rβ1 receptor results in reduced STAT3-mediated signaling compared to reference polypeptides lacking one or more amino acid substitutions. In some embodiments, STAT3 and / or STAT4 signaling is determined by assays selected from the group consisting of gene expression assays, phosphoflow signaling assays, and enzyme-linked immunosorbent assays (ELISA).

[0030] In some embodiments, the administered composition results in cell-type biased signaling of downstream signaling mediated by interleukin-12 (IL-12) and / or interleukin-23 (IL-23) compared to a reference polypeptide lacking one or more amino acid substitutions. In some embodiments, cell-type biased signaling includes a reduced ability of the recombinant polypeptide to stimulate IL-12-mediated signaling in NK cells. In some embodiments, cell-type biased signaling includes a substantially unmodified ability of the recombinant polypeptide to stimulate IL-12 signaling in CD8+ T cells. In some embodiments, the administered composition results in a reduced ability of the recombinant polypeptide to stimulate IL-12-mediated signaling in NK cells, but substantially retains its ability to stimulate IL-12 signaling in CD8+ T cells. In some embodiments, the administered composition substantially retains the ability of the recombinant polypeptide to stimulate INFγ expression in CD8+ T cells. In some embodiments, the administered composition enhances anti-tumor immunity in the tumor microenvironment.

[0031] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject has or is suspected of having a condition related to IL-12p40-mediated signaling. In some embodiments, IL-12p40-mediated signaling is IL-12-mediated signaling or IL-23-mediated signaling. In some embodiments, the condition is cancer, an immune disorder, or a chronic infection. In some embodiments, the immune disorder is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, moderate to severe psoriasis vulgaris, psoriatic arthritis, Crohn's disease, ulcerative colitis, and graft-versus-host disease.

[0032] In some embodiments, various methods for treating a condition in a subject requiring such treatment are provided herein, wherein the condition is a cancer selected from the group consisting of acute myelomatous leukemia, anaplastic lymphoma, astrocytoma, B-cell carcinoma, breast cancer, colon cancer, ependymoma, esophageal cancer, glioblastoma, glioma, leiomyosarcoma, liposarcoma, liver cancer, lung cancer, mantle cell lymphoma, melanoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, ovarian cancer, pancreatic cancer, peripheral T-cell lymphoma, renal cancer, sarcoma, gastric cancer, carcinoma, mesothelioma, and sarcoma.

[0033] In some embodiments, the composition is administered to the subject individually as a first treatment or in combination with a second treatment. In some embodiments, the second treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first and second treatments are administered concurrently. In some embodiments, the first treatment is administered simultaneously with the second treatment. In some embodiments, the first and second treatments are administered sequentially. In some embodiments, the first treatment is administered before the second treatment. In some embodiments, the first treatment is administered after the second treatment. In some embodiments, the first treatment is administered before and / or after the second treatment. In some embodiments, the first and second treatments are administered alternately. In some embodiments, the first and second treatments are administered together in a single formulation.

[0034] In another embodiment, some embodiments of the present disclosure relate to kits for carrying out the methods disclosed herein. Some embodiments relate to kits for methods for modulating IL-12p40-mediated signaling in subjects, wherein the kit comprises one or more of the recombinant polypeptides of the present disclosure; recombinant nucleic acids of the present disclosure; recombinant cells of the present disclosure; and pharmaceutical compositions of the present disclosure, as well as instructions for carrying out the methods disclosed herein. Some embodiments relate to kits for methods for treating conditions in subjects requiring such treatment, wherein the kit comprises one or more of the recombinant polypeptides of the present disclosure; recombinant nucleic acids of the present disclosure; recombinant cells of the present disclosure; and pharmaceutical compositions of the present disclosure, as well as instructions for carrying out the methods disclosed herein.

[0035] A further aspect of the present disclosure is the use of one or more of the nucleic acid molecules, recombinant cells, or pharmaceutical compositions of the present disclosure for the treatment of individuals having or suspected to have a condition related to disruption of IL-12p40-mediated signaling.

[0036] The above summary is for illustrative purposes only and is not intended to limit the scope of this disclosure. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, purposes, and features of this disclosure will be fully apparent from the drawings, detailed description, and claims. [Brief explanation of the drawing]

[0037] [Figure 1A] Figures 1A-1E show the structure of the four-component IL-23 complex. Figure 1A: Schematic of cytokine composition and receptor use of the IL-12 family. [Figure 1B] Figures 1A-1B: Side view of the IL-23 receptor complex. [Figure 1C-1E] Figures 1C-1E: Enlarged views highlighting the interactions between the three interaction sites between IL-23 and the receptor subunit.

[0038] [Figure 2A]Figures 2A–2H schematically summarize the results from experiments conducted to demonstrate that IL-12p40 plays a conserved role in IL-12 and IL-23 signaling. Figure 2A: IL-12p40 directly binds to IL-12Rβ1. Surface plasmon resonance (SPR) sensorgram showing the binding of IL-12Rβ1 to immobilized IL-12p40. The dissociation constant (KD) was determined using a steady-state affinity model. [Figure 2B-2C] Figures 2B-2C: IL-12 and IL-23 induce different patterns of STAT phosphorylation in CD4+ T cells. CD4+ T cells were activated with 2.5 μg αCD3, 5 μg αCD28, and 100 IU / mL rhIL-2 for 2 days, rested overnight, stimulated with IL-12 or IL-23 for 20 minutes, fixed, and permeabilized, and STAT phosphorylation was evaluated by flow cytometry. (D-F) The covalent interface of IL-12p40 regulates IL-12 and IL-23 signaling. [Figure 2D] Figure 2D: Ribbon diagram showing the interaction between IL-12p40 and IL-12Rβ1. The inset shows the location of the amino acid targeted for mutagenesis. [Figure 2E-2F] Figure 2E: The IL-12p40 mutant induces altered IL-12pSTAT4 signaling in CD4+ T cells. The ability to stimulate STAT4 signaling in CD4+ T cell blasts by co-expressing the IL-12p40 variant with IL-12p35 was tested. Figure 2F: The IL-12p40 mutant induces altered IL-23pSTAT3 signaling in CD4+ T cells. The ability to stimulate STAT3 signaling in CD4+ T cell blasts by co-expressing the IL-12p40 variant with IL-23p19 was tested. [Figure 2G] Figure 2G: Ribbon diagram of IL-12p40, including an inset showing amino acids at the IL-12Rβ1 interface. [Figure 2H] Figure 2H: STAT4 signaling by the IL-12p40 variant. The ability of the IL-12p40 variant to stimulate STAT4 signaling in CD4+ T cell blasts was tested by co-expressing it with IL-12p35.

[0039] [Figure 3A] Figures 3A–3D summarize experiments demonstrating that IL-12p40 regulates mouse IL-12 STAT4 signaling. Figure 3A: Cell type and activation state-dependent expression of IL-12Rβ1. Flow cytometry plots showing IL-12Rβ1 expression levels measured by mouse IL-12p40 tetramer staining in mouse NK cells (CD3-NK1.1+) or CD8+ T cells (CD3+CD8+). Red lines indicate 200 nM tetramer staining, and gray areas represent streptavidin staining only. Single-cell suspensions from the spleen and lymph nodes of C57 / BL6 mice were stained directly (ex vivo) with IL-12p40 tetramer, or after 2 days of stimulation with 2.5 μg / mL αCD3, 5 μg / mL αCD28, and 100 IU / mL rmIL-2 (blast cells). [Figure 3B] Figure 3B shows the sequence alignment of the human IL-12p40 polypeptide (SEQ ID NO: 1) and the mouse IL-12p40 polypeptide (SEQ ID NO: 2). In the alignment, conserved positions are shown in gray shading, and mutagenic target positions are shown with asterisks. [Figure 3C-3D] Figures 3C-3D: IL-12p40 mutations regulate IL-12 signaling in CD8+ T cell blasts. Dose-response (Figure 3C) and representative histogram at peak concentration (Figure 3D) of phospho-STAT4 staining after 20 minutes of stimulation with the indicated IL-12 variants (2×Ala:E81AF82A, 3×Ala:E81AF82AK106A, 4×Ala:E81AF82AK106AK217A). Dose-response values ​​show the mean and standard error of biological dual measurements, representing two or more independent experiments.

[0040] [Figure 4A-4B]Figures 4A–4C schematically summarize results from experiments conducted to demonstrate that three exemplary IL-12 partial agonists, according to some non-limiting embodiments of this disclosure, induce cell-type-specific responses based on differential IL-12Rβ1 expression. Figure 4A: IL-12 partial agonists promote IFNγ production by antigen-specific CD8+ T cells. Representative histogram (left) and quantification (right) of intracellular IFNγ in OT-ICD8+ T cells (CD3+CD8+). OT-I splenocytes were stimulated for 48 hours with 1 μg / mL OVA peptide, 100 IU / mL IL-2, and 1 μM IL-12 variant. GolgiStop was added during the last 4 hours to prevent further cytokine secretion. Figure 4B: IL-12 partial agonists show attenuation of IFNγ induction in NK cells. Purified NK cells were stimulated with 50 ng / mL IL-18 containing 1 μM IL-12 variant for 48 hours. GolgiStop was added during the last 4 hours to prevent further cytokine secretion. [Figure 4C] Figure 4C: IL-12 partial agonists exhibit cell type-biased activity. The ratio of αIFNγ AF647 MFI in T cells / NK cells, standardized to wild-type IL-12, is shown for IL-12 and partial agonists. Bar graphs show the mean and standard error of biological dual measurements, representing two or more independent experiments. MFI, mean fluorescence intensity.

[0041] [Figure 4D] Figures 4D–4G schematically summarize results from experiments conducted to demonstrate that further exemplary IL-12 partial agonists in some non-limiting embodiments of this disclosure induce cell type-specific responses based on differential IL-12Rβ1 expression. Figure 4D: IL-12p40 mutation modulates IL-12 signaling in CD8+ T cell blasts. Dose response by phosphoSTAT4 staining after 20 minutes of stimulation with the shown IL-12 variant. Dose response shows mean and standard error of biological double measurements. [Figure 4E-4F]Figure 4E: IL-12 partial agonists promote IFNγ production by antigen-specific CD8+ T cells. Quantification of intracellular IFNγ in OT-ICD8+ T cells (CD3+CD8+). OT-I splenocytes were stimulated for 48 hours with 1 μg / mL OVA peptide, 100 IU / mL IL-2, and 1 μM IL-12 variant. GolgiStop was added during the last 4 hours to prevent further cytokine secretion. Figure 4F: IL-12 partial agonists show attenuation of IFNγ induction in NK cells. Purified NK cells were stimulated for 48 hours with 50 ng / mL IL-18 containing 1 μM IL-12 variant. GolgiStop was added during the last 4 hours to prevent further cytokine secretion. [Figure 4G] Figure 4G: IL-12 partial agonists exhibit cell type-biased activity. The ratio of αIFNγ AF647 MFI in T cells / NK cells, standardized to wild-type IL-12, is shown for IL-12 and partial agonists. Bar graphs show the mean and standard error of biological dual measurements, representing two or more independent experiments. MFI, mean fluorescence intensity.

[0042] [Figure 5A-5B] Figures 5A–5C schematically summarize the results from experiments conducted to demonstrate that the exemplary IL-12 partial agonists described in Figures 4A–4C above promote antigen-specific tumor cell death. Figures 5A–5B: Supernatant from OT-I effectors generated in the presence of IL-12 partial agonists enhances MHC-I upregulation against B16F10 melanoma cells. Dose-response of H2-Kb surface expression after overnight incubation with OT-I effector supernatant (Figure 5A) and representative histogram (Figure 5B). Arrows indicate dilution of the supernatant shown in the representative histogram. OT-I effectors were generated by co-culturing splenocytes with 1 μg / mL OVA peptide, 100 IU / mL IL-2, and 1 μM IL-12 variant for 72 hours. Figures 5C–5D: IL-12 partial agonists enhance the efficacy of antigen-specific tumor cell death. [Figure 5C-5D]Figure 5C: Schematic of the specific cell death assay. A 1:1 mixture of wild-type cells and OVA-GFP-expressing B16F10 cells was incubated with OT-I effectors in various ratios, and antigen-specific cell death was measured using the frequency of OVA-GFP+ cells. (Figure 5D) Dose-response curves showing specific cell death of OT-1 effectors generated in the absence of IL-12 or in the presence of the indicated IL-12 variant. Data are expressed as the mean and standard error of biological dual measurements and represent two or more independent experiments.

[0043] [Figure 6A-6B] Figures 6A–6I provide a schematic summary of results from experiments conducted to characterize mouse IL-12 signaling on NK cells. Figure 6A: IL-12 partial agonists induce a decrease in pSTAT4 signaling in NK cells. MACS-purified NK cells were mixed with carrier cells loaded with CellTrace Violet and stimulated with IL-12 agonists for 20 minutes. Figure 6B: IL-18 is required for IL-12-mediated induction of IFNγ in NK cells. MACS-purified NK cells were stimulated with 50 ng / mL of IL-18 and 1 nM of IL-12, as shown. [Figure 6C-6D] Figure 6C: IL-12 induces dose-dependent IFNγ production in NK cells. NK cells were stimulated with dose-controlled IL-12 as shown in Figure 4B, and IFNγ induction at 48 hours was analyzed by intracellular cytokine staining. Figure 6D: IL-12 agonists induce dose-dependent IFNγ expression in NK cells, as shown in Figure 4B. [Figure 6E-6G]Figure 6E: 3×Ala and 4×Ala IL-12 partial agonists reduced IFNγ secretion by NK cells compared to IL-12. Analysis of IFNγ in the supernatant of NK cell cultures by ELISA, related to Figure 4B. Figures 6F-6G: Quantitative PCR (qPCR) of lfng (Figure 6F) and Tigit (Figure 6G) from NK cells stimulated for 8 hours with 50 ng / mL IL-18 and 1 μM IL-12. Ct values ​​were standardized against Gapdh and expressed as induction factors relative to unstimulated control. Bar graphs show the mean ± standard deviation of technical triple measurements. [Figure 6H-6I] Figure 6H: Pre-activation of IL-2 upregulates IL-12Rβ1 on NK cells. MACS-purified NK cells were stimulated with 1000 IU / mL of IL-2 for 48 hours, and IL-12Rβ1 expression levels were identified by staining with 200 nM p40 tetramer (red) or streptavidin control (gray) as shown in Figure 3A. Figure 6I: IL-2 enhances IFNγ induction in NK cells, but there is no synergistic effect with IL-12 partial agonists. MACS-purified NK cells were activated with 1000 IU / mL of IL-2, 50 ng / mL of IL-18, and 1 μM of IL-12 agonist for 48 hours. The dashed line shows IFNγ staining in NK cells stimulated with IL-18 alone. Unless otherwise specified, data are shown as mean ± standard deviation of biological dual measurements and represent two or more experiments.

[0044] [Figure 7A] Figures 7A–7F schematically summarize the results from experiments conducted to characterize various exemplary human IL-12 partial agonists of this disclosure. Figure 7A: Cell type-dependent and activation state-dependent expression of IL-12Rβ1 in human PBMCs. Flow cytometry plots showing IL-12Rβ1 expression levels measured by p40 tetramer staining in human NK cells (CD3-CD56+) or CD8+ T cells (CD3+CD8+). The red line indicates 200 nM tetramer staining, and the gray clusters represent streptavidin staining only. In the case of T cell blasts, PBMCs were stimulated for 2 days with 2.5 μg / mL αCD3, 5 μg / mL αCD28, and 100 IU / mL IL-2. [Figure 7B] Figure 7B: NK cell and T cell gating scheme. [Figure 7C-7D] Figures 7C-7D: Phosphoflow cytometry of CD8+ T cell blasts stimulated with IL-12 partial agonist for 20 minutes. Figure 7C: Dose-response curve of pSTAT4 signaling in human CD8+ T cell blasts. Figure 7D: Histograms show pSTAT4 staining with 8 nM (IL-12) or 1 μM (2 × Ala:E81A / F82A, 3 × Ala:E81A / F82A / K106A). [Figures 7E-7F] Figure 7E: IL-12 partial agonists support IFNγ secretion by CD8+ T cells. MACS-isolated CD8+ T cells were stimulated with 2 μg / mL αCD3, 0.5 μg / mL αCD28, and 5 ng / mL IL-2 with or without an IL-12 agonist. After 48 hours, the supernatant was analyzed for IFNγ ELISA. The dashed line shows IFNγ levels in the absence of IL-12. Figure 7F: IL-12 partial agonists demonstrate attenuation of IFNγ production by NK cells. MACS-isolated NK cells were stimulated with 100 ng / mL IL-18 for 48 hours with or without an IL-12 agonist, and the supernatant was assayed for IFNγ by ELISA. Conditions in which IFNγ above background levels was not detected are listed as "nd" under "not measured". Data are presented as mean ± standard deviation of biological dual measurements and represent two independent experiments.

[0045] [Figure 7G-7I]Figures 7G-7I provide a schematic summary of results from experiments conducted to demonstrate the bias of the human IL-12 partial agonist W37AE81AF82A towards T cells. Figure 7G: Phosphoflow cytometry of CD8+ T cell blasts stimulated with the IL-12 partial agonist for 20 minutes. Figure 7H: The IL-12 partial agonist supports IFNγ secretion by CD8+ T cells. MACS-isolated CD8+ T cells were stimulated with 2 μg / mL αCD3, 0.5 μg / mL αCD28, and 5 ng / mL IL-2, with or without the IL-12 agonist. After 48 hours, the supernatant was analyzed for IFNγ by ELISA. The dashed line indicates IFNγ levels in the absence of IL-12. Figure 7I: The IL-12 partial agonist shows attenuation of IFNγ production by NK cells. MACS-isolated NK cells were stimulated with 100 ng / mL of IL-18 for 48 hours, with or without an IL-12 agonist, and the supernatant was assayed for IFNγ by ELISA. Data are expressed as the mean ± standard deviation of two independent biological dual measurements.

[0046] [Figures 8A-8D] Figures 8A-8E provide a schematic summary of the results of experiments conducted to verify the expression of mouse IL-12 agonists from mammalian cells. Figure 8A: Purification of IL-12 from Expi293F cells. (A) Representative S200 size exclusion chromatography (SEC) of Ni-NTA purified mouse IL-12. mAU: milliabsorbance units. Figure 8B: SDS-PAGE of IL-12 after Ni-NTA affinity purification and SEC under reduced (R) and non-reduced (NR) conditions. Figures 8C-8F: Characterization of mouse IL-12 variants expressed in mammals. Figures 8C-8D: pSTAT4 staining of CD8+ T cell blasts after 20 minutes of cytokine stimulation. Histograms show pSTAT4 staining at 8 nM for IL-12 and 1 μM for partial agonists. [Figures 8E-8F]Figure 8E: Mammalian-expressed IL-12 partial agonists promote IFNγ production by antigen-specific CD8+ T cells. Representative histograms (left) and quantifications (right) of intracellular IFNγ in OT-ICD8+ T cells (CD3+CD8+) after 48 hours of stimulation with 1 μg / mL OVA peptide (257-264), 0.5 μg / mL αCD28, 100 IU / mL IL-2, and 1 μM IL-12 variant. Figure 8F: Mammalian-expressed IL-12 partial agonists show attenuation of IFNγ induction in NK cells. Purified NK cells were stimulated for 48 hours with 50 ng / mL IL-18 containing 1 μM IL-12 variant.

[0047] [Figures 9A-9C] Figures 9A–9J provide a schematic summary of the results of experiments conducted to demonstrate that IL-12 partial agonists induce cell-type specific responses in vivo. Figure 9A: Schematic of the experimental design. CD8+ T cells from OT-I TCR transgenic mice (Thy1.2) were transplanted into congenic recipient mice (Thy1.1) on day 0. The following day, the mice were subcutaneously immunized with 50 μg of OVA (257–264) in Freund's incomplete adjuvant (IFA), and daily intraperitoneal injections of 30 μg of cytokine were initiated. After 5 days of cytokine treatment, the mice were euthanized, and serum IFNγ was analyzed by ELISA, and cell type profiling of the outflow lymph nodes was performed by flow cytometry. Figure 9B: IL-12 causes weight loss, but partial agonists do not. Mouse weight was monitored daily and standardized against day 1 weight before cytokine treatment was initiated. Figure 9C: As measured by serum ELISA on day 6, IL-12 increases systemic IFNγ, but partial agonists do not. The dashed line represents measurements from unimmunized mice in this panel and subsequent panels. [Figure 9D-9G]Figures 9D-9E: Immunization increases the frequency of PD-1+ OT-I T cells independently of cytokine treatment. Figure 9D: Representative FACS plot showing PD-1 expression in OT-I+ T cells identified as CD3+ CD8+ Thy1.2+. Figure 9E: Quantification of PD-1+ cells as frequency of OT-I+ T cells. Figure 9F: IL-12 expands OT-I T cells, but partial agonists do not. OT-T cells were identified as Thy1.2+ and expressed as the frequency of total CD8+ T cells. Data were analyzed by the Kruskal-Wallis test using Dunn's multiple comparisons. Figure 9G: IL-12 increases the frequency of LAG-3+ NK cells, but partial agonists do not. Data were analyzed by the Kruskal-Wallis test using Dunn's multiple comparisons. 9H~J: IL-12 partial agonists preferentially increase the frequency of CD25+-expressing OT-I T cells with reduced activity against NK cells, compared to IL-12. [Figure 9H-9J] Figure 9H: Representative FACS plots showing CD25 expression in OT-I T cells (top) and NK cells (bottom). Figure 9I: Quantification of CD25+ OT-I T cells. Figure 9J: Quantification of CD25+ NK cells. Data were analyzed by one-way ANOVA using Tukey's multiple comparisons. Data are expressed as mean ± standard deviation of n=5 mice / group and represent two independent experiments.

[0048] [Figure 10A-10B]Figures 10A–10G schematically summarize the results of experiments conducted to demonstrate that IL-12 partial agonists support an MC-38 antitumor response without inducing IL-12-related toxicity. Figure 10A: Schematic of the experimental design. 5 × 10⁵ MC-38 cells in Matrigel were transplanted into mice on day 0. As shown, from day 7 onwards, mice were injected daily with PBS (n=10), 1 μg IL-12 (n=10), 30 μg IL-12 (n=9), 30 μg 2 × Ala (n=9), or 30 μg 3 × Ala (n=10). Figure 10B: IL-12 induces weight loss in tumor-bearing mice, but partial agonists do not. Body weight was normalized to 7 days prior to cytokine treatment. Mice administered with a 30 μg dose of IL-12 died from cytokine toxicity between days 13 and 15. [Figure 10C-10D] Figure 10C: IL-12 enhances systemic IFNγ, while partial agonists do not. Serum IFNγ ELISA at day 10, n=5 mice / group. Figure 10D: IL-12 reduces motor function, while partial agonists do not. Cumulative displacement of MC-38-carrying mice after cytokine treatment. Quantification of 30-second video captured at day 16. Cumulative displacement was calculated as the sum of ΔX and ΔY over time. Data are shown as mean ± standard deviation for n=5 mice / group. [Figures 10E-10F] Figure 10E: IL-12 and partial agonists attenuate MC-38 tumor growth. Tumor volume was compared on day 20 using the Kruskal-Wallis test with Dunn's multiple comparisons. Figure 10F: IL-12 and partial agonists extend survival in MC-38-carrying mice. Kaplan-Meier curves for mice treated with PBS or IL-12 variants. P-values ​​for log-rank tests were corrected for multiple comparisons using the Holm-Sidak method. [Figure 10G] Figure 10G: Individual tumor growth curves of MC-38-carrying mice. Growth curves of PBS-treated mice are shown in gray for comparison with cytokine-treated mice, which are shown in color. Data are expressed as mean ± standard deviation and represent two independent experiments. [Modes for carrying out the invention]

[0049] Detailed explanation of disclosure This disclosure generally relates to compositions and methods for selectively modulating interleukin-12 (IL-12) and interleukin-23 (IL-23) mediated signaling pathways in subjects. In particular, this disclosure provides novel IL-12p40 compositions based on new insights into how IL-12p40 interacts with its congener receptor IL-12Rβ1. As will be described in more detail below, IL-12p40-mediated signaling can be modulated by regulating STAT3-mediated and / or STAT4-mediated signaling. More specifically, in some embodiments, this disclosure provides a novel set of IL-12p40 polypeptide variants having regulated binding affinity to the interleukin-12 receptor beta-1 subunit (IL-12Rβ1). This disclosure also provides compositions and methods useful for producing such IL-12p40 polypeptides, methods for regulating IL-12p40-mediated signaling in subjects, and methods for treating conditions associated with disruption of downstream signaling of the IL-12p40 receptor.

[0050] Interleukins IL-12 and IL-23 are heterodimeric cytokines that share the IL-12p40 cytokine subunit and the IL-12Rβ1 cell surface receptor. As described in the examples below, experiments were planned and carried out to determine the X-ray crystal structure of the complete IL-23 receptor complex, revealing a common modular interaction between IL-12p40 and IL-12Rβ1 shared by IL-12 and IL-23. Based on this new structural understanding, several L-12p40 variants with mutations at the interface with IL-12Rβ1 were generated and tested for their ability to induce STAT3 and STAT4 signaling. This approach identified a range of IL-12p40 variants that can generate stepwise STAT4 signaling in the context of IL-12 and stepwise STAT3 signaling in the context of IL-23. In the case of IL-12, numerous recombinant IL-12p40 polypeptides described herein have been identified and confirmed to confer cell-type biased IL-12p40 signaling, e.g., a reduced ability of recombinant polypeptides to stimulate IL-12-mediated signaling in NK cells. In several other embodiments, cell-type-based IL-12p40 signaling involves a reduced ability of recombinant polypeptides to stimulate IL-12 signaling in NK cells, while substantially retaining their ability to stimulate IL-12 signaling in CD8+ T cells. These novel cytokine agonists may have therapeutic utility by maintaining the antitumor effects of cytotoxic T cells while mitigating the toxicity associated with NK cell activation.

[0051] definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have meanings that are generally understood by those skilled in the art to which this disclosure relates. In some cases, terms that have generally understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from those generally understood in the art. Many of the techniques and methods described or referenced herein are well understood by those skilled in the art and are commonly used using conventional methodologies.

[0052] The singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. For example, the term “cell” includes one or more cells, and mixtures thereof. “A and / or B” is used herein to include all alternatives of “A,” “B,” “A or B,” and “A and B.”

[0053] The term "approximately," as used herein, means roughly. Where the degree of approximation is not clear from the context, "approximately" means within ±10% of the given value, or, in all cases including the given value, rounded to the nearest significant figure. If a range is specified, it includes boundary values.

[0054] As used herein, the terms “administer” and “administer” refer to the delivery of a bioactive composition or formulation by routes of administration including, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or combinations thereof. This term includes, but not limited to, administration by a healthcare professional and self-administration.

[0055] The terms “cell,” “cell culture,” and “cell line” refer not only to specific target cells, cell cultures, or cell lines, but also to the offspring or potential offspring of such cells, cell cultures, or cell lines, regardless of the number of translocations or passages in culture. It should be understood that not all offspring are exactly identical to the parent cells. This is because certain modifications may occur in the next generation due to either mutation (e.g., intentional or accidental mutation) or environmental influence (e.g., methylation or other epigenetic modifications), and therefore the offspring may not actually be identical to the parent cells, but as long as the offspring retain the same function as the original cells, cell culture, or cell line, they are still included within the scope of the terms used herein.

[0056] The terms “effective dose,” “therapeutic dose,” or “pharmaceutically effective dose” of the recombinant polypeptides covered by this disclosure generally refer to an amount sufficient to achieve the described purpose of the composition (e.g., to achieve the effect it is administered, to treat a disease, to reduce a signaling pathway, or to reduce one or more symptoms of a disease or health condition) compared to the absence of the composition. An example of an “effective dose” is an amount sufficient to contribute to the treatment, prevention, or reduction of symptoms of a disease, also known as a “therapeutic dose.” “Reduction” of symptoms means a decrease in the severity or frequency of symptoms, or the elimination of symptoms. The exact amount of a composition containing the "therapeutic dose" depends on the therapeutic purpose and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0057] As used herein, the term "IL-12p40" means wild-type IL-12p40, whether natural or recombinant. Therefore, IL-12p40 polypeptide refers to any IL-12p40 polypeptide, but is not limited to recombinant-produced IL-12p40 polypeptide, synthetically produced IL-12p40 polypeptide, and IL-12p40 extracted from cells or tissues. The amino acid sequence of wild-type human IL-12p40 precursor is shown in SEQ ID NO: 1, which is a 328-amino acid protein with a 22-amino acid signal peptide at the N-terminus, which can be removed to produce a mature 306-amino acid protein. The amino acid sequence of mature human IL-12p40 is shown in SEQ ID NO: 26. The amino acid sequence of wild-type mouse (Mus musculus) IL-12p40 precursor is shown in SEQ ID NO: 2. This is a 335-amino acid protein with a 22-amino acid signal peptide at the N-terminus, which can be removed to produce a mature 313-amino acid protein. The amino acid sequence of mature mouse IL-12p40 is provided in SEQ ID NO: 27. For the purposes of this disclosure, all amino acid numbering is based on the precursor polypeptide (or preprotein) sequence of the IL-12p40 protein shown in SEQ ID NO: 1 (human IL-12p40) or SEQ ID NO: 2 (mouse IL-12p40). However, those skilled in the art will understand that mature proteins are often used to prepare recombinant polypeptide constructs.

[0058] As used herein, the term “variant” of IL-12p40 polypeptide refers to a polypeptide that has one or more amino acid substitutions, deletions, and / or insertions compared to the amino acid sequence of a reference IL-12p40 polypeptide, e.g., wild-type IL-12p40 polypeptide. Therefore, the term “IL-12p40 polypeptide variant” includes naturally occurring allele variants or alternative splice variants of IL-12p40 polypeptide. For example, a polypeptide variant includes substitutions of one or more amino acids in the amino acid sequence of the parent IL-12p40 polypeptide by similar, homologous, or dissimilar amino acids. Many criteria exist for determining which amino acids are similar or homologous (Gunnar von Heijne, Sequence Analysis in Molecular Biology, pp. 123-39 (Academic Press, New York, NY 1987)).

[0059] As used herein, the term “operatably linked” means a physical or functional linkage between two or more elements, such as polypeptide sequences or polynucleotide sequences, that enables them to function in the intended manner. For example, an operatable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that enables the expression of the polynucleotide of interest. In this sense, “operatably linked” refers to an arrangement in which a regulatory region and a coding sequence are transcribed such that the regulatory region is effective in regulating the transcription or translation of the coding sequence of interest. That is, if a promoter can mediate the transcription of a nucleic acid sequence, it is operatably linked to the nucleic acid sequence. It should be understood that operatably linked elements may be continuous or discontinuous. In the context of polypeptides, “operatably linked” refers to a physical linkage (e.g., directly or indirectly) between amino acid sequences (e.g., different segments, modules, or domains) to provide the described polypeptide activity. In this disclosure, various segments, regions, or domains of the recombinant polypeptides of this disclosure may be operably linked to maintain the proper folding, processing, targeting, expression, binding, and other functional properties of the recombinant polypeptide within a cell. Unless otherwise specified, the various modules, domains, and segments of the recombinant polypeptides of this disclosure are operably linked to one another. The operably linked modules, domains, and segments of the recombinant polypeptides of this disclosure may be continuous or discontinuous (e.g., linked to one another via linkers).

[0060] As used herein, the term “percent identity” refers to two or more sequences or subsequences that are identical or have a specific percentage of identical nucleotides or amino acids (e.g., approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity, across a comparison window or specified region) when measured using the BLAST or BLAST2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection. See, for example, the NCBI website, ncbi.nlm.nih.gov / BLAST. Such sequences are said to be “substantially identical.” This definition may also refer to or apply to sequence complements. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Sequence identity can be calculated using publicly available techniques and widely accessible computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., J Mol Biol 215:403, 1990). Sequence identity can also be measured using sequence analysis software such as the Sequence Analysis Software Package from the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) with default parameters.

[0061] As used herein, the term “pharmaceutically acceptable excipient” refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administering the compound of interest. Thus, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, physiological salines, solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders that are suitable for pharmacopoeial administration. Auxiliary active compounds (e.g., antibiotics and additional therapeutic agents) may also be incorporated into the composition.

[0062] As used herein, the terms “recombinant” or “modified” nucleic acid molecules refer to nucleic acid molecules modified by human intervention. As a non-limiting example, cDNA is a recombinant DNA molecule, which is any nucleic acid molecule produced by an in vitro polymerase reaction, or to which a linker is attached, or incorporated into a vector such as a cloning vector or expression vector. As a non-limiting example, a recombinant nucleic acid molecule may be 1) synthesized or modified in vitro by, for example, chemical or enzymatic techniques (e.g., by the use of chemical nucleic acid synthesis, or by replication, polymerization, exonuclease digestion, endonuclease digestion, ligation, reverse transcription, transcription, base modification (e.g., including methylation), or recombination (including homologous and site-directed recombination); 2) containing a binding nucleotide sequence that is not originally bound; 3) modified using molecular cloning techniques to lack one or more nucleotides from a naturally occurring nucleic acid molecular sequence; and / or 4) manipulated using molecular cloning techniques to have one or more sequence changes or rearrangements from a naturally occurring nucleic acid sequence. As a non-limiting example, cDNA is a recombinant DNA molecule, which is any nucleic acid molecule that is produced by an in vitro polymerase reaction, or to which a linker is attached, or incorporated into a vector such as a cloning vector or an expression vector. Another non-limiting example of recombinant nucleic acid and recombinant protein is the IL-12p40 polypeptide variant disclosed herein.

[0063] As used herein, “individual” or “subject” includes humans (e.g., human individuals) and animals such as non-human animals. In some embodiments, “individual” or “subject” is a patient under the care of a physician. Thus, a subject may be a human patient or individual who has, is at risk of having, or is suspected of having, one or more symptoms of the disease of interest (e.g., cancer) and / or the disease. A subject may also be an individual diagnosed at the time of diagnosis or thereafter as being at risk of the condition of interest. The term “non-human animal” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, e.g., sheep, dogs, cattle, chickens, and non-mammals, e.g., amphibians, reptiles, etc.

[0064] For all purposes, including providing written descriptions, as will be understood by those skilled in the art, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. The scopes described are readily recognizable as sufficiently described and enabling the decomposition of the same scope into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can readily be decomposed into lower thirds, middle thirds, upper thirds, etc. Furthermore, as will be understood by those skilled in the art, all expressions such as “maximum,” “at least,” “greater than,” and “less than” include the listed numbers, which can be decomposed into the subscopes discussed above. Finally, as will be understood by those skilled in the art, a scope includes individual members. Thus, for example, the group having 1 to 3 articles refers to the group having 1, 2, or 3 articles. Similarly, the group with 1 to 5 articles refers to the group having 1, 2, 3, 4, or 5 articles, etc.

[0065] The term “vector” is used herein to refer to a nucleic acid molecule or sequence that can transfer or transport another nucleic acid molecule. The nucleic acid molecule to be transferred is generally ligated, for example, inserted, into the vector nucleic acid molecule. Generally, vectors are replicable when bound with appropriate regulatory elements. The term “vector” includes cloning vectors and expression vectors, as well as viral vectors and integration vectors. An “expression vector” is a vector that contains a regulatory region and is therefore capable of expressing DNA sequences and fragments in vitro and / or in vivo. A vector may contain a sequence that directs autonomous replication within a cell, or it may contain a sequence sufficient to enable integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses. In some embodiments, a vector is a gene delivery vector. In some embodiments, a vector is used as a gene delivery vehicle for transferring a gene into a cell.

[0066] The aspects and embodiments of the disclosure described herein are understood to include the terms “comprising,” “consisting,” and “consisting essentially of.” As used herein, “comprising” is synonymous with “containing,” “containing,” or “characterized by,” and is inclusive or unrestricted and does not exclude additional unlisted elements or process steps. As used herein, “consisting” excludes elements, processes, or components not specified in the claimed composition or method. As used herein, “consisting essentially of” does not exclude materials or processes that do not substantially affect the basic and novel features of the claimed composition or method. Any use of the term “comprising” in this specification, particularly in descriptions of the components of a composition or the process steps of a method, is understood to include compositions and methods that are essentially of and consist of the described components or processes.

[0067] For example, headings such as (a), (b), and (i) are provided simply to make the specification and claims easier to read. The use of headings in the specification or claims does not require that the processes or elements be performed in alphabetical or numerical order, or in the order in which they are presented.

[0068] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intervening value up to one-tenth of the lower limit, between the upper and lower limits of that range, and any other intervening values ​​within the stated range are included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges and are included in the disclosure according to the limits specifically excluded in the stated range. If the stated range includes one or both limits, the range excluding one or both of those limits is also included in this disclosure.

[0069] Some ranges are presented herein with numbers preceded by the term “approximately.” The term “approximately” is used herein to provide literal support for the exact number that precedes it, as well as for any number that is close to or approximates the number that precedes it. In determining whether a number is close to or approximates a specifically stated number, the close or approximate unspecified number may be a number that, in the context in which it is presented, provides a substantially equivalent to the specifically stated number.

[0070] For clarity, it is understood that some features of the Disclosure described in relation to separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the Disclosure described in relation to a single embodiment may be provided separately or in any suitable partial combination. All combinations of embodiments relating to the Disclosure are specifically encompassed by the Disclosure and are disclosed herein as if every combination were individually and expressly disclosed. Furthermore, all partial combinations of various embodiments and their elements are also specifically encompassed by the Disclosure and are disclosed herein as if all such partial combinations were individually and expressly disclosed herein.

[0071] Interleukin-12 subunit p40 (IL-12p40) Cytokines are secreted factors that regulate various aspects of physiology through the multimerization of cell surface receptors and the induction of the JAK-STAT signaling pathway. Interleukin-12 (IL-12) and interleukin-23 (IL-23) are heterodimeric cytokines produced by antigen-presenting cells in response to pathogen-associated molecular patterns, regulating the activation and differentiation of multiple lymphocyte populations. Despite using a common IL-12p40 subunit and IL-12 receptor beta-1 (IL-12Rβ1), IL-12 and IL-23 play non-overlapping roles in the immune system.

[0072] IL-12 signals via the receptor complex of IL-12Rβ1 and IL-12Rβ2 expressed on NK cells and T cells (Figure 1A). Dimerization of the IL-12 receptor induces activation of receptor-associated Janus kinase (JAK) molecules, which phosphorylate each other and residues on the intracellular domain of IL-12Rβ2, which function as a docking site for the SH2-containing signal transducer and activator 4 (STAT4). Subsequently, the receptor-associated STAT4 protein translocates to the nucleus after phosphorylation, where it promotes IFNγ expression and the polarization of CD4+ T cells into the T helper 1 (Th1) phenotype. Given the similarities between immunity to intracellular pathogens and cancer, therapeutic approaches that stimulate the Th1 response have been investigated in the context of cancer immunotherapy, indirectly through the selection of vaccine adjuvants and epitopes, or directly through the administration of IL-12. Despite its promising results in preclinical models, the therapeutic efficacy of IL-12 administration is limited due to toxicity associated with NK cell-mediated IFNγ production.

[0073] As schematically shown in Figure 1, IL-12 shares its IL-12p40 subunit with IL-23, which signals via a receptor complex formed by IL-12Rβ1 and the IL-23 receptor (IL-23R). As a co-receptor for IL-12 and IL-23, IL-12Rβ1 is expressed in T cells, NK cells, and monocytes, while IL-23R expression is limited to γδ T cells and CD4+ T cells. Despite the sharing of IL-12Rβ1, IL-12 and IL-23 have different phenotypic effects. In CD4+ T cells, IL-23 signaling promotes STAT3 phosphorylation and stabilization of the IL-17-producing Th17 lineage. Th17 cells and IL-23 signaling play important roles in the immune response to extracellular pathogens, and abnormal Th17 activity is associated with several autoimmune diseases. In fact, genetic deficiencies in either IL-23p19 or IL-12p40 protect mice from experimental autoimmune encephalomyelitis and colitis. Clinically, IL-23-targeted antagonist antibodies are approved for the treatment of moderate to severe plaque psoriasis, psoriatic arthritis, Crohn's disease, and ulcerative colitis, but many of these antibodies block both IL-12 and IL-23 signaling, leading to complications such as an increased risk of infection.

[0074] Given the clinical importance of IL-12 and IL-23 signaling, novel strategies are needed to specifically modulate this critical cytokine axis. However, the ability to modify novel cytokine variants has been limited due to a lack of structural information on how IL-12 and IL-23 bind to their receptors and initiate downstream signaling. To address this, experiments were conducted to elucidate the crystal structure of the complete IL-23 receptor complex (IL-23p19 / IL-12p40 / IL-23R / IL-12Rβ1), revealing that IL-12p40 directly engages with IL-12Rβ1. Since both IL-12Rβ1 and IL-12p40 are shared between IL-12 and IL-23, this interface represents a critical function of complex assembly in initiating signaling for both IL-12 and IL-23. Next, using the new insights gained from the crystal structure, a panel of IL-12 and IL-23 partial agonists that modulate STAT signaling was designed. As shown below, many IL-12 agonists can maintain IFNγ induction by CD8+ T cells and tumor cell death, but they have been shown to induce a decrease in IFNγ production from NK cells. Therefore, by limiting the activity of IL-12 to antigen-specific T cells, IL-12 partial agonists may have therapeutic utility by reducing the toxicity associated with IFNγ production by NK cells.

[0075] As described in more detail below, we conducted experiments to determine the 3.4 Å resolution crystal structure of the quaternary IL-23 receptor complex, revealing that IL-12p40 engages with the co-receptor IL-12Rβ1. This mechanism of receptor assembly is specific to the cytokine superfamily and demonstrates the shared role of IL-12p40 in IL-12 and IL-23 receptor assembly. Using insights from this newly established structure, additional experiments were conducted to design and test a panel of IL-12 partial agonists. This panel supports antigen-specific CD8+ T cells with reduced activity against NK cells by leveraging differences in IL-12Rβ1 expression across cell types. This disclosure provides novel molecules useful for modulating IL-12p40-mediated signaling and a novel approach for modifying cell-type-selective cytokine agonists.

[0076] Disclosure structure A. Recombinant IL-12p40 polypeptide As outlined above, some embodiments of the present disclosure relate to a novel set of IL-12p40 polypeptide variants that exhibit altered binding affinity to IL-12Rβ1 and possess characteristics of partial agonism of tissue-specific downstream signaling mediated via interleukin-12 (IL-12) and / or interleukin-23 (IL-23). ​​For example, in some embodiments of the present disclosure, the IL-12p40 polypeptide variants disclosed herein confer a reduced ability to stimulate IL-12-mediated signaling in NK cells. In some other embodiments, the IL-12p40 polypeptide variants disclosed herein confer a reduced ability to stimulate IL-12 signaling in NK cells, while substantially retaining the ability to stimulate IL-12 signaling in CD8+ T cells.

[0077] In one embodiment, several embodiments of the present disclosure relate to a recombinant polypeptide comprising: (a) an amino acid sequence having at least 70% sequence identity with the IL-12p40 polypeptide having the amino acid sequence of SEQ ID NO: 1; and further (b) one or more amino acid substitutions in the sequence of SEQ ID NO: 1.

[0078] Non-limiting exemplary embodiments of recombinant polypeptides disclosed herein may include one or more of the following features: In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having 100% sequence identity with the sequence of SEQ ID NO: 1.

[0079] In some embodiments, the amino acid sequences of the recombinant polypeptides disclosed herein further include one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid sequences of the recombinant polypeptides further include about 1 to about 14 amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the recombinant polypeptide further includes about 1 to about 5, about 2 to about 8, about 3 to about 10, about 4 to about 12, about 5 to about 15, about 3 to about 5, about 7 to about 5, or about 3 to about 12 amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the recombinant polypeptide further includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 1.

[0080] In some embodiments, the amino acid sequences of the recombinant polypeptides disclosed herein further include one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid sequences of the recombinant polypeptides further include at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acid sequences at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO: 1. The exemplary IL-12p40 polypeptide variants of this disclosure may include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the recombinant polypeptide may include one, two, three, four, or five amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the recombinant polypeptide disclosed herein includes one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X81, X82, X106, X217, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the recombinant polypeptide disclosed herein includes a combination of amino acid substitutions at positions corresponding to amino acid residues X39, X40, X81, and X82 of SEQ ID NO: 1.

[0081] In accordance with this and other aspects of the Disclosure, such amino acid substitutions in the IL-12p40 polypeptide result in an IL-12p40 variant with a modified binding affinity to IL-12Rβ1 compared to the binding affinity of the parental IL-12p40 polypeptide lacking such substitutions. For example, the IL-12p40 polypeptide variants disclosed herein may have increased or decreased affinity to IL-12Rβ1, or may have an affinity for IL-12Rβ1 that is identical or similar to that of wild-type IL-12p40. The IL-12p40 polypeptide variants disclosed herein may also include conservative modifications and substitutions (e.g., those having minimal effect on the secondary or tertiary structure of the IL-12p40 variant) at other positions of IL-12p40. Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and by Argos in EMBO J, 8:779-785 (1989). For example, amino acids belonging to one of the following groups exhibit conservative changes: Group I: Ala, Pro, Gly, Gln, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Val, Ile, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0082] In some embodiments, amino acid substitutions in the amino acid sequence of the recombinant IL-12p40 polypeptide disclosed herein are independently selected from the group consisting of alanine (A) substitutions, arginine (R) substitutions, asparagine (N) substitutions, aspartic acid (D) substitutions, leucine (L) substitutions, lysine (K) substitutions, phenylalanine (F) substitutions, lysine substitutions, glutamine (Q) substitutions, glutamic acid (E) substitutions, serine (S) substitutions, and threonine (T) substitutions, and any combination thereof. Non-limiting examples of amino acid substitutions in the recombinant IL-12p40 polypeptide disclosed herein are provided in Table 1 below. [Table 1]

[0083] In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 70% sequence identity with the sequence of SEQ ID NO: 1, and further comprises amino acid substitutions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with the sequence of SEQ ID NO: 1, and further comprises amino acid substitutions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the recombinant polypeptide further includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 of SEQ ID NO: 1.

[0084] In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 in SEQ ID NO: 1. In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, E81, F82, K106, K217, and K219 in SEQ ID NO: 1. In some embodiments, the amino acid sequences of the recombinant polypeptides disclosed herein further include one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of E81, F82, K106, K217, and K219 in SEQ ID NO: 1. In some embodiments, the amino acid sequences of the recombinant polypeptides disclosed herein further include combinations of amino acid substitutions at positions corresponding to amino acid residues W37, P39, D40, E81, and F82 in SEQ ID NO: 1. In some embodiments, the amino acid sequence includes amino acid substitutions corresponding to amino acid residues E81, F82, K106, K217, and K219 of SEQ ID NO: 1. In some embodiments, the polypeptide of the present disclosure has at least 70% sequence identity with SEQ ID NO: 1 and further includes amino acid substitutions corresponding to the following: (a) W37A; (b) P39A; (c) D40A; (d) E81A; (e) F82A; (f) K106A; (g) D109A; (h) K217A; (i) K219A; (j) E81A / F82A; (k) W37A / E81A / F82A; (l) E81A / F82A / K106A; (m) E81A / F82A / K106A / K2 19A, (n)E81A / F82A / K106A / K217A, (o)81A / F82A / K106A / E108A / D115A, (p)E81F / F82A, (q)E81K / F82A, (r)E81L / F82A, (s)E81H / F82A, ( t)E81S / F82A, (u)E81A / F82A / K106N, (v)E81A / F82A / K106Q, (w)E81A / F82A / K106T, (x)E81A / F82A / K106R, or (y)P39A / D40A / E81A / F82A.In some embodiments, the polypeptides of the present disclosure have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 1, and further include amino acid substitutions corresponding to the following amino acid substitutions: (a) W37A; (b) P39A; (c) D40A; (d) E81A; (e) F82A; (f) K106A; (g) D109A; (h) K217A; (i) K219A; (j) E81A / F82A; (k) W37A / E81A / F82A; (l) E 81A / F82A / K106A, (m)E81A / F82A / K106A / K219A, (n)E81A / F82A / K106A / K217A, (o)81A / F82A / K106A / E108A / D115A, (p)E81F / F82A, (q)E81K / F82A, (r)E81L / F 82A, (s)E81H / F82A, (t)E81S / F82A, (u)E81A / F82A / K106N, (v)E81A / F82A / K10 6Q, (w)E81A / F82A / K106T, (x)E81A / F82A / K106R, or (y)P39A / D40A / E81A / F82A. In some embodiments, the polypeptides of the present disclosure comprise an amino acid sequence having 100% sequence identity with SEQ ID NO: 1, and further comprise amino acid substitutions corresponding to the following: (a) W37A; (b) P39A; (c) D40A; (d) E81A; (e) F82A; (f) K106A; (g) D109A; (h) K217A; (i) K219A; (j) E81A / F82A; (k) W37A / E81A / F82A; (l) E81A / F82A / K106A; (m) E81A / F82A / K106A / K219A, (n)E81A / F82A / K106A / K217A, (o)81A / F82A / K106A / E108A / D115A, (p)E81F / F82A, (q)E81K / F82A, (r)E81L / F82A, (s)E81H / F82A, (t)E81S / F82A, (u)E81A / F82A / K106N, (v)E81A / F82A / K106Q, (w)E81A / F82A / K106T, (x)E81A / F82A / K106R, or (y)P39A / D40A / E81A / F82A.In some embodiments, the recombinant polypeptide of the present disclosure comprises an IL-12p40 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-8 and 13-16, and an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity.

[0085] In another embodiment, some embodiments of the present disclosure relate to a recombinant polypeptide comprising: (a) an amino acid sequence having at least 70% sequence identity with the IL-12p40 polypeptide having the amino acid sequence of SEQ ID NO: 2; and further (b) one or more amino acid substitutions in the sequence of SEQ ID NO: 2. Non-limiting exemplary embodiments of the recombinant polypeptide of this embodiment may include one or more of the following features: In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence of SEQ ID NO: 2. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having 100% sequence identity with the sequence of SEQ ID NO: 2.

[0086] In some embodiments, the amino acid sequence of the recombinant polypeptide further includes one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the recombinant polypeptide further includes about 1 to about 14 amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the recombinant polypeptide further includes about 1 to about 5, about 2 to about 8, about 3 to about 10, about 4 to about 12, about 5 to about 15, about 3 to about 5, about 7 to about 5, or about 3 to about 12 amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the recombinant polypeptide further includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 amino acid sequences at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 2.

[0087] In some embodiments, the amino acid sequences of the recombinant polypeptides disclosed herein further include one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X39, X40, X81, X82, X106, X217, and X219 in SEQ ID NO: 2. In some embodiments, the amino acid sequences of the recombinant polypeptides further include at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X39, X40, X81, X82, X106, X217, and X219 in SEQ ID NO: 2. In some embodiments, the amino acid sequences of the recombinant polypeptides further include at least one, at least two, at least three, at least four, or at least five amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X39, X40, X81, X82, X106, X217, and X219 in SEQ ID NO: 2. In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of X81, X82, X106, and X217 in SEQ ID NO: 2. In some embodiments, the amino acid sequence includes a combination of amino acid substitutions at positions corresponding to amino acid residues X81, X82, and X106 in SEQ ID NO: 2. In some embodiments, the amino acid sequence includes a combination of amino acid substitutions at positions corresponding to amino acid residues X81, X82, X106, and X217 in SEQ ID NO: 2.

[0088] In accordance with this and other embodiments of the present disclosure, such amino acid substitutions in the IL-12p40 polypeptide result in an IL-12p40 variant with a modified binding affinity to IL-12Rβ1 compared to the binding affinity of the parental IL-12p40 polypeptide lacking such substitutions. For example, the IL-12p40 polypeptide variants disclosed herein may have increased or decreased affinity to IL-12Rβ1, or may have an affinity for IL-12Rβ1 that is identical or similar to that of wild-type IL-12p40. The IL-12p40 polypeptide variants disclosed herein may also include conservative modifications and substitutions (e.g., those having minimal effect on the secondary or tertiary structure of the IL-12p40 variant) at other positions of IL-12p40. Such conservative substitutions include those described in Dayhoff 1978 (mentioned above) and Argos 1989 (mentioned above). For example, amino acids belonging to one of the following groups exhibit conservative changes: Group I: Ala, Pro, Gly, Gln, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Val, Ile, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0089] In some embodiments, amino acid substitutions in the amino acid sequence of the recombinant IL-12p40 polypeptide disclosed herein are independently selected from the group consisting of alanine (A) substitutions, arginine (R) substitutions, asparagine (N) substitutions, aspartic acid (D) substitutions, leucine (L) substitutions, lysine (K) substitutions, phenylalanine (F) substitutions, lysine substitutions, glutamine (Q) substitutions, glutamic acid (E) substitutions, serine (S) substitutions, and threonine (T) substitutions, and any combination thereof. In some embodiments, amino acid substitutions in the amino acid sequence of the recombinant IL-12p40 polypeptide disclosed herein include alanine substitutions. Non-limiting examples of amino acid substitutions in the recombinant IL-12p40 polypeptide disclosed herein are provided in Table 2 below. [Table 2]

[0090] In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 70% sequence identity with the sequence of SEQ ID NO: 2, and further comprises amino acid substitutions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and E219 of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with the sequence of SEQ ID NO: 2, and further comprises amino acid substitutions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and E219 of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the recombinant polypeptide further includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and E219 of SEQ ID NO: 2.

[0091] In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and E219 in SEQ ID NO: 2. In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, E81, F82, K106, K217, and E219 in SEQ ID NO: 2. In some embodiments, the amino acid sequence is located at positions corresponding to amino acid residues selected from the group consisting of E81, F82, K106, and K217 in SEQ ID NO: 2. In some embodiments, the amino acid sequence includes a combination of amino acid substitutions at positions corresponding to amino acid residues E81, F82, and K106 in SEQ ID NO: 2. In some embodiments, the amino acid sequence includes a combination of amino acid substitutions at positions corresponding to amino acid residues E81, F82, K106, and K217 of SEQ ID NO: 2. In some embodiments, the polypeptide of the present disclosure includes an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2, and further includes amino acid substitutions corresponding to the following: (a) W37A; (b) P39A; (c) D40A; (d) E81A; (e) F82A; (f) K106A; (g) D109A; (h) K217A; (i) E219A; (j) E81A / F82A; (k) W37A / E81A / F82A; (l) E81A / F82A / K106A, (m)E81A / F82A / K106A / K217A, (n)E81F / F82A, (o)E81K / F82A, (p)E81L / F82A, (q)E81H / F82A, (r)E81S / F82A , (s)E81A / F82A / K106N, (t)E81A / F82A / K106Q, (u)E81A / F82A / K106T, (v)E81A / F82A / K106R, or (w)P39A / D40A / E81A / F82A.

[0092] In some embodiments, the polypeptides of the present disclosure have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 2, and further include amino acid substitutions corresponding to the following: (a) W37A; (b) P39A, (c) D40A, (d) E81A, (e) F82A, (f) K106A, (g) D109A, (h) K217A, (i) E219A, (j) E81A / F82A, (k)W37A / E81A / F82A, (l)E81A / F82A / K106A, (m)E81A / F82A / K1 06A / K217A, (n)E81F / F82A, (o)E81K / F82A, (p)E81L / F82A, (q)E81H / F8 2A, (r)E81S / F82A, (s)E81A / F82A / K106N, (t)E81A / F82A / K106Q, (u)E 81A / F82A / K106T, (v)E81A / F82A / K106R, or (w)P39A / D40A / E81A / F82A. In some embodiments, the polypeptides of the present disclosure comprise an amino acid sequence having 100% sequence identity with SEQ ID NO: 2, and further comprise amino acid substitutions corresponding to the following: (a) W37A; (b) P39A; (c) D40A; (d) E81A; (e) F82A; (f) K106A; (g) D109A; (h) K217A; (i) E219A; (j) E81A / F82A; (k) W37A / E81A / F82A; (l) E81A / F8 2A / K106A, (m)E81A / F82A / K106A / K217A, (n)E81F / F82A, (o)E81K / F82A, (p)E81L / F82A, (q)E81H / F82A, (r)E81S / F82A, ( s)E81A / F82A / K106N, (t)E81A / F82A / K106Q, (u)E81A / F82A / K106T, (v)E81A / F82A / K106R, or (w)P39A / D40A / E81A / F82A. In some embodiments, the recombinant polypeptide of the present disclosure comprises an IL-12p40 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 9-11 and 17-25, and an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity.

[0093] In some embodiments, amino acid substitutions in the sequence of the recombinant IL-12p40 polypeptide disclosed herein result in a modified affinity of the recombinant IL-12p40 polypeptide to IL-12Rβ1 and modulate the binding of IL-12p40 to IL-12Rβ1. The term “modulate” with respect to the binding activity of the IL-12p40 polypeptide refers to a change in the binding affinity of the polypeptide to IL-12Rβ1. Modulation includes both an increase (e.g., induction, stimulation) and a decrease (e.g., reduction, inhibition) of the polypeptide, or other effects that affect the binding affinity of the polypeptide. In some embodiments, the amino acid substitution increases the IL-12Rβ1 binding affinity of the recombinant IL-12p40 polypeptide compared to a reference IL-12p40 polypeptide lacking the amino acid substitution. In some embodiments, the amino acid substitution decreases the IL-12Rβ1 binding affinity of the recombinant IL-12p40 polypeptide compared to a reference IL-12p40 polypeptide lacking the amino acid substitution.

[0094] The binding activity of the recombinant polypeptides of this disclosure, including the IL-12p40 polypeptide variant described herein, can be measured by any suitable method known in the art. For example, the binding activity of the IL-12p40 polypeptide variant disclosed herein and its homologous ligands (e.g., IL-12Rβ1, IL-p35, and IL-22p19) can be determined by scatchard analysis (Munsen et al. Analyt. Biochem. 107:220-239, 1980). Specific binding can also be evaluated using techniques known in the art, including, but not limited to, competitive ELISA, Biacore® assays, and / or KinExA® assays. Polypeptides that preferentially or specifically bind to a target ligand are a well-understood concept in the art, and methods for determining such specific or preferential binding are also known in the art.

[0095] Various assay formats can be used to select recombinant IL-12p40 polypeptides that bind to target ligands (e.g., IL-12Rβ1, IL-p35, and / or IL-22p19). For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore® (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet® (ForteBio, Inc., Menlo Park, CA), and Western blot analysis are some of the many assays that can be used to identify polypeptides that specifically react with the receptor or its ligand-binding moiety, or that specifically bind to a homologous ligand or binding partner. Generally, specific or selective binding reactions occur at least twice the background signal or noise, more commonly more than 10 times the background, more than 20 times the background, more commonly more than 50 times the background, more than 75 times the background, more than 100 times the background, more typically more than 500 times the background, more typically more typically more than 1000 times the background, and more typically more than 10,000 times the background.

[0096] Those skilled in the art will understand that binding affinity can also be used as a measure of the strength of a non-covalent interaction between two binding partners, for example, an IL-12p40 polypeptide and an IL-12Rβ1 polypeptide. In some cases, binding affinity is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules is the dissociation constant (K). D It can be quantified by measuring K. D This can be determined, for example, by measuring the dynamics of complex formation and dissociation using surface plasmon resonance (SPR) spectroscopy (Biacore). The rate constants corresponding to the association and dissociation of monovalent complexes are the association rate constant k, respectively. a (or k on ) and the dissociation rate constant k d (or k off ) is called K D is, formula K D =kd / k a by k a and k d are associated. The value of the dissociation constant can be determined directly by well-known methods and can also be calculated for complex mixtures, for example, by the method shown in Caceci et al. (Byte 9: 340-362, 1984). For example, K D can be established using a double-filter nitrocellulose filter binding assay as disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays for evaluating the binding ability of the IL-12p40 polypeptide variant of the present disclosure to the target receptor are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays shown in the examples. The binding kinetics and binding affinity of the IL-12p40 polypeptide variant can be evaluated using standard assays known in the art, such as surface plasmon resonance (SPR), for example, the Biacore™ system or KinExA. In some embodiments, the binding affinity of the IL-12p40 polypeptide variant of the present disclosure for IL-12Rβ1, IL-p35, and / or IL-23p19 is determined by a solid-phase receptor binding assay (Matrosovich MN et al., Methods Mol Biol. 865:71-94, 2012). In some embodiments, the binding affinity of the IL-12p40 polypeptide variant of the present disclosure for IL-12Rβ1, IL-p35, and / or IL-23p19 is determined by a surface plasmon resonance (SPR) assay.

[0097] In some embodiments, amino acid substitutions in the sequence of the recombinant IL-12p40 polypeptide disclosed herein reduce the IL-12Rβ1 binding affinity of the recombinant IL-12p40 polypeptide by about 10% to about 100% compared to a reference IL-12p40 polypeptide lacking the amino acid substitution. In some embodiments, the recombinant IL-12p40 polypeptide has a binding affinity to IL-12Rβ1 that is reduced by at least 10%, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or at least about 95%, compared to a reference IL-12p40 polypeptide lacking the amino acid substitution. In some embodiments, recombinant IL-12p40 polypeptides have a binding affinity to IL-12Rβ1 that is reduced by about 10% to about 50%, e.g., about 20% to about 70%, about 30% to about 80%, about 40% to about 90%, about 50% to about 100%, about 20% to about 50%, about 40% to about 70%, about 30% to about 60%, about 40% to about 100%, about 20% to about 80%, or about 10% to about 90%, compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the binding affinity of the IL-12p40 polypeptide variants of this disclosure to IL-12Rβ1, IL-p35, and / or IL-23p19 is determined by surface plasmon resonance (SPR) assay.

[0098] In some embodiments, the recombinant IL-12p40 polypeptides disclosed herein, when combined with the IL-12p35 polypeptide, exhibit reduced ability to stimulate STAT4 signaling compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the ability of the IL-12p40 polypeptide variant to stimulate STAT4 signaling is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 95% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the ability of the IL-12p40 polypeptide variant to stimulate STAT4 signaling is reduced by about 10% to about 100% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the ability of IL-12p40 polypeptide variants to stimulate STAT4 signaling is reduced by approximately 10% to 50%, 20% to 70%, 30% to 80%, 40% to 90%, 50% to 100%, 20% to 50%, 40% to 70%, 30% to 60%, 40% to 100%, 20% to 80%, or 10% to 90% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions.

[0099] In some embodiments, the recombinant IL-12p40 polypeptides disclosed herein, when combined with the IL-12p35 polypeptide, exhibit reduced ability to stimulate STAT3 signaling compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the ability of recombinant IL-12p40 polypeptide variants to stimulate STAT3 signaling is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 95% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the ability of recombinant IL-12p40 polypeptide variants to stimulate STAT3 signaling is reduced by about 10% to about 100% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the ability of IL-12p40 polypeptide variants to stimulate STAT4 signaling is reduced by approximately 10% to 50%, 20% to 70%, 30% to 80%, 40% to 90%, 50% to 100%, 20% to 50%, 40% to 70%, 30% to 60%, 40% to 100%, 20% to 80%, or 10% to 90% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions.

[0100] In principle, there are no specific limitations on the assays and methods that can be used to determine STAT3 signaling and / or STAT4 signaling. Exemplary methodologies suitable for the compositions and methods disclosed herein include, but are not limited to, phosphoflow signaling assays, enzyme-linked immunosorbent assays (ELISAs), and any techniques known in the art for assaying downstream gene expression. In some embodiments, the regulation of STAT3 signaling and / or STAT4 signaling can be determined by phosphoflow signaling assays, such as the phosphoflow cytometry assays described in Examples 4 and 5.

[0101] In some embodiments, the recombinant IL-12p40 polypeptide variants disclosed herein provide cell type-biased signaling of downstream signaling mediated by IL-12p40 compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the recombinant IL-12p40 polypeptide variants disclosed herein provide cell type-biased signaling of downstream signaling mediated by IL-12. In some embodiments, the recombinant IL-12p40 polypeptide variants disclosed herein provide cell type-biased signaling of downstream signaling mediated by IL-23. In some embodiments, the recombinant IL-12p40 polypeptide variants disclosed herein provide cell type-biased signaling of downstream signaling mediated by IL-12 and IL-23.

[0102] In the case of IL-12, as described in more detail below, the specific partial agonist IL-12p40 variant of this disclosure exhibits selectivity for T cells versus NK cells and is therefore predicted to be less toxic than natural IL-12, which is being clinically developed for cancer by many companies, with its toxicity being the limiting factor. While not bound by theory, these IL-12 partial agonists are thought to have therapeutic utility in cancer immunotherapy by decoupled from the toxicity associated with cytokine pleiotropy. As shown in Example 4 below, the specific IL-12 partial agonists disclosed herein exhibit reduced affinity for IL-12Rβ1, which retains activity for antigen-specific CD8+ T cells but shows reduced (e.g., impaired) stimulation of NK cells. Since NK cell-mediated IFNγ is thought to be the cause of IL-12 toxicity, these novel agonists are predicted to maintain the antitumor effect of IL-12 stimulation with lower toxicity. In the case of IL-23, the partial agonist IL-12p40 variant of the present disclosure is thought to have therapeutic utility in the treatment of autoimmune diseases by enabling stepwise control of IL-23 signaling.

[0103] Complementing current therapeutic approaches that rely on antibody blockade of IL-12p40, which inhibits IL-12 and IL-23 signaling, the partial agonist IL-12p40 variants of the present disclosure can be used to specifically modulate IL-23 signaling without affecting IL-12 by modulating the affinity of IL-23 for IL-12Rβ1.

[0104] Accordingly, some embodiments of the present disclosure provide recombinant IL-12p40 polypeptides that confer cell-type biased signaling of IL-12-mediated downstream signaling compared to a reference IL-12 polypeptide lacking amino acid substitutions, where cell-type biased signaling includes a reduced ability of the recombinant polypeptide to stimulate IL-12-mediated signaling in NK cells. In some embodiments, the ability of the recombinant IL-12p40 polypeptide variants disclosed herein to stimulate IL-12-mediated signaling in NK cells is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 95% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, cell-type biased signaling includes the substantially unmodified ability of the recombinant polypeptide to stimulate IL-12 signaling in CD8+ T cells. In some embodiments, the ability of the recombinant IL-12p40 polypeptide variants disclosed herein to stimulate IL-12-mediated signaling in CD8+ T cells remains unchanged, and is the same or substantially the same as, for example, a reference IL-12p40 polypeptide lacking an amino acid. In some embodiments, the recombinant IL-12p40 polypeptide variants disclosed herein confer a reduced ability of the T cell polypeptide to stimulate IL-12 signaling in NK cells, but substantially retain the ability to stimulate IL-12 signaling in CD8+ T cells, promoting antigen-specific death of target cells, as described in Example 5 below.

[0105] B. Nucleic acid In one embodiment, various nucleic acid molecules comprising nucleotide sequences encoding the recombinant IL-12p40 polypeptide of the Disclosure are provided herein, including expression cassettes and expression vectors, the expression vectors comprising these nucleic acid molecules operably ligated to heterologous nucleic acid sequences, such as regulatory sequences enabling in vivo expression of the recombinant IL-12p40 polypeptide in host cells or ex vivo cell-free expression systems.

[0106] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to both RNA molecules and DNA molecules, including nucleic acid molecules containing cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules may be double-stranded or single-stranded (e.g., sense strand or antisense strand). Nucleic acid molecules may contain non-conventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein refer interchangeably to sequences of polynucleotide molecules. Polynucleotide sequences and polypeptide sequences disclosed herein are indicated by reference using standard letter abbreviations for nucleotide bases and amino acids in 37 CFR §1.82, incorporating by reference WIPO standard ST.25 (1998), Appendix 2, Tables 1-6.

[0107] The nucleic acid molecules of this disclosure may be nucleic acid molecules of any length, including nucleic acid molecules that are generally about 0.5Kb to about 20Kb, for example about 0.5Kb to about 20Kb, about 1Kb to about 15Kb, about 2Kb to about 10Kb, or about 5Kb to about 25Kb, for example about 10Kb to about 15Kb, about 15Kb to about 20Kb, about 5Kb to about 20Kb, about 5Kb to about 10Kb, or about 10Kb to about 25Kb.

[0108] In some embodiments disclosed herein, the nucleic acid molecules of the Disclosure comprise a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of a recombinant polypeptide disclosed herein and an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity. In some embodiments, the nucleic acid molecules of the Disclosure comprise a nucleotide sequence encoding a polypeptide comprising: (a) the IL-12p40 polypeptide having the amino acid sequence of SEQ ID NO: 1 and an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity; and further (b) one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid sequence includes amino acid sequences corresponding to amino acid residues X81, X82, X106, X217, and X219 of SEQ ID NO: 1. In some embodiments, the amino acid sequence includes combinations of amino acid substitutions at positions corresponding to amino acid residues X39, X40, X81, and X82 of SEQ ID NO: 1. In some embodiments, the nucleic acid molecule of the present disclosure comprises a nucleotide sequence encoding a polypeptide having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 1, and further comprises a nucleotide sequence encoding a polypeptide further comprising amino acid substitutions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 of SEQ ID NO: 1. In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, E81, F82, K106, K217, and K219 of SEQ ID NO: 1.In some embodiments, the amino acid sequence includes amino acid substitutions corresponding to amino acid residues E81, E82, K106, K217, and K219 of SEQ ID NO: 1. In some embodiments, the amino acid sequence includes combinations of amino acid substitutions at positions corresponding to amino acid residues P39, D40, E81, and F82 of SEQ ID NO: 1. In some embodiments, the nucleic acid molecules of the present disclosure include a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1, and further comprising amino acid substitutions corresponding to the following: (a) W37A, (b) P39A, (c) D40A, (d) E81A, (e) F82A, (f) K106A, (g) D109A, (h) K217A, (i) K219A, (j) E81A / F82A, (k) W37A / E81A / F82A, (l) E81A / F82A / K106A, (m) E8 1A / F82A / K106A / K219A, (n)E81A / F82A / K106A / K217A, (o)81A / F82A / K10 6A / E108A / D115A, (p)E81F / F82A, (q)E81K / F82A, (r)E81L / F82A, (s)E81 H / F82A, (t)E81S / F82A, (u)E81A / F82A / K106N, (v)E81A / F82A / K106Q, (w )E81A / F82A / K106T, (x)E81A / F82A / K106R, or (y)P39A / D40A / E81A / F82A. In some embodiments, the nucleic acid molecule of the present disclosure comprises an IL-12p40 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-8 and 13-16, and a nucleotide sequence encoding a polypeptide having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity of the amino acid sequence.

[0109] In some embodiments, the nucleic acid molecule of the present disclosure comprises a nucleotide sequence encoding a polypeptide comprising: (a) an IL-12p40 polypeptide having the amino acid sequence of SEQ ID NO: 2 and an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity; and further (b) one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO: 2. In some embodiments, the polypeptide further comprises additional amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO: 2. In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of X81, X82, X106, and X217 of SEQ ID NO: 2. In some embodiments, the amino acid sequence includes a combination of amino acid substitutions at positions corresponding to amino acid residues X81, X82, and X106 of SEQ ID NO: 2. In some embodiments, the amino acid sequence includes a combination of amino acid substitutions at positions corresponding to amino acid residues X81, X82, X106, and X217 of SEQ ID NO: 2. In some embodiments, the nucleic acid molecule of the present disclosure includes a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 2, and further comprising amino acid substitutions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and E219 of SEQ ID NO: 2. In some embodiments, the amino acid sequence includes additional amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, E81, F82, K106, K217, and E219 in SEQ ID NO: 2. In some embodiments, the amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of E81, F82, K106, and K217 in SEQ ID NO: 2.In some embodiments, the amino acid sequence includes a combination of amino acid substitutions at the positions corresponding to amino acid residues E81, F82, and K106 of SEQ ID NO: 2. In some embodiments, the amino acid sequence includes a combination of amino acid substitutions at the positions corresponding to E81, F82, K106, and K217 of SEQ ID NO: 2.

[0110] In some embodiments, the nucleic acid molecules of the present disclosure include a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 2, and further comprising amino acid substitutions corresponding to the following: (a) W37A; (b) P39A, (c) D40A, (d) E81A, (e) F82A, (f) K106A, (g) D109A, (h) K217A, (i) K219A, (j) E81A / F82A, (k) W37A / E81A / F 82A, (l)E81A / F82A / K106A, (m)E81A / F82A / K106A / K217A, (n)E81A / F82A, (o)81K / F82A, (p)E81L / F82A, (q)E81H / F82A, (r)E81s / F82A, (s)E81A / F82A / K106N, (t)E81A / F82A / K106Q, (u)E81A / F82A / K106T, (v)E81A / F82A / K106R, or (w)P39A / D40A / E81A / F82A. In some embodiments, the nucleic acid molecules of the present disclosure include an IL-12p40 polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs. 9-11 and 17-25, and a nucleotide sequence encoding a polypeptide having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with the amino acid sequence.

[0111] In some embodiments, nucleotide sequences are incorporated into an expression cassette or expression vector. An expression cassette is generally understood to include a construct of genetic material comprising a coding sequence and sufficient regulatory information to direct the correct transcription and / or translation of the coding sequence in recipient cells in vivo and / or ex vivo. Generally, an expression cassette can be inserted into a vector to target a desired host cell and / or individual. Thus, in some embodiments, an expression cassette of the present disclosure comprises a coding sequence of a recombinant polypeptide disclosed herein, which is operably linked to an expression regulatory element such as a promoter and, optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0112] In some embodiments, a nucleotide sequence is incorporated into an expression vector. Those skilled in the art will understand that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells and can be used for the purpose of transformation, for example, for the introduction of heterologous DNA into host cells. Thus, in some embodiments, the vector may be a replicon such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted to result in replication of the inserted segment. In some embodiments, the expression vector may be an integration vector.

[0113] In some embodiments, the expression vector may be a viral vector. As will be understood by those skilled in the art, the term “viral vector” is broadly used to refer typically to a nucleic acid molecule (e.g., a transfer plasmid) containing a virus-derived nucleic acid element that facilitates the movement of nucleic acid molecules or their integration into a cell’s genome, or to a viral particle that mediates the movement of nucleic acids. Viral particles typically contain various viral components in addition to nucleic acids, and sometimes host cell components as well. The term viral vector may refer to either a virus or viral particle that can move nucleic acids into a cell, or the moved nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. In some embodiments, viral vectors are baculovirus vectors, retrovirus vectors, or lentivirus vectors. The term “retrovirus vector” refers to a viral vector or plasmid containing structural and functional genetic elements or parts thereof, primarily derived from retroviruses. The term “lentivirus vector” refers to a viral vector or plasmid containing structural and functional genetic elements or parts thereof, primarily derived from lentiviruses, which belong to the retrovirus genus.

[0114] Accordingly, vectors, plasmids, or viruses comprising one or more nucleic acid molecules encoding any recombinant polypeptide or IL-12p40 polypeptide variant disclosed herein are also provided herein. Nucleic acid molecules may be contained within a vector, for example, which can direct their expression in cells transformed / transduced with the vector. Vectors suitable for use in eukaryotic and prokaryotic cells are known in the art, commercially available, or readily prepared by those skilled in the art.

[0115] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Appropriate methods for transforming or transfecting cells can be found in Sambrook et al. (2012, cited above) and other standard molecular biology laboratory manuals, and include, for example, calcium phosphate transfection, DEAE-dextran-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.

[0116] Viral vectors available for use in this disclosure include, for example, baculovirus vectors, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpesviruses, Simian virus 40 (SV40), and bovine papillomavirus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY). For example, the chimeric receptors disclosed herein can be produced in eukaryotic cells, such as mammalian cells (e.g., COS cells, NIH3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). When selecting an expression system, care must be taken to ensure that the components are compatible with each other. Those skilled in the art can make such a decision. Furthermore, if guidance is needed when selecting an expression system, those skilled in the art can refer to P. Jones, “Vectors: Cloning Applications”, John Wiley and Sons, New York, NY, 2009).

[0117] The nucleic acid molecules provided may contain sequences that are naturally occurring, or sequences that are different from naturally occurring sequences but, due to the degenerate nature of the gene code, may encode the same polypeptide, such as an antibody. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., those produced by phosphoamidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, the nucleic acid molecules may be double-stranded or single-stranded (e.g., either a sense strand or an antisense strand).

[0118] Nucleic acid molecules are not limited to sequences that encode polypeptides (e.g., antibodies), but may also include some or all of non-coding sequences located upstream or downstream of coding sequences (e.g., the coding sequence of a chimeric receptor). Those skilled in molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can be produced, for example, by treating genomic DNA with restriction endonucleases or by performing polymerase chain reactions (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0119] In another embodiment, cell cultures and media containing at least one recombinant cell disclosed herein are provided herein. Generally, the media may be any suitable medium for culturing the cells described herein. Techniques for transforming a wide variety of the aforementioned cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures containing at least one recombinant cell disclosed herein are also within the scope of this application. Suitable methods and systems for generating and maintaining cell cultures are known in the art.

[0120] C. Recombinant cells and cell cultures The recombinant nucleic acids of this disclosure can be introduced into cells such as human T lymphocytes to produce recombinant cells containing nucleic acid molecules. The introduction of the nucleic acid molecules of this disclosure into cells can be achieved by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery.

[0121] Therefore, in some embodiments, nucleic acid molecules can be delivered by viruses or nonviral delivery vehicles known in the art. For example, nucleic acid molecules can be stably incorporated into the genome of recombinant cells, replicate episomalally, or exist in recombinant cells as minicircle expression vectors for transient expression. Therefore, in some embodiments, nucleic acid molecules are maintained and replicated as episomal units within recombinant host cells. In some embodiments, nucleic acid molecules are stably incorporated into the genome of recombinant cells. Stable integration can be achieved using classical random genome recombination techniques or more precise techniques, such as guide RNA-directed CRISPR / Cas9 genome editing, DNA-induced endonuclease genome editing using NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing. t ranscription u ctivator- l ike e ffector n This can be achieved using methods such as ucleases. In some embodiments, nucleic acid molecules are present in recombinant cells as minicircle expression vectors for transient expression.

[0122] Nucleic acid molecules can be encapsulated in viral capsids or lipid nanoparticles, or delivered by viral or nonviral delivery means and methods known in the art, such as electroporation. For example, the introduction of nucleic acids into cells can be achieved by viral transduction. In non-limiting examples, baculoviruses or adeno-associated viruses (AAVs) can be modified to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all known serotypes can infect cells of multiple diverse tissue types. AAVs can be transduced in vivo into a wide range of species and tissues without evidence of toxicity, generating relatively mild innate and adaptive immune responses.

[0123] Lentiviral vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue affinity, including target cell types for important gene and cell therapies; (iv) no viral protein expression after vector transduction; (v) the ability to deliver complex genetic elements such as polycistron sequences or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively simple system for vector manipulation and production.

[0124] In some embodiments, host cells can be modified (e.g., transfected, transformed, or otherwise modified) using the vector construct of the Application, which may be a viral vector or vector for homologous recombination that contains a nucleic acid sequence homologous to a portion of the host cell's genome, or an expression vector for the polypeptide of interest. The host cell may be either an untransformed cell or a cell that has already been transfected with at least one nucleic acid molecule.

[0125] In some embodiments, recombinant cells are prokaryotic or eukaryotic cells. In some embodiments, the cells are in vivo. In some embodiments, the cells are ex vivo. In some embodiments, the cells are in vitro. In some embodiments, recombinant cells are eukaryotic cells. In some embodiments, recombinant cells are animal cells. In some embodiments, animal cells are mammalian cells. In some embodiments, animal cells are human cells. In some embodiments, cells are non-human primate cells. In some embodiments, recombinant cells are immune system cells, such as lymphocytes (e.g., T cells or NK cells), or dendritic cells. In some embodiments, immune cells include B cells, monocytes, natural killer (NK) cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, regulatory T cells, and helper T cells (T). H ), cytotoxic T cells (T CTL ), or other T cells. In some embodiments, the immune system cells are T lymphocytes. In some embodiments, the cells can be obtained by leukocyte apheresis performed on a sample obtained from a subject. In some embodiments, the subject is a human subject. In some embodiments, the human subject is a patient.

[0126] Non-limiting examples of suitable cell lines include Trichoplusia ni cells, Spodotera frugiperda insect cells, Expi293F cells, N-acetylglucosaminyltransferase I (GnTI) deficient HEK293 cells, HEK-293T (ATCC #CRL-3216), HT-29 (ATCC #HTB-38), Panc-1 (ATCC #CRL-1469), HepG2 (ATCC #HB-8065), and EC4 cells.

[0127] In another embodiment, cell cultures and culture media comprising at least one recombinant cell disclosed herein are provided herein. Generally, the culture media may be any suitable medium for culturing the cells described herein. Techniques for transforming a wide variety of the aforementioned cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures comprising at least one recombinant cell disclosed herein are also within the scope of this application. Suitable methods and systems for generating and maintaining cell cultures are known in the art.

[0128] Method for producing D.IL-12p40 polypeptide In another embodiment, some embodiments of the present disclosure relate to various methods for producing recombinant polypeptides of the present disclosure, the methods including: (a) providing one or more recombinant cells of the present disclosure; and culturing the recombinant cells in a culture medium so that the cells produce polypeptides encoded by recombinant nucleic acid molecules. Accordingly, recombinant polypeptides produced by the methods disclosed herein are also within the scope of the present disclosure.

[0129] Non-limiting exemplary embodiments of the disclosed method for producing recombinant polypeptides may include one or more of the following features: In some embodiments, the method further includes isolating and / or purifying the produced polypeptide. In some embodiments, the method for producing recombinant polypeptides of the disclosure further includes isolating and / or purifying the produced polypeptide. In some embodiments, the method for producing polypeptides of the disclosure further includes modifying the structure of the produced polypeptide to extend its half-life.

[0130] In some embodiments, modifications include one or more modifications selected from the group consisting of fusion to a human Fc antibody fragment, fusion to albumin, and PEGylation. For example, any of the recombinant polypeptides disclosed herein can be prepared as a fusion or chimeric polypeptide comprising a recombinant polypeptide and a heterologous polypeptide (e.g., a polypeptide that is neither IL-12p40 nor a variant thereof). Exemplary heterologous polypeptides can extend the circulating half-life of the chimeric polypeptide in vivo and thus further enhance the properties of the recombinant polypeptides disclosed herein. In various embodiments, the heterologous polypeptide that extends the circulating half-life may be serum albumin, such as human serum albumin, or the Fc region of an IgG subclass of an antibody lacking an IgG heavy chain variable region. The exemplary Fc region may contain mutations that inhibit complement binding and Fc receptor binding, or it may be soluble and, for example, able to bind to complement, or able to lyse cells via another mechanism such as antibody-dependent complement lysis (ADCC).

[0131] In some embodiments, the “Fc region” may be a native or synthetic polypeptide homologous to the C-terminal domain of IgG produced by the digestion of IgG with papain. The molecular weight of IgG Fc is approximately 50 kDa. The recombinant fusion polypeptides of this disclosure may include the entire Fc region or a smaller portion thereof that retains the ability to extend the cyclic half-life of the chimeric polypeptide in which it is a part. Furthermore, the full-length or fragmented Fc region may be variants of the wild-type molecule; that is, they may include mutations that may or may not affect the function of the polypeptide. As will be further discussed below, native activity is not necessarily required or desirable in all cases. In some embodiments, the recombinant fusion protein (e.g., the IL-12p40 partial agonist or antagonist described herein) includes an IgG1, IgG2, IgG3, or IgG4 Fc region.

[0132] The Fc region can be "soluble" or "insoluble," but is typically insoluble. Insoluble Fc regions typically lack a high-affinity Fc receptor binding site and a C'1q binding site. The high-affinity Fc receptor binding site of mouse IgG Fc contains a Leu residue at position 235 of IgG Fc. Therefore, the Fc receptor binding site can be disrupted by mutation or deletion of Leu235. For example, substituting Leu235 with Glu inhibits the ability of the Fc region to bind to the high-affinity Fc receptor. The mouse C'1q binding site can be functionally disrupted by mutation or deletion of the Glu318, Lys320, and Lys322 residues of IgG. For example, substituting Glu318, Lys320, and Lys322 with Ala residues prevents IgG1 Fc from instructing antibody-dependent complement lysis. In contrast, the soluble IgG Fc region possesses a high-affinity Fc receptor binding site and a C'1q binding site. The high-affinity Fc receptor binding site contains a Leu residue at position 235 of IgG Fc, and the C'1q binding site contains Glu318, Lys320, and Lys322 residues of IgG1. Soluble IgG Fc has wild-type residues or conserved amino acid substitutions at these sites. Soluble IgG Fc can target cells for antibody-dependent cell-mediated cytotoxicity or complement-directed cell lysis (CDC). Appropriate mutations in human IgG are also known (see, e.g., Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2:125, 1994).

[0133] In other embodiments, the recombinant fusion polypeptide may include the recombinant IL-12p40 polypeptide of the Disclosure and a polypeptide that functions as an antigen tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated, highly specific anti-FLAG antibody. In some embodiments, the recombinant fusion polypeptide further includes a C-terminal c-myc epitope tag.

[0134] In other embodiments, the recombinant fusion polypeptide comprises the recombinant IL-12p40 polypeptide of the Disclosure and a heterologous polypeptide that functions to enhance the expression of the IL-12p40 polypeptide, such as an Aga2p agglutinin subunit, or to direct its cellular localization.

[0135] In other embodiments, a fusion polypeptide comprising the recombinant IL-12p40 polypeptide and an antibody or its antigen-binding moiety can be generated. The antibody or antigen-binding component of the chimeric protein can act as a targeting moiety. For example, this can be used to localize the chimeric protein to a specific subset of cells or target molecules. Methods for generating cytokine-antibody chimeric polypeptides are known in the art.

[0136] In some embodiments, the recombinant IL-12p40 polypeptide of the present disclosure can be modified with one or more polyethylene glycol (PEG) molecules to extend its half-life. As used herein, the term "PEG" means polyethylene glycol molecule. In a typical form, PEG is a linear polymer having terminal hydroxyl groups, with the formula HO-CH2CH2-(CH2CH2O) n It has a -CH2CH2-OH group, where n is approximately 8 to 4000.

[0137] Generally, "n" is not a discrete value but constitutes a range with a roughly Gaussian distribution around the mean. Terminal hydrogens may be substituted with capping groups such as alkyl or alkanol groups. PEG may have at least one hydroxyl group, more preferably a terminal hydroxyl group. This hydroxyl group may be attached to a linker moiety that can react with the peptide to form a covalent bond. Numerous derivatives of PEG exist in the art. PEG molecules covalently attached to the recombinant IL-12p40 polypeptide of this disclosure may have an average molecular weight of about 10,000, 20,000, 30,000, or 40,000 daltons. The PEGylated reagent may be a linear or branched molecule and may exist alone or in tandem. The PEGylated IL-12p40 polypeptide of this disclosure may have tandem PEG molecules attached to the C-terminus and / or N-terminus of the peptide. As used herein, the term "PEGylated" means the covalent attachment of one or more PEG molecules to a molecule such as the IL-12p40 polypeptide of this disclosure, as described above. In some embodiments, recombinant polypeptides of the present disclosure, such as IL-12p40(p40) variant polypeptides, may be PEGylated at one or more positions corresponding to W37, P39, D40, K80, K106, E108, D115, H216, and K217 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0138] E. Pharmaceutical Compositions The recombinant polypeptides, nucleic acids, recombinant cells, and / or cell cultures of this disclosure can be incorporated into compositions comprising pharmaceutical compositions. Such compositions generally comprise one or more recombinant polypeptides, nucleic acids, recombinant cells, and / or cell cultures provided and described herein, as well as pharmaceutically acceptable excipients, such as carriers. In some embodiments, the pharmaceutical compositions of this disclosure are formulated to treat, prevent, or improve diseases such as cancer, or to mitigate or delay the onset of diseases.

[0139] Accordingly, one aspect of the present disclosure relates to a pharmaceutical composition comprising one or more of the following: (a) recombinant polypeptides of the present disclosure; (b) recombinant nucleic acids of the present disclosure; (c) recombinant cells of the present disclosure; and (d) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises (a) recombinant polypeptides of the present disclosure and (b) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises (a) recombinant cells of the present disclosure and (b) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises (a) recombinant nucleic acids of the present disclosure and (b) a pharmaceutically acceptable carrier. In some embodiments, the recombinant nucleic acids are encapsulated in a viral capsid or lipid nanoparticles.

[0140] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and stored in a manner protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium, and includes, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and using surfactants such as sodium dodecyl sulfate. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, the composition will commonly include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, and / or sodium chloride. Sustained absorption of the injectable composition is achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0141] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent containing, if necessary, one or a combination of the components listed above, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which give the active ingredient powder, in addition to any additional desired components from its pre-sterilized filtered solution.

[0142] In some embodiments, the recombinant polypeptides covered by this disclosure are prepared using controlled-release formulations, including carriers such as implants and microencapsulation delivery systems, that protect the recombinant polypeptides from rapid elimination from the body. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Such formulations can be prepared using standard techniques. Liposome suspensions (containing liposomes that target infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0143] As described in more detail below, the recombinant polypeptides of this disclosure may also be modified to achieve an extended duration of action, such as by PEGylation, acylation, Fc fusion, or binding to molecules such as albumin. In some embodiments, the recombinant polypeptides may be further modified to extend their half-lives in vivo and / or ex vivo. Non-limiting examples of known strategies and methodologies suitable for modifying the recombinant polypeptides of this disclosure include (1) chemical modification of the recombinant polypeptides described herein with highly soluble polymers such as polyethylene glycol ("PEG") to prevent the recombinant polypeptides from contacting proteases, and (2) covalently linking or binding the recombinant polypeptides described herein with stable proteins such as albumin. Thus, in some embodiments, the recombinant polypeptides of this disclosure may be fused to stable proteins such as albumin. Human albumin, for example, is known to be one of the most effective proteins to enhance the stability of polypeptides fused thereto, and many such fusion proteins have been reported.

[0144] Acylation In some embodiments, one or both components of the dimerized IL-12 or IL-23 polypeptide, including the IL-12p40 polypeptide variant polypeptide of this disclosure, can be acylated by binding to fatty acid molecules described in Resh (2016) Progress in Lipid Research 63:120-131. Examples of fatty acids that can be bound include myristate, palmitate, and palmitoleic acid. Myristoylic acid is typically bound to the N-terminal glycine, but lysine may also be myristoylated. Palmitoylation is usually achieved by enzymatic modification of the free cysteine-SH group, as the DHHC protein catalyzes S-palmitoylation. Palmitoylation of serine and threonine residues is usually achieved enzymatically using the PORCN enzyme.

[0145] Acetylation In some embodiments, IL-12 or IL-23, including the IL-12p40 variant polypeptide of the present disclosure, are acetylated at the N-terminus of either or both of the dimeric IL-12 or IL-23 molecules by enzymatic reaction with an N-terminal acetyltransferase and, for example, acetyl-CoA. Alternatively, or in addition to N-terminal acetylation, a subunit of the IL-12(p35 / p40) variant or IL-23(p19 / p40) variant polypeptide of the present disclosure is acetylated at one or more lysine residues by enzymatic reaction with, for example, a lysine acetyltransferase. See, for example, Choudhary et al. (2009) Science 325 (5942):834L2 ortho840.

[0146] Fc fusion In some embodiments, when a dimeric IL-12(p35 / p40) variant or IL-23(p19 / p40) variant polypeptide may be provided in the form of an Fc fusion, each component of the dimeric molecule is provided on an individual subunit of the dimeric Fc molecule. In some embodiments, the IL-12p40 fusion protein may incorporate an Fc region derived from an IgG subclass of an antibody lacking the IgG heavy chain variable region. The "Fc region" may be a native or synthetic polypeptide homologous to the C-terminal domain of IgG produced by the digestion of IgG with papain. The molecular weight of IgG Fc is approximately 50 kDa. The variant conjugate polypeptide may contain the entire Fc region or a smaller portion (which is part of the chimeric polypeptide) that retains the ability to extend the cyclic half-life of the chimeric polypeptide. Furthermore, the full-length or fragmented Fc region may be a variant of the wild-type molecule; that is, they may contain mutations that affect or do not affect the function of the polypeptide; as will be further discussed below, intrinsic activity is not always necessary or desirable. In certain embodiments, the Fc fusion protein (e.g., IL-12p35 or IL-23p19, and IL-12p40 variants) includes an IgG1, IgG2, IgG3, or IgG4Fc region. The exemplary Fc region may contain mutations that inhibit complement binding and Fc receptor binding, or it may be soluble, i.e., able to bind to complement, or it may be able to lyse cells via another mechanism such as antibody-dependent complement lysis (ADCC).

[0147] In some embodiments, IL-12p35 or IL-23p19 and p40 variant fusion proteins contain a functional domain of an Fc-fusion chimeric polypeptide molecule. Fc-fusion conjugates have been shown to extend the systemic half-life of biopharmaceuticals, thus reducing the frequency of biopharmaceutical administration. Fc binds to neonatal Fc receptors (FcRn) on endothelial cells lining the inside of blood vessels; upon binding, the Fc-fusion molecule is protected from degradation and re-released into circulation, maintaining the molecule in circulation for a longer period. This Fc binding is thought to be the mechanism by which endogenous IgG retains its long plasma half-life. More recent Fc-fusion technologies annex a single copy of a biopharmaceutical to the Fc region of an antibody to optimize the pharmacokinetic and pharmacodynamic properties of the biopharmaceutical compared to conventional Fc-fusion conjugates. A “Fc region” useful for the preparation of Fc fusions can be a natural or synthetic polypeptide homologous to the C-terminal domain of IgG produced by the digestion of IgG with papain. The molecular weight of IgGFc is approximately 50 kDa. The IL-12p40 variant may contain the entire Fc region or a smaller portion (which is part of the chimeric polypeptide) that retains the ability to extend the cyclic half-life of the chimeric polypeptide. Furthermore, the full-length or fragmented Fc region may be a variant of the wild-type molecule. In typical practice, each monomer of the dimeric Fc contains components of the dimeric IL-12(p35 / p40) variant or the IL-23(p19 / p40) variant polypeptide.

[0148] Knob / HoleFc Conjugate In some embodiments, dimeric IL-12(p35 / p40) variants or IL-23(p19 / p40) variant polypeptides can be provided in the form of an Fc fusion, where each component of the dimeric molecule is provided on individual subunits of the dimeric Fc molecule, and when the dimeric Fc molecule subunits are modified to have a "knob-into-hole modification", each subunit of IL-12 (i.e., p35 and p40 variants) or IL-23 (p19 and p40 variants) is expressed on a "knob" or "hole" Fc subunit as a fusion protein (optionally including an intervening linker sequence between the p35 or p19 sequence and the Fc subunit sequence), and the p40 variant polypeptide is expressed on a complementary "knob" or "hole" Fc subunit. Knob-into-hole modifications are described in detail in Ridgway, et al. (1996) Protein Engineering 9(7):617-621 and U.S. Patent No. 5,731,168. Generally, knob-into-hole modifications refer to modifications at the interface between two immunoglobulin heavy chains in the CH3 domain, where i) in the CH3 domain of the first heavy chain, an amino acid residue is substituted with an amino acid residue having a larger side chain (e.g., tyrosine or tryptophan) to form a projection ("knob") from the surface, and ii) in the CH3 domain of the second heavy chain, an amino acid residue is substituted with an amino acid residue having a smaller side chain (e.g., alanine or threonine) to create a cavity ("hole") at the interface of the second CH3 domain, within which the protruding side chain ("knob") of the first CH3 domain is received by the cavity of the second CH3 domain. In one embodiment, the "knob-into-hole modification" includes amino acid substitution T366W and optionally amino acid substitution S354C on one side of the antibody heavy chain, and amino acid substitutions T366S, L368A, Y407V, and optionally Y349C on the other side of the antibody heavy chain.Furthermore, the Fc domain can be modified by introducing cysteine ​​residues at positions S354 and Y349, resulting in stable disulfide crosslinks between the two antibody heavy chains in the Fc region (CARTer, et al. (2001) Immunol Methods 248, 7-15). The knob-into-hole configuration is used to promote the expression of a first polypeptide (e.g., the p40 variant of this disclosure) on a first Fc monomer having a "knob" modification and a second polypeptide (p19 or p35) on a second Fc monomer having a "hole" modification, or vice versa, thereby promoting the expression and surface presentation of heterodimer IL-12 (p35 / p40) variant or IL-23 (p19 / p40) variant polypeptide Fc fusion constructs.

[0149] PEGylation In some embodiments, the pharmaceutical compositions of the Disclosure comprise one or more PEGylated reagents. As used herein, the term “PEGylated” refers to the modification of a protein by covalently bonding polyethylene glycol (PEG) to the protein, and “PEGylated” refers to a protein to which PEG has been bound. A range of PEGs or PEG derivatives having sizes in any range from about 10,000 daltons to about 40,000 daltons can be bonded to the recombinant polypeptides of the Disclosure using various chemistry methods. In some embodiments, the average molecular weight of the PEGs or PEG derivatives is about 1 kD to about 200 kD, for example, about 10 kD to about 150 kD, about 50 kD to about 100 kD, about 5 kD to about 100 kD, about 20 kD to about 80 kD, about 30 kD to about 70 kD, about 40 kD to about 60 kD, about 50 kD to about 100 kD, about 100 kD to about 200 kD, or about 150 kD to about 200 kD. In some embodiments, the average molecular weight of PEG or PEG derivatives is about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, or about 80 kD. In some embodiments, the average molecular weight of PEG or PEG derivatives is about 40 kD. In some embodiments, the PEGylating reagent is selected from methoxypolyethylene glycol succinimidyl propionate (mPEG-SPA), mPEG succinimidyl butyrate (mPEG-SBA), mPEG succinimidyl succinate (mPEG-SS), mPEG succinimidyl carbonate (mPEG-SC), mPEG succinimidyl glutarate (mPEG-SG), mPEG-N-hydroxyl succinimide (mPEG-NHS), mPEG torecylate, and mPEG aldehyde. In some embodiments, the PEGylating reagent is polyethylene glycol. For example, the PEGylation reagent is polyethylene glycol with an average molecular weight of 20,000 daltons, covalently bonded to the N-terminal methionine residue of the recombinant polypeptide of this disclosure.In some embodiments, the PEGylation reagent is polyethylene glycol with an average molecular weight of about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, or about 80 kD, covalently bonded to the N-terminal methionine residue of the recombinant polypeptide of the Disclosure. In some embodiments, the PEGylation reagent is polyethylene glycol with an average molecular weight of about 40 kD, covalently bonded to the N-terminal methionine residue of the recombinant polypeptide of the Disclosure.

[0150] Accordingly, in some embodiments, the recombinant polypeptides of the Disclosure are chemically modified with one or more polyethylene glycol moieties, e.g., PEGylated, or similarly modified, e.g., PASlated. In some embodiments, the PEG or PAS molecule is attached to one or more amino acid side chains of the disclosed recombinant polypeptide. In some embodiments, the PEGylated or PASlated polypeptide contains a PEG or PAS moiety on only one amino acid. In other embodiments, the PEGylated or PASlated polypeptide contains a PEG or PAS moiety attached to two or more amino acids, e.g., two or more, five or more, ten or more, fifteen or more, or twenty or more different amino acid residues. In some embodiments, the PEG or PAS chains are 2000, greater than 2000, 5000, greater than 5000, 10,000, greater than 10,000, greater than 10,000, 20,000, greater than 20,000, and 30,000 Da. PAS-modified polypeptides can be directly bonded to PEG or PAS (e.g., without linking groups) via amino groups, sulfhydryl groups, hydroxyl groups, or carboxyl groups. In some embodiments, the recombinant polypeptides of the present disclosure are covalently bonded to polyethylene glycol having an average molecular weight in the range of about 1 kD to about 200 kD, for example, about 10 kD to about 150 kD, about 50 kD to about 100 kD, about 5 kD to about 100 kD, about 20 kD to about 80 kD, about 30 kD to about 70 kD, about 40 kD to about 60 kD, about 50 kD to about 100 kD, about 100 kD to about 200 kD, or about 150 kD to about 200 kD. In some embodiments, the recombinant polypeptides of the Disclosure are covalently bonded to polyethylene glycol having an average molecular weight of about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, or about 80 kD. In some embodiments, the recombinant polypeptides of the Disclosure are covalently bonded to polyethylene glycol having an average molecular weight of about 40 kD.

[0151] Site-specific PEGylated site uptake In some embodiments, the recombinant polypeptides of the Disclosure (e.g., IL-12p40 variants) may be modified by the incorporation of unnatural amino acids having unnatural amino acid side chains that promote site-specific binding (e.g., PEGylation), as described, for example, U.S. Patent Nos. 7,045,337; 7,915,025; Dieters, et al. (2004) Bioorganic and Medicinal Chemistry Letters 14(23):5743-5745; Best, M (2009) Biochemistry 48(28): 6571-6584. In some embodiments, cysteine ​​residues may be incorporated at various positions within the recombinant polypeptides of the Disclosure to promote site-specific PEGylation via cysteine ​​side chains, as described, for example, Dozier and Distefano (2015) International Journal of Molecular Science 16(10): 25831-25864.

[0152] In certain embodiments, the Disclosure provides an IL-12p40 variant polypeptide comprising the incorporation of one or more amino acids (e.g., cysteine ​​or a non-natural amino acid) that enables site-specific PEGylation of the Disclosure, wherein the amino acid substitution for the site-specific PEGylation site is not at the interface with the p40 / p35(IL-12) or p40 / p19(IL-23) interface.

[0153] In some embodiments, site-directed amino acid modification is incorporated at IL-12p40 amino acid positions other than amino acid residues W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 of SEQ ID NO: 1 (i.e., residues W15, P17, D18, A19, K58, E59, F60, K84, E86, D93, H194, K195, L196, and K197, when numbered according to a mature IL-12p40 protein lacking a signal peptide). In some embodiments, the Disclosure provides compositions comprising human p40 variants that include site-specific amino acid substitutions enabling site-specific binding (e.g., PEGylation) at amino acid positions W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to Sequence ID No. 1.

[0154] In some embodiments, site-directed amino acid modification is incorporated at IL-12p40 amino acid positions other than amino acid residues W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 of SEQ ID NO: 2 (i.e., residues W15, P17, D18, A19, K58, E59, F60, K84, E86, D93, H194, K195, L196, and K197, when numbered according to a mature IL-12p40 protein lacking a signal peptide). In some embodiments, the Disclosure provides compositions comprising human p40 variants that include site-specific amino acid substitutions enabling site-specific binding (e.g., PEGylation) at amino acid positions W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219, numbered according to Sequence ID No. 2.

[0155] IL-12 and IL-23 partial agonists via site-specific PEGylation sites at the interface In some embodiments, the interaction between the IL-12p40 protein and the p35 or p19 protein can be regulated by incorporating site-directed PEGylation at the amino acid positions described herein on the IL-12p40 interface. This includes residues 37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 of SEQ ID NO: 1 or 2 (i.e., mature IL-12p40 protein lacking the signal peptide, i.e., residues W15, P17, D18, A19, K58, E59, F60, K84, E86, D93, H194, K195, when numbered according to the sequence of SEQ ID NO: 26 or 27). The incorporation of non-natural amino acids (or cysteine ​​residues) that promote site-directed PEGylation at one or more positions corresponding to L196 and K197) provides an IL-12p40 variant polypeptide with regulated binding to the p19 and / or p35 subunits, thus yielding a variant IL-12p40(p35 / p40) variant or IL-23(p19 / p40) variant molecule with partial agonist activity. When PEG molecules are incorporated at an interface, the PEG is typically a low molecular weight PEG species ranging from approximately 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 12 kDa, 15 kDa, or 20 kDa, so as not to completely disrupt the binding between the IL-12p40 variant and the p19 or p35 protein, and thus not to remove its activity.

[0156] Method of disclosure A therapeutic composition, such as a recombinant polypeptide (e.g., an IL-12p40 polypeptide variant), nucleic acid, recombinant cell, or pharmaceutical composition described herein, can be administered to treat a target in the treatment of related diseases such as cancer, immune disorders, and chronic infections. In some embodiments, the recombinant polypeptides, IL-12p40 polypeptide variants, nucleic acids, recombinant cells, and pharmaceutical compositions described herein can be incorporated into therapeutic agents for use in treating individuals who have, are suspected of having, or are at high risk of developing one or more autoimmune diseases or conditions related to disruption in IL-12p40 signaling. Exemplary autoimmune diseases or conditions include, but are not limited to, cancer, immune disorders, and chronic infections. In some embodiments, the individual is a patient under the care of a physician.

[0157] Accordingly, in one embodiment, several embodiments of the present disclosure relate to a method for modulating IL-12p40-mediated signaling in a subject, the method comprising administering to a subject a composition comprising one or more of the following: (a) the polypeptide of the present disclosure; (b) the recombinant nucleic acid of the present disclosure; (c) the recombinant cell of the present disclosure; and (d) the pharmaceutical composition of the present disclosure. In some embodiments, the composition comprises a therapeutically effective amount of the recombinant IL-12p40 polypeptide of the present disclosure. In some embodiments, the composition comprises a therapeutically effective amount of the recombinant nucleic acid of the present disclosure. As described above, IL-12p40 is a covalent subunit of interleukin 12 and interleukin 23. Accordingly, in some embodiments, a method for modulating IL-12-mediated signaling in a subject is provided herein. In some embodiments, the method for modulating IL-12 signaling disclosed herein further comprises administering to a subject the IL-12p35 polypeptide of the IL-12 complex. In some embodiments, the method further comprises administering to a subject a nucleic acid molecule encoding the IL-12p35 subunit of the IL-12 complex. In some embodiments, the nucleic acid encoding the IL-12p35 polypeptide is encoded by a different nucleic acid molecule (e.g., a vector). In some embodiments, the IL-12p40 polypeptide and the IL-12p35 polypeptide are encoded by nucleic acid sequences operably linked to each other within a single expression cassette (e.g., a polycistronic expression cassette).

[0158] In some other embodiments, the disclosure provides methods for modulating IL-23-mediated signaling in a subject. In some embodiments, the methods for modulating IL-23 signaling disclosed herein further include administering the subject with the IL-23p19 subunit of the IL-23 complex. In some embodiments, the method further includes administering the subject with a nucleic acid encoding the IL-12p35 polypeptide of the IL-12 complex. In some embodiments, the nucleic acid molecule encoding the IL-12p35 polypeptide is encoded by a different nucleic acid molecule (e.g., a vector). In some embodiments, the IL-12p40 polypeptide and the IL-23p19 polypeptide are encoded by nucleic acid sequences operably linked to each other within a single expression cassette (e.g., a polycistronic expression cassette).

[0159] In another embodiment, some embodiments of the present disclosure relate to methods for treating a condition in a subject requiring such treatment, the methods comprising administering to a subject a composition comprising one or more of the following: (a) the IL-12p40 polypeptide of the present disclosure; (b) recombinant nucleic acid of the present disclosure; (c) recombinant cells of the present disclosure; (d) pharmaceutical compositions of the present disclosure. In some embodiments, the composition comprises a therapeutically effective amount of recombinant IL-12p40 polypeptide of the present disclosure. In some embodiments, the composition comprises a therapeutically effective amount of recombinant nucleic acid of the present disclosure. In some embodiments, the therapeutic method disclosed herein further comprises the administration of the IL-12p35 subunit of the IL-12 complex. In some embodiments, the therapeutic method disclosed herein further comprises the administration of the IL-23p19 subunit of the IL-23 complex. In some embodiments, the therapeutic method disclosed herein further comprises administering to a subject a nucleic acid molecule encoding the IL-12p35 subunit of the IL-12 complex and / or a nucleic acid molecule encoding the IL-23p19 subunit of the IL-23 complex.

[0160] In some embodiments, the disclosed pharmaceutical compositions are formulated to suit their intended route of administration. The recombinant polypeptides of this disclosure may be administered orally or by inhalation, but are more likely to be administered via parenteral routes. Examples of parenteral routes include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral administration may contain the following components: sterile diluents, e.g., water for injection, saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid (EDTA); buffers, e.g., acetates, citrates, or phosphates; and substances for adjusting tonicity, e.g., sodium chloride or dextrose. The pH can be adjusted using acids or bases, e.g., monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (e.g., to a pH of about 7.2 to 7.8, e.g., 7.5). Parenteral formulations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0161] The dosage, toxicity, and therapeutic effects of such recombinant polypeptides in this disclosure are determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, LD 50 (Lethal dose for 50% of the population) and ED 50 The dose that is effective in 50% of the population can be determined. The dose-to-toxicity ratio is the therapeutic index, or LD50. 50 / ED 50 This can be expressed as a ratio. Compounds exhibiting a high therapeutic index are generally suitable. Compounds exhibiting toxic side effects may be used, but care must be taken to design a delivery system that targets such compounds to the site of the affected tissue, thereby minimizing potential damage to uninfected cells and thus reducing side effects.

[0162] Data obtained from cell culture assays and animal studies can be used when preparing dose ranges for use in humans. The doses of such compounds are preferably ED with little or no toxicity. 50 The circulating concentration range includes [specific component]. The dose may vary within this range depending on the form of administration and the route of administration used. For any compound used in the methods of this disclosure, the therapeutically effective dose can first be estimated from a cell culture assay. The dose is determined in animal models by cell culture IC 50 The preparation may be adjusted to achieve a circulating plasma concentration range including (for example, the concentration of the test compound that achieves half of the maximum symptom inhibition). Using such information, a more accurate determination of a useful dose in humans can be made. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0163] The therapeutically effective dose (e.g., effective dose) of the recombinant polypeptide of interest in this disclosure depends on the polypeptide selected. For example, a single dose in the range of about 0.001 to 0.1 mg / kg patient body weight may be administered. In some embodiments, doses of about 0.005, 0.01, or 0.05 mg / kg may be administered. In some embodiments, 600,000 IU / kg may be administered (IU can be determined by a lymphocyte proliferation bioassay and expressed in international units (IU)). The composition may be administered once daily or less, or once per week or more (including once every other day). Those skilled in the art will understand certain factors that influence the dose and timing required to effectively treat the subject, including, but not limited to, the severity of the disease, previous treatments, general health status and / or the age of the subject, and other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective dose of the recombinant polypeptide of interest in this disclosure may include a single treatment or a series of treatments. In some embodiments, the composition may be administered every 8 hours for 5 days, followed by a rest period of 2 to 14 days, for example, 9 days, followed by administration every 8 hours for 5 days.

[0164] Non-limiting exemplary embodiments of the disclosed methods for modulating IL-12p40-mediated signaling in subjects and / or for treating conditions in subjects requiring such modulation may include one or more of the following features:

[0165] In some embodiments, the administered composition results in a modification of the binding affinity of the recombinant IL-12p40 polypeptide to IL-12Rβ1 compared to the binding affinity of a reference polypeptide lacking amino acid substitutions. In some embodiments, the administered composition results in a decrease in the binding affinity of the recombinant IL-12p40 polypeptide to IL-12Rβ1 compared to the binding affinity of a reference polypeptide lacking amino acid substitutions. In some embodiments, the recombinant IL-12p40 polypeptide has a binding affinity to IL-12Rβ1 that is reduced by about 10% to about 100% compared to the binding affinity of a reference polypeptide lacking amino acid substitutions. In some embodiments, the recombinant IL-12p40 polypeptide has a binding affinity to IL-12Rβ1 that is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 95% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, recombinant IL-12p40 polypeptides have a binding affinity to IL-12Rβ1 that is reduced by approximately 10% to 50%, 20% to 70%, 30% to 80%, 40% to 90%, 50% to 100%, 20% to 50%, 40% to 70%, 30% to 60%, 40% to 100%, 20% to 80%, or 10% to 90% compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the binding affinity of the IL-12p40 polypeptide variants of this disclosure to IL-12Rβ1 is determined by a surface plasmon resonance (SPR) assay.

[0166] In some embodiments, the administered composition results in a decrease in STAT4 signaling compared to a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, STAT4 signaling in subjects is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 95% compared to administration of a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, STAT4 signaling in subjects is reduced by about 10% to about 100% compared to administration of a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, STAT4 signaling in subjects was reduced by approximately 10% to 50%, 20% to 70%, 30% to 80%, 40% to 90%, 50% to 100%, 20% to 50%, 40% to 70%, 30% to 60%, 40% to 100%, 20% to 80%, or 10% to 90% compared to administration of a reference IL-12p40 polypeptide lacking amino acid substitutions.

[0167] In some embodiments, the administered composition results in a decrease in STAT3 signaling compared to administration of a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, STAT3 signaling in subjects is reduced by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 95% compared to administration of a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, STAT3 signaling in subjects is reduced by about 10% to about 100% compared to administration of a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, STAT4 signaling in subjects was reduced by approximately 10% to 50%, 20% to 70%, 30% to 80%, 40% to 90%, 50% to 100%, 20% to 50%, 40% to 70%, 30% to 60%, 40% to 100%, 20% to 80%, or 10% to 90% compared to administration of a reference IL-12p40 polypeptide lacking amino acid substitutions.

[0168] In some embodiments, the administered composition results in cell type biased signaling of downstream signaling mediated via IL-12p40 compared to a composition containing a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the administered composition results in cell type biased signaling of downstream signaling mediated via IL-12 compared to a composition containing a reference polypeptide lacking amino acid substitutions. In some embodiments, the administered composition results in cell type biased signaling of downstream signaling mediated via IL-23 compared to a composition containing a reference polypeptide lacking amino acid substitutions. In some embodiments, the administered composition results in cell type biased signaling of downstream signaling mediated via IL-12 and IL-23 compared to a composition containing a reference polypeptide lacking amino acid substitutions.

[0169] In some embodiments, the administered composition results in cell-type biased IL-12 signaling compared to a composition containing a reference IL-12p40 polypeptide lacking amino acid substitutions, where cell-type biased signaling results in reduced IL-12-mediated signaling in NK cells. In some embodiments, IL-12-mediated signaling in NK cells is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or at least about 95% compared to a composition containing a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, cell-type biased signaling includes substantially unmodified IL-12 signaling in CD8+ T cells. In some embodiments, the administered composition results in unmodified IL-12-mediated signaling in CD8+ T cells, e.g., the same or substantially the same IL-12-mediated signaling, compared to a composition containing a reference IL-12p40 polypeptide lacking amino acid substitutions. In some embodiments, the administered composition results in a decrease in IL-12 signaling in NK cells, but substantially retains IL-12 signaling in CD8+ T cells. In some embodiments, the administered composition substantially retains the ability of recombinant polypeptides to stimulate INFγ expression in CD8+ T cells. In some embodiments, the administered composition enhances anti-tumor immunity in the tumor microenvironment.

[0170] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject has or is suspected of having a condition related to IL-12p40 mediated signaling. In some embodiments, the subject has or is suspected of having a condition related to IL-12 mediated signaling. In some embodiments, the subject has or is suspected of having a condition related to IL-23 mediated signaling. In some embodiments, the condition is cancer, an immune disorder, or a chronic infection.

[0171] The term cancer generally refers to the presence of cells that possess characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic ability, rapid growth and proliferation rate, and specific characteristic morphological features. While cancer cells are often observed aggregated in tumors, such cells may exist alone within animal subjects or may be non-tumorogenic cancer cells, such as leukemia cells. Thus, the term "cancer" can encompass references to solid tumors, soft tissue tumors, or metastatic lesions. As used herein, the term "cancer" includes pre-malignant and malignant cancers. In some embodiments, cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.

[0172] In some embodiments, the Specified Methods for Treating Conditions in Subjects Requiring It, wherein the condition is a cancer selected from the group consisting of acute myelomatous leukemia, anaplastic lymphoma, astrocytoma, B-cell carcinoma, breast cancer, colon cancer, ependymoma, esophageal cancer, glioblastoma, glioma, leiomyosarcoma, liposarcoma, liver cancer, lung cancer, mantle cell lymphoma, melanoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, ovarian cancer, pancreatic cancer, peripheral T-cell lymphoma, renal cancer, sarcoma, gastric cancer, carcinoma, mesothelioma, and sarcoma.

[0173] In some embodiments, the immune disease is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, moderate to severe plaque psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis, and graft-versus-host disease. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject has or is suspected of having a condition related to disruption of IL-12p40-mediated signaling. In some embodiments, the subject has or is suspected of having a condition related to disruption of IL-12-mediated signaling. In some embodiments, the subject has or is suspected of having a condition related to disruption of IL-23-mediated signaling.

[0174] Additional therapy As described above, any one of the recombinant polypeptides, nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein may be administered in combination with one or more additional (e.g., adjunct) therapeutic agents, such as chemotherapeutic agents, anticancer agents, or anticancer therapies. “Combined” administration with one or more additional therapeutic agents includes simultaneous administration and sequential administration in any order. In some embodiments, one or more additional therapeutic agents, chemotherapeutic agents, anticancer agents, or anticancer therapies are selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, and surgery. “Chemotherapy” and “anticancer agent” are used interchangeably herein. Various types of anticancer agents can be used. Non-exclusive examples include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxins, antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormone therapies, soluble receptors, and other antineoplastic agents.

[0175] Topoisomerase inhibitors are another class of anticancer agents available herein. Topoisomerases are essential enzymes that maintain the topology of DNA. Inhibition of type I or type II topoisomerases disrupts both DNA transcription and replication by disrupting proper DNA supercoiling. Some type I topoisomerase inhibitors include camptothecin, irinotecan, and topotecan. Examples of type II inhibitors include amsacrin, etoposide, phosphate etoposide, and teniposide. These are semi-synthetic derivatives of epipodophyllotoxin, an alkaloid naturally present in the roots of the American mayapple (Podophyllum peltatum).

[0176] Antitumor drugs include immunosuppressants such as dactinomycin, doxorubicin, epirubicin, bleomycin, mechloretamine, cyclophosphamide, chlorambucil, and ifosfamide. Antitumor compounds generally function by chemically modifying the DNA of cells.

[0177] Alkylating agents can alkylate many nucleophilic functional groups under conditions present in cells. Cisplatin, carboplatin, and oxaliplatin are alkylating agents. They impair cellular function by forming covalent bonds with amino, carboxyl, sulfhydryl, and phosphate groups of biologically important molecules.

[0178] Vinca alkaloids bind to specific sites on tubulin and inhibit its aggregation into microtubules (during the M phase of the cell cycle). Vinca alkaloids include vincristine, vinblastine, vinorelbine, and vindesine.

[0179] In some embodiments, the therapeutic methods described herein further include the administration of a compound that inhibits one or more immune checkpoint molecules. In some embodiments, the one or more immune checkpoint molecules include one or more of CTLA4, PD-1, PD-L1, A2AR, B7-H3, B7-H4, TIM3, and any combination thereof. In some embodiments, the compound that inhibits one or more immune checkpoint molecules includes an antagonistic antibody. In some embodiments, the antagonistic antibody is ipilimumab, nivolumab, pembrolizumab, durvalumab, atezolizumab, tremelimumab, or avelumab.

[0180] Antimetabolites are similar to purines (azathiopurine, mercaptopurine) or pyrimidines, and these substances prevent their uptake into DNA during the "S" phase of the cell cycle, halting normal development and division. Antimetabolites also affect RNA synthesis.

[0181] Plant alkaloids and terpenoids are derived from plants and inhibit cell division by interfering with microtubule function. Microtubules are essential for cell division; without them, cell division cannot occur. Major examples include vinca alkaloids and taxanes. Podophyllotoxin is a plant-derived compound that has been reported to aid digestion and is also used in the production of two other cell growth inhibitors, etoposide and teniposide. These prevent cells from entering the G1 phase (initiation of DNA replication) and the S phase (DNA replication).

[0182] The taxane group includes paclitaxel and docetaxel. Paclitaxel is a natural product originally known as taxol, initially obtained from the bark of the Pacific yew tree. Docetaxel is a semi-synthetic analog of paclitaxel. Taxanes enhance microtubule stability and prevent chromosome segregation in late gestation.

[0183] In some embodiments, anticancer agents include Remicade, docetaxel, celecoxib, melphalan, dexamethasone (Decadron®), steroids, gemcitabine, cisplatin, temozolomide, etoposide, cyclophosphamide, temodal, carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate, gefitinib (Iressa®), taxol, taxotere, fluorouracil, leucovorin, irinotecan, Xeloda, CPT-11, interferon alpha, PEGylated interferon alpha (e.g., PEG You can choose from INTRON-A), capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicin, cytarabine, doxetaxol, paclitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin, busulfan, prednisone, bortezomib (Velcade®), bisphosphonate, arsenic trioxide, vincristine, doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estrene phosphate sodium (Emcyt®), sulindac, etoposide, and any combination thereof.

[0184] In other embodiments, the anticancer agent can be selected from bortezomib, cyclophosphamide, dexamethasone, doxorubicin, interferon alfa, lenalidomide, melphalan, PEGylated interferon alfa, prednisone, thalidomide, or vincristine.

[0185] In some embodiments, the therapeutic methods described herein further include immunotherapy. In some embodiments, the immunotherapy includes the administration of one or more checkpoint inhibitors. Accordingly, some embodiments of the therapeutic methods described herein include the further administration of compounds that inhibit one or more immune checkpoint molecules. In some embodiments, the compounds that inhibit one or more immune checkpoint molecules include antagonistic antibodies. In some embodiments, the antagonistic antibody is ipilimumab, nivolumab, pembrolizumab, durvalumab, atezolizumab, tremelimumab, or avelumab.

[0186] In some embodiments, one or more anticancer therapies include radiotherapy. In some embodiments, radiotherapy may include the administration of radiation to kill cancer cells. Radiation induces cell death by interacting with intracellular molecules such as DNA. Radiation can also damage cell membranes, nuclear membranes, and other organelles. Depending on the type of radiation, the mechanism of DNA damage may differ as well as the relative biological effects. For example, heavy particles (i.e., protons and neutrons) directly damage DNA, resulting in a larger relative biological effect. Electromagnetic radiation results in indirect ionization, acting primarily through short-lived hydroxyl free radicals produced by the ionization of cellular water. Clinical applications of radiation include external beam radiation (from an external source) and brachytherapy (using a radiation source implanted or inserted into the patient). External beam radiation consists of X-rays and / or gamma rays, while brachytherapy uses radionuclides that decay to emit alpha or beta particles along with gamma rays. Radiation as intended herein also includes, for example, the directed delivery of radioisotopes to cancer cells. This specification also considers other forms of DNA damage factors, such as microwave and UV irradiation.

[0187] Radiation can be delivered in a single dose or in a series of small doses in a dose-splitting schedule. The doses of radiation intended herein range from about 1 to about 100 Gy, including, for example, about 5 to about 80, about 10 to about 50 Gy, or about 10 Gy. The total dose can be applied by a splitting method. For example, the method may include split individual doses of 2 Gy. The dose range of radioisotopes varies considerably, depending on the half-life of the isotope and the intensity and type of radiation emitted. When radiation involves the use of radioisotopes, the isotopes can be bound to targeted substances, such as therapeutic antibodies, that deliver radionucleotides to target tissue (e.g., tumor tissue).

[0188] The surgical procedures described herein include excisions in which all or part of the cancerous tissue is physically removed, extracted, and / or destroyed. Tumor excision means the physical removal of at least part of the tumor. In addition to tumor excision, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery). Removal of precancerous or normal tissue is also intended herein.

[0189] Accordingly, in some embodiments, the composition is administered to the subject individually as a first treatment or in combination with a second treatment. In some embodiments, the second treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first treatment is administered simultaneously with the second treatment. In some embodiments, the first and second treatments are administered sequentially. In some embodiments, the first treatment is administered before the second treatment. In some embodiments, the first treatment is administered after the second treatment. In some embodiments, the first treatment is administered before and / or after the second treatment. In some embodiments, the first and second treatments are administered alternately. In some embodiments, the first and second treatments are administered together in a single formulation.

[0190] Combinations of IL-12 / IL-23 containing the IL-12p40 variant and adjuvant therapy This disclosure provides the use of IL-12 or IL-23 comprising the variant IL-12p40 subunit described herein, which may be administered to a subject in combination with one or more additional active substances ("adjuncts"). Such further combinations are interchangeably referred to as "adjunct combinations" or "adjunct combination therapies," and therapeutic agents used in combination with IL-12 or IL-23 comprising the variant IL-12p40 subunit of this disclosure are referred to as "adjunct agents." As used herein, the term "adjunct agents" includes agents that can be administered or introduced separately, e.g., substances that are separately formulated for another administration (e.g., may be provided in a kit), and / or therapeutic agents that can be administered or introduced in combination with the IL-12p40 variant of this disclosure.

[0191] As used herein, the term "in combination with" refers to the administration of a first agent to a subject with at least one additional agent (i.e., a second, third, fourth, fifth, etc.) when used in relation to the administration of multiple agents to a subject. For the purposes of the present invention, if the biological effect resulting from the administration of the first agent is sustained in the subject upon administration of the second agent, and the therapeutic effects of the first and second agents overlap, then one agent (e.g., IL-12 or IL-23 containing the variant IL-12p40 subunit) is considered to be administered in combination with a second agent (e.g., an immune checkpoint pathway modulator). For example, PD1 immune checkpoint inhibitors (e.g., nivolumab or pembrolizumab) are typically administered by intravenous infusion every two or three weeks, while IL-12 or IL-23 species containing the variant p40 subunit of this disclosure may be administered more frequently, for example, daily, twice daily, or weekly. However, if the administration of a first drug (e.g., pembrolizumab) provides a long-lasting therapeutic effect, and the administration of a second drug (e.g., an IL-12 (p35 / p40) variant or an IL-23 (p19 / p40) variant) provides a therapeutic effect while the first drug is still effective, then the second drug is considered to have been administered in combination with the first drug, even if the first drug may have been administered at a considerable time after the second drug (e.g., several days or weeks later). In one embodiment, if the first and second drugs are administered simultaneously (within 30 minutes of each other), concurrently, or consecutively, the first drug is considered to have been administered in combination with the second drug. In some embodiments, if the first and second drugs are administered within about 24 hours of each other, preferably within about 12 hours of each other, preferably within about 6 hours of each other, preferably within about 2 hours of each other, or preferably within about 30 minutes of each other, the first drug is considered to have been administered "concurrently" with the second drug. The term “in combination with” is also understood to apply to situations in which the first and second drugs are co-formulated into a single pharmaceutically acceptable formulation, and the co-formulation is administered to the subject.In certain embodiments, the IL-12(p35 / p40) variant or IL-23(p19 / p40) variant polypeptide and the adjuvant are administered or applied sequentially, for example, one agent is administered before one or more agents. In other embodiments, the IL-12(p35 / p40) variant or IL-23(p19 / p40) variant polypeptide and the adjuvant are administered simultaneously, for example, two or more agents are administered simultaneously or nearly simultaneously; the two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether the agents are administered sequentially or simultaneously, they are considered to have been administered in combination for the purposes of this disclosure.

[0192] Further embodiments include methods or models for determining the optimal amount of drugs in a combination. The optimal amount may be, for example, the amount that achieves the optimal effect in a subject or target population, or the amount that achieves a therapeutic effect while minimizing or eliminating side effects associated with one or more drugs. In some embodiments, a method involving a combination of an IL-12(p35 / p40) variant or IL-23(p19 / p40) variant polypeptide and an adjunct agent known or determined to be effective in treating or preventing a disease, disorder, or condition described herein (e.g., cancerous condition) in a subject (e.g., human) or target population, wherein the amount of one drug is potentiated while keeping the amount of the other drug constant. By manipulating the amounts of drugs in this way, clinicians can determine the most effective ratio of drugs to treat a particular disease, disorder, or condition, or to eliminate or mitigate adverse effects that are acceptable under certain circumstances.

[0193] Additional or adjunct medications In some embodiments, one or more additional (e.g., adjunct) therapeutic agents include chemotherapeutic agents. In some embodiments, the adjunct agent is a "cocktail" of multiple chemotherapeutic agents. In some embodiments, the chemotherapeutic agents or cocktail are administered in combination with one or more physical methods (e.g., radiotherapy). The term "chemotherapeutic agent" is not particularly limited to, but includes alkylating agents, e.g., thiotepa and cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and picosulfan; aziridines, e.g., benzodopa, carbocon, metredopa, and uredopa; ethyleneimines and methylamelamines, e.g., altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards, e.g., thiorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenestrine, prednimustine, trophosphamide, and uracil mustard; nitroureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; and antibiotics, e.g., acrasinomycete. Syn, actinomycin, autoramycin, azaserin, bleomycin, e.g., bleomycin A2, kactinomycin, calicheamicin, carabicin, kaminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, and derivatives, e.g., demethoxy-daunorubicin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin N, esolubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, N-methylmitomycin C; mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU);Folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate, dideazatetrahydrofolate, and folic acid; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, Testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., floric acid; acegraton; aldofosphamide glycoside; aminolevulinic acid; amsacrin; bestrabusil; bisanthren; edatraxate; defoamin; demecolsin; diazicon; elformitin; eriptinium acetate; etogluside; gallium nitrate; hydroxyurea; lentinan; ronidamin; mitogluazone; mitoxantrone; mopidamol; nitracrin; pentostatin; fenamet; pirarubicin; podofi Phosphate; 2-ethylhydrazide; procarbazine; razoxane; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel, nab-paclitaxel, and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate Platinum and platinum-coordinate complexes, e.g., cisplatin, oxaplatin, carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelvine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamycin; capecitabine; taxanes, e.g., paclitaxel, docetaxel, cabazitaxel;This includes carminomycin, adriamycin, e.g., 4'-epiaadriamycin, 4-adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthalene acetate; colchicine and any pharmaceutically acceptable salts, acids, or any derivatives thereof.

[0194] The term “chemotherapeutic agent” also includes antihormone agents that act to modulate or inhibit hormonal effects on tumors, such as antiestrogens, e.g., tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxyfen, keoxyfen, onapristone, and toremifene; and antiandrogens, e.g., flutamide, nilutamide, bicalutamide, leuprolide, goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0195] In some embodiments, the adjuvant is one or more chemical or biological agents identified in the Art as useful for treating neoplastic diseases, but is not limited to, cytokines or cytokine antagonists, e.g., IL-2, INFγ, or anti-epidermal growth factor receptors, irinotecan; tetrahydrofolate antimetabolites, e.g., pemetrexed; antibodies against tumor antigens, monoclonal antibody-toxin complexes, T-cell adjuvants, bone marrow transplants, or antigen-presenting cells (e.g., dendritic cell therapy), antitumor vaccines, replication-competent viruses, signaling inhibitors (e.g., Gleevec® or Herceptin®), or immunomodulators, non-steroidal agents for achieving additive or synergistic suppression of tumor growth. Lloyd's anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 (COX-2) inhibitors, steroids, TNF antagonists (e.g., Remicade® and Enbrel®), interferon-β1a (Avonex®), and interferon-β1b (Betaseron®), as well as one or more combinations of the above carried out in known chemotherapy regimens, including, but not limited to, TAC, FOLFOX, TPC, FEC, ADE, FOLFOX-6, EPOCH, CHOP, CMF, CVP, BEP, OFF, FLOX, CVD, TC, FOLFIRI, PCV, FOLFOXIRI, ICE-V, XELOX, and others readily recognizable to those skilled in the art.

[0196] In some embodiments, the IL-12(p35 / p40) variant or the (IL-23)p19 / p40 variant is administered in combination with a BRAF / MEK inhibitor, a kinase inhibitor such as sunitinib, a PARP inhibitor such as olaparib, an EGFR inhibitor such as osimertinib (Ahn, et al. (2016) J Thorac Oncol 11:S115), an IDO inhibitor such as epacadostat, and an oncolytic virus such as talimogenel aherparepvec (T-VEC).

[0197] Combination with therapeutic antibodies In some embodiments, the “adjunct agent” is a therapeutic antibody (including, but not limited to, bispecific and trispecific antibodies that bind to one or more tumor-associated antigens, such as bispecific T-cell engagers (BITEs), biaffinity retargeting (DART) constructs, and trispecific killer engagers (TriKE) constructs).

[0198] In some embodiments, the therapeutic antibody is HER2 (e.g., trastuzumab, pertuzumab, adtrastuzumab emtansine), nectin-4 (e.g., enfortumab), CD79 (e.g., polatuzumab vedotin), CTLA4 (e.g., ipilumumab), CD22 (e.g., moxetumomab pasdotox), CCR4 (e.g., magumizumab), IL23p19 (e.g., tildrakizumab), PDL1 (e.g., durvalumab, avelumab, atezolizumab), IL17a (e.g., ixekizumab) ), CD38 (e.g., daratuzumab), SLAMF7 (e.g., elotuzumab), CD20 (e.g., rituximab, tositumomab, ibritumomab, and ofatumumab), CD30 (e.g., brentuximab vedotin), CD33 (e.g., gemtuzumab ozogamicin), CD52 (e.g., alemtuzumab), EpCam, CEA, fpA33, TAG-72, CAIX, PSMA, PSA, folate-binding protein, GD2 (e.g., dinuntuximab), GD3, IL6 (e.g., siltuximab), GM2, Le Y An antibody that binds to at least one tumor antigen selected from the group consisting of VEGF (e.g., bevacizumab), VEGFR, VEGFR2 (e.g., ramucirumab), PDGFRα (e.g., olatumumab), EGFR (e.g., cetuximab, panitumumab, and nesitumumab), ERBB2 (e.g., trastuzumab), ERBB3, MET, IGF1R, EPHA3, MUC-1, TRAILR1, TRAILR2, RANKLRAP, tenasicin, integrin αVβ3, and integrin α4β1.

[0199] In some embodiments, the antibodies are bispecific antibodies targeting first and second tumor antigens such as HER2 and HER3 (abbreviated as HER2×HER3), FAP×DR-5 bispecific antibody, CEA×CD3 bispecific antibody, CD20×CD3 bispecific antibody, EGFR-EDV-miR16 trispecific antibody, gp100×CD3 bispecific antibody, Ny-eso×CD3 bispecific antibody, EGFR×CMet bispecific antibody, BCMA×CD3 bispecific antibody, EGFR-EDV bispecific antibody, CLEC12A×CD3 bispecific antibody, HER2×HER3 bispecific antibody, Lgr5×EGFR bispecific antibody, PD1×CTLA-4 bispecific antibody, CD123×CD3 bispecific antibody, gpA33×CD3 bispecific antibody, B7-H3×C These include D3 bispecific antibody, LAG-3 x PD1 bispecific antibody, DLL4 x VEGF bispecific antibody, cadherin-P x CD3 bispecific antibody, BCMA x CD3 bispecific antibody, DLL4 x VEGF bispecific antibody, CD20 x CD3 bispecific antibody, Ang-2 x VEGF-A bispecific antibody, CD20 x CD3 bispecific antibody, CD123 x CD3 bispecific antibody, SSTR2 x CD3 bispecific antibody, PD1 x CTLA-4 bispecific antibody, HER2 x HER2 bispecific antibody, GPC3 x CD3 bispecific antibody, PSMA x CD3 bispecific antibody, LAG-3 x PD-L1 bispecific antibody, CD38 x CD3 bispecific antibody, HER2 x CD3 bispecific antibody, GD2 x CD3 bispecific antibody, and CD33 x CD3 bispecific antibody. Such therapeutic antibodies may be further conjugated to one or more chemotherapeutic agents (e.g., antibody-drug conjugates or ADCs) directly, via linkers, particularly acids and bases, or via enzymatically unstable linkers.

[0200] Combination with physical methods In some embodiments, the adjunct agents are one or more non-pharmacological treatments (e.g., local or total body radiotherapy or surgery). As an example, the Disclosure envisions a treatment method in which a radiotherapy step is performed before or after treatment using a treatment method comprising an IL-12(p35 / p40) variant or an IL23(p19 / p40) variant and one or more adjunct agents. In some embodiments, the Disclosure further envisions the use of an IL12p35 / p40 variant or an IL23p19 / p40 variant in combination with surgery (e.g., tumor resection). In some embodiments, the Disclosure further envisions the use of an IL-12p40 variant in combination with bone marrow transplantation, peripheral blood stem cell transplantation, or other types of transplantation therapy.

[0201] Combination with immune checkpoint modulators In some embodiments, the adjunct agent is an immune checkpoint modulator for treating and / or preventing neoplastic diseases, as well as diseases, disorders, or conditions associated with neoplastic diseases in a subject. Those skilled in the art will understand the term “immune checkpoint pathway” as a biological response triggered by the binding of a first molecule (e.g., a protein such as PD1) expressed on antigen-presenting cells (APCs) to a second molecule expressed on immune cells (e.g., T cells), which modulates the immune response through stimulation (e.g., upregulation of T cell activity) or inhibition (e.g., downregulation of T cell activity). The molecules involved in the formation of binding partners that modulate the immune response are called “immune checkpoints.” The biological responses modulated by such immune checkpoint pathways are mediated by intracellular signaling pathways that lead to downstream immune-effective pathways such as cell activation, cytokine production, cell migration, cytotoxic factor secretion, and antibody production. Immune checkpoint pathways are generally triggered by the binding of primary cell surface expression molecules to secondary cell surface molecules associated with the immune checkpoint pathway (e.g., PD1 binding to PDL1, CTLA4 binding to CD28, etc.). Activation of immune checkpoint pathways can lead to stimulation or inhibition of the immune response.

[0202] Immune checkpoints whose activation results in the inhibition or downregulation of the immune response are referred to herein as “negative immune checkpoint pathway modulators.” The inhibition of the immune response resulting from the activation of negative immune checkpoint modulators reduces the host immune system’s ability to recognize foreign antigens, such as tumor-associated antigens. The term negative immune checkpoint pathway includes, but is not limited to, biological pathways regulated by the binding of PD1 to PDL1, PD1 to PDL2, and CTLA4 to CDCD80 / 86. Examples of such negative immune checkpoint antagonists include, but are not limited to, antagonists (e.g., antagonist antibodies) that bind to T cell inhibitory receptors, including PD1 (also known as CD279), TIM3 (T cell membrane protein 3; also known as HAVcr2), BTLA (B and T lymphocyte attenuator; also known as CD272), VISTA(B7-H5) receptor, LAG3 (lymphocyte activation gene 3; also known as CD233), and CTLA4 (cytotoxic T lymphocyte-associated antigen 4; also known as CD152).

[0203] In one embodiment, an immune checkpoint pathway whose activation results in the stimulation of an immune response is referred to herein as a “positive immune checkpoint pathway modulator.” Therefore, the term positive immune checkpoint pathway modulator includes, but is not limited to, biological pathways modulated by the binding of ICOSL to ICOS(CD278), B7-H6 to NKp30, CD155 to CD96, OX40L to OX40, CD70 to CD27, CD40 to CD40L, and GITRL to GITR. Molecules that stimulate positive immune checkpoints (e.g., natural or synthetic ligands for components of binding pairs that stimulate an immune response) are useful for upregulating the immune response. Examples of such positive immune checkpoint agonists include, but are not limited to, agonist antibodies that bind to T cell activating receptors such as ICOS (e.g., JTX-2011, Jounce Therapeutics), OX40 (e.g., MEDI6383, Medimmune), CD27 (e.g., valrirumab, Celldex Therapeutics), CD40 (e.g., dasetuzumab CP-870, 893, Roche, Chi Lob7 / 4), HVEM, CD28, CD137 4-1BB, CD226, and GITR (e.g., MEDI1873, Medimmune, INCAGN1876, Agenus).

[0204] Those skilled in the art will understand the term “immune checkpoint pathway modulator” as a molecule that inhibits or stimulates the activity of immune checkpoint pathways in biological systems, including immunocompetent mammals. Immune checkpoint pathway modulators can exert their effects by binding to immune checkpoint proteins (e.g., immune checkpoint proteins expressed on the surface of antigen-presenting cells (APCs) such as cancer cells and / or immune T effector cells) or by influencing upstream and / or downstream responses of immune checkpoint pathways. For example, immune checkpoint pathway modulators may modulate the activity of SHP2, a tyrosine phosphatase involved in PD-1 and CTLA-4 signaling. Those skilled in the art will understand that the term “immune checkpoint pathway modulator” encompasses both immune checkpoint pathway modulators that can at least partially downregulate the function of inhibitory immune checkpoints (referred herein as “immune checkpoint pathway inhibitors” or “immune checkpoint pathway antagonists”) and immune checkpoint pathway modulators that can at least partially upregulate the function of stimulating immune checkpoints (referred herein as “immune checkpoint pathway effectors” or “immune checkpoint pathway agonists”).

[0205] Immune responses mediated by immune checkpoint pathways are not limited to T cell-mediated immune responses. For example, the KIR receptor on NK cells modulates the immune response against tumor cells mediated by NK cells. Tumor cells express a molecule called HLA-C, which inhibits the KIR receptor on NK cells, causing a reduction or anti-tumor immune response. Administration of drugs that antagonize the binding of HLA-C to the KIR receptor, such as anti-KIR3mab (e.g., lirilumab, BMS), inhibits the ability of HLA-C to bind to the NK cell inhibitory receptor (KIR), thereby restoring the ability of NK cells to detect and attack cancer cells. Thus, the immune response mediated by the binding of HLA-C to the KIR receptor is an example of a negative immune checkpoint pathway in which its inhibition leads to the activation of a non-T cell-mediated immune response.

[0206] In one embodiment, the immune checkpoint pathway modulator is a negative immune checkpoint pathway inhibitor / antagonist. In another embodiment, the immune checkpoint pathway modulator used in combination with an IL12p35 / p40 variant or an IL23p19 / p40 variant is a positive immune checkpoint pathway agonist. In yet another embodiment, the immune checkpoint pathway modulator used in combination with an IL12p35 / p40 variant or an IL23p19 / p40 variant is an immune checkpoint pathway antagonist.

[0207] Those skilled in the art will understand the term “negative immune checkpoint pathway inhibitor” as an immune checkpoint pathway modulator that interferes with the activation of negative immune checkpoint pathways that result in upregulation or enhancement of the immune response. Exemplary negative immune checkpoint pathway inhibitors include, but are not limited to, programmed death-1 (PD1) pathway inhibitors, programmed death ligand-1 (PDL1) pathway inhibitors, TIM3 pathway inhibitors, and anticytotoxic T lymphocyte antigen 4 (CTLA4) pathway inhibitors.

[0208] In one embodiment, an immune checkpoint pathway modulator is a negative immune checkpoint pathway antagonist ("PD1 pathway inhibitor") that inhibits the binding of PD1 to PDL1 and / or PDL2. PD1 pathway inhibitors stimulate a variety of favorable immune responses, including reversal of T cell depletion, restoration of cytokine production, and expansion of antigen-dependent T cells. PD1 pathway inhibitors are recognized as effective against a variety of cancers and have received USFDA approval for the treatment of various cancers, including melanoma, lung cancer, kidney cancer, Hodgkin lymphoma, head and neck cancer, bladder cancer, and urothelial carcinoma.

[0209] In some embodiments, PD1 pathway inhibitors include monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2. Antibody PD1 pathway inhibitors are well known in the art. Examples of commercially available PD1 pathway inhibitors that are monoclonal antibodies that interfere with the binding of PD1 to PDL1 and / or PDL2 include nivolumab (Opdivo®, BMS-936558, MDX1106, commercialized by BristolMyers Squibb, Princeton NJ), pembrolizumab (Keytruda® MK-3475, lambrolizumab, commercialized by Merck and Company, Kenilworth NJ), and atezolizumab (Tecentriq®, Genentech / Roche, South San Francisco CA). Additional antibody PD1 pathway inhibitors are in clinical development and include, but are not limited to, durvalumab (MEDI4736, Medimmune / AstraZeneca), pidilizumab (CT-011, CureTech), PDR001 (Novartis), BMS-936559 (MDX1105, Bristol Myers Squibb), avelumab (MSB0010718C, Merck Serono / Pfizer), and SHR-1210 (Incyte). Additional antibody PD1 pathway inhibitors are described in U.S. Patents 8,217,149, 8,168,757, 8,008,449, and 7,943,743.

[0210] PD1 pathway inhibitors are not limited to antagonist antibodies. Non-antibody biological PD1 pathway inhibitors are also in clinical development, with AMP-224, a PD-L2IgG2a fusion protein, and AMP-514, a PDL2 fusion protein, being developed by Amplimmune and Glaxo SmithKline. Aptamer compounds have also been documented as useful PD1 pathway inhibitors (Wang, et al. (2018) 145:125-130).

[0211] In some embodiments, PD1 pathway inhibitors include peptidyl PD1 pathway inhibitors, such as those described in U.S. Patent No. 9,422,339 and U.S. Patent No. 8,907,053 by Sasikumar et al. CA-170 (AUPM-170, Aurigene / Curis) has been reported as an orally bioavailable small molecule targeting immune checkpoints PD-L1 and VISTA. Pottayil Sasikumar, et al. Oral immune checkpoint antagonists targeting PD-L1 / VISTA or PD-L1 / Tim3 for cancer therapy. [Abstract]. Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl): Abstract No. 4861. CA-327 (AUPM-327, Aurigene / Curis) has been reported to be an orally bioavailable small molecule that inhibits the immune checkpoint programmed death ligand-1 (PDL1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3).

[0212] In some embodiments, PD1 pathway inhibitors include small molecule PD1 pathway inhibitors. Examples of small molecule PD1 pathway inhibitors useful in carrying out the present invention include Sasikumar, et al., 1,2,4-oxadiazole and thiadiazole compounds as immunomodulators (published as PCT / IB2016 / 051266, WO2016142833A1), Sasikumar, et al. 3-substituted-1,2,4-oxadiazole and thiadiazole (published as PCT / IB2016 / 051343, WO2016142886A2), BMS-1166, Chupak LS and Zheng X. Compounds useful as immunomodulators. Bristol-Myers Squibb Co. (2015), WO2015 / 034820A1, EP3041822B1;WO2015 / 034820A1; and Chupak, et al. Compounds useful Compounds useful as immunomodulators. Bristol-Myers Squibb Co. (2015), WO2015 / 160641A2, WO2015 / 160641A2, Chupak, et al. are described in the art, including Bristol-Myers Squibb Co., Sharpe, et al. Modulators of the immunoinhibitory receptor PD-1 and methods of use thereof are described in WO2011082400A2 and U.S. Patent No. 7,488,802.

[0213] In some embodiments, combinations of IL-12p35 / p40 variants or IL-23p19 / p40 variants with one or more PD1 immune checkpoint modulators have been useful in treating neoplasms in which PD1 pathway inhibitors have demonstrated clinical efficacy in humans, including but not limited to FDA approval for the treatment of diseases, melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, renal cell carcinoma, bladder cancer, ovarian cancer, endometrial cancer, cervical cancer, uterine sarcoma, gastric cancer, esophageal cancer, DNA mismatch repair deficiency colorectal cancer, DNA mismatch repair deficiency endometrial cancer, hepatocellular carcinoma, breast cancer, Merkel cell carcinoma, thyroid cancer, Hodgkin lymphoma, follicular carcinoma, diffuse large B-cell lymphoma, mycosis, and peripheral T-cell lymphoma. In some embodiments, combinations of IL12p35 / p40 variants or IL23p19 / p40 variants with PD1 immune checkpoint modulators are useful in treating tumors characterized by high levels of PDL1 expression, where the tumor has a tumor mutational burden, high levels of CD8+ T cells in the tumor, signs of IFNγ-associated immune activation, and a lack of metastatic disease, particularly liver metastases.

[0214] In some embodiments, the IL-12p35 / p40 variant or the IL-23p19 / p40 variant is administered in combination with a negative immune checkpoint pathway antagonist that inhibits the binding of CTLA4 to CD28 ("CTLA4 pathway inhibitor"). Examples of CTLA4 pathway inhibitors are known in the art (see, for example, U.S. Patents 6,682,736, 6,984,720, and 7,605,238).

[0215] In some embodiments, IL12p35 / p40 variants or IL23p19 / p40 variants are administered in combination with a negative immune checkpoint pathway antagonist ("BTLA pathway inhibitor") that inhibits the binding of BTLA to HVEM. Numerous approaches targeting the BTLA / HVEM pathway using anti-BTLA antibodies and antagonistic HVEM-Ig have been evaluated, and such approaches suggest promising utility in many diseases, disorders, and conditions, including transplantation, infection, tumors, and autoimmune diseases (see, e.g., Wu, et al., (2012) Int. J. Biol. Sci. 8:1420-30).

[0216] In some embodiments, the IL-12p35 / p40 variant or the IL-23p19 / p40 variant is administered in combination with a negative immune checkpoint pathway antagonist ("TIM3 pathway inhibitor") that inhibits the ability of TIM3 to bind to the TIM3 activating ligand. Examples of TIM3 pathway inhibitors are known in the art, and representative non-limiting examples are described in U.S. Patent Publication PCT / US2016 / 021005, published September 15, 2016; Lifke, et al.; U.S. Patent Publication US2016 / 0257749A1 (F. Hoffman-LaRoche), published September 8, 2016; Karunsky, U.S. Patent No. 9,631,026; Karunsky, Sabatos-Peyton, et al., U.S. Patent No. 8,841,418; U.S. Patent No. 9,605,070; Takayanagi, et al., U.S. Patent No. 8,552,156.

[0217] In some embodiments, IL-12 or IL-23 containing the variant p40 subunit is administered in combination with both LAG3 and PD1 inhibitors, as it has been suggested that blocking LAG3 and PD1 synergistically reverses the anergy between tumor-specific CD8+ T cells and virus-specific CD8+ T cells in chronic infections. IMP321 (ImmuFact) has been evaluated in melanoma, breast cancer, and renal cell carcinoma. See, for general information, Woo et al., (2012) Cancer Res 72:917-27; Goldberg et al., (2011) Curr. Top. Microbiol. Immunol. 344:269-78; Pardoll (2012) Nature Rev. Cancer 12:252-64; Grosso et al., (2007) J. Clin. Invest. 117:3383-392.

[0218] In some embodiments, IL-12 or IL-23 containing the variant p40 subunit is administered in combination with an A2aR inhibitor. A2aR stimulates CD4+ T cells to T Reg By promoting cell growth, it inhibits the T cell response. A2aR is particularly important in tumor immunity because tumors have a high rate of cell death due to cell turnover, and dying cells release adenosine, a ligand for A2aR. Furthermore, A2aR deletion has been associated with an enhanced, sometimes pathological, inflammatory response to infection. Inhibition of A2aR can be achieved by administering molecules such as antibodies that block adenosine binding, or by adenosine analogs. Such agents can be used in combination with IL12p35 / p40 variants and IL23p19 / p40 variants for use in the treatment of cancer and disorders such as Parkinson's disease.

[0219] In some embodiments, IL-12 or IL-23 containing the variant p40 subunit is administered in combination with an IDO (indoleamine 2,3-dioxygenase) inhibitor. IDO downregulates the tryptophan oxidation-mediated immune response, resulting in inhibition of T cell activation and induction of T cell apoptosis, creating an environment in which tumor-specific cytotoxic T lymphocytes become functionally inactive, no longer function, or unable to attack the subject's cancer cells. Indoximod (NewLink Genetics) is an IDO inhibitor that has been evaluated for metastatic breast cancer.

[0220] As described above, the present invention provides a method for treating neoplasmic diseases (e.g., cancer) in mammals by administering an IL12p35 / p40 variant or an IL23p19 / p40 variant in combination with an agent that modulates at least one immune checkpoint pathway, including an immune checkpoint pathway modulator that modulates two, three, or more immune checkpoint pathways.

[0221] In some embodiments, IL12p35 / p40 variants or IL23p19 / p40 variants are administered in combination with immune checkpoint modulators capable of modulating multiple immune checkpoint pathways. Multiple immune checkpoint pathways can be modulated by the administration of multifunctional molecules capable of acting as modulators of multiple immune checkpoint pathways. Examples of such multiple immune checkpoint pathway modulators include, but are not limited to, bispecific or multispecific antibodies. Examples of multispecific antibodies capable of acting as modulators or multiple immune checkpoint pathways are known in the art. For example, U.S. Patent Publication 2013 / 0156774 describes bispecific and multispecific agents (e.g., antibodies) and methods of use thereof for targeting cells co-expressing PD1 and TIM3. Furthermore, double blockade of BTLA and PD1 has been shown to enhance antitumor immunity (Pardoll, (April 2012) Nature Rev. Cancer 12:252-64). This disclosure, though not limited to, envisions the use of IL12p35 / p40 variants and / or IL23p19 / p40 variants in combination with immune checkpoint pathway modulators that target multiple immune checkpoint pathways, including bispecific antibodies that bind to both PD1 and LAG3. Thus, antitumor immunity can be enhanced at multiple levels, and combination strategies can be developed considering various mechanisms.

[0222] In some embodiments, the IL-12p35 / p40 variant or the IL-23p19 / p40 variant may be administered in combination with two, three, four, or more checkpoint pathway modulators. Such combinations may be advantageous in that the immune checkpoint pathways have different mechanisms of action, which provides an opportunity to attack the underlying disease, disorder, or condition from multiple different therapeutic angles.

[0223] It should be noted that the therapeutic response to immune checkpoint pathway inhibitors often appears considerably later than the response to conventional chemotherapy, such as tyrosine kinase inhibitors. In some cases, it may take more than six months after the initiation of treatment with immune checkpoint pathway inhibitors before objective indicators of the therapeutic response can be observed. Therefore, whether or not treatment with immune checkpoint pathway inhibitors in combination with the IL-12p35 / p40 variant or IL-23p19 / p40 variant of this disclosure is necessary should often be determined considering the longer tumor growth arrest time compared to conventional chemotherapy. The desired response may be any outcome that is deemed desirable under the circumstances. In some embodiments, the desired response is prevention of disease, disability, or condition progression, while in other embodiments, the desired response is regression or stabilization of one or more characteristics of the disease, disability, or condition (e.g., reduction in tumor size). In yet another embodiment, the desired response is reduction or elimination of one or more adverse effects associated with one or more drugs in the combination.

[0224] Cell therapy and methods as adjunct agents In some embodiments, the methods of the present disclosure may involve the administration of an IL-12(p35 / p40) variant or an IL-23(p19 / p40) variant in combination with an adjunct agent in the form of cell therapy for the treatment of neoplasms, autoimmune diseases, or inflammatory diseases. Examples of cell therapies suitable for use in combination with the methods of the present disclosure include, but are not limited to, one or more activated CAR-T cells, modified TCR cells, tumor-infiltrating lymphocytes (TILs), and modified T cell products. Since modified T cell products are generally activated ex vivo before administration to a subject and thus provide upregulated levels of CD25, cell products containing such activated modified T cell types are suitable for further support via the administration of the IL-12p40 variant described herein.

[0225] CAR-T cells In some embodiments of the methods of this disclosure, the adjunct agent is a “chimeric antigen receptor T cell” (CAR-T cell), which generally refers to a T cell that has been recombinantly modified to express a chimeric antigen receptor. Those skilled in the art will know that a chimeric antigen receptor (CAR) generally refers to a chimeric polypeptide comprising a plurality of functional domains arranged from the amino terminus to the carboxy terminus, in the order of (a) an antigen-binding domain (ABD); (b) a transmembrane domain (TD); and (c) one or more cytoplasmic signaling domains (CSD), where the aforementioned domains may optionally be linked by one or more spacer domains. The CAR may also further comprise a signal peptide sequence on the cell surface of a cell transformed with an expression vector containing the nucleic acid sequence encoding the CAR, which is conventionally removed during post-translational processing and presentation of the CAR. CARs useful for carrying out the present invention are prepared according to principles well known in the art. For example, see Eshhaar et al. U.S. Patent No. 7,741,465B1; Sadelain, et al (2013) Cancer Discovery 3(4):388-398; Jensen and Riddell (2015) Current Opinions in Immunology 33:9-15; Gross, et al. (1989) PNAS USA) 86(24):10024-10028; Curran, et al. (2012) J Gene Med 14(6):405-15. Examples of commercially available CAR-T cell products that can be modified to take up the orthogonal receptors of the present invention include axicabtageneciloleucel (commercially available from Gilead Pharmaceuticals as YesCARTa®) and tisagenlecleucel (commercially available from Novartis as Kymriah®).

[0226] Those skilled in the art will understand that the term antigen-binding domain (ABD) refers to a polypeptide that specifically binds to an antigen expressed on the surface of a target cell. An ABD can be any polypeptide that specifically binds to one or more cell surface molecules (e.g., tumor antigens) expressed on the surface of a target cell. In some embodiments, the ABD is a polypeptide that specifically binds to a cell surface molecule associated with tumor cells, selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Rα2, CD19, mesoserine, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP. In some embodiments, ABD is an antibody (including one or more molecules such as VHH, scFv, etc., as described above) that specifically binds to at least one cell surface molecule associated with tumor cells (i.e., at least one tumor antigen), where the cell surface molecule associated with tumor cells is selected from the group consisting of GD2, BCMA, CD19, CD33, CD38, CD70, GD2, IL3Rα2, CD19, mesoserine, Her2, EpCam, Muc1, ROR1, CD133, CEA, EGRFRVIII, PSCA, GPC3, Pan-ErbB, and FAP. Examples of CAR-T cells useful as adjuvant agents in carrying out the methods of this disclosure include, but are not limited to, CAR-T cells expressing CARs containing ABD, further comprising at least one of the following: anti-GD2 antibody, anti-BCMA antibody, anti-CD19 antibody, anti-CD33 antibody, anti-CD38 antibody, anti-CD70 antibody, anti-GD2 antibody and IL3Rα2 antibody, anti-CD19 antibody, anti-mesothelin antibody, anti-Her2 antibody, anti-EpCam antibody, anti-Muc1 antibody, anti-ROR1 antibody, anti-CD133 antibody, anti-CEA antibody, anti-PSMA antibody, anti-EGRFRVIII antibody, anti-PSCA antibody, anti-GPC3 antibody, anti-Pan-ErbB antibody, and anti-FAP antibody.

[0227] The cytoplasmic domain of a CAR polypeptide includes one or more intracellular signaling domains. In one embodiment, the intracellular signaling domain includes a cytoplasmic sequence of a T cell receptor (TCR), a co-receptor that initiates signaling after engagement with an antigen receptor, and functional derivatives and sub-fragments thereof. A cytoplasmic signaling domain, such as one derived from the T cell receptor zeta chain, is used as part of the CAR to generate stimulating signals for T lymphocyte proliferation and effector function after engagement of the chimeric receptor with a target antigen. Examples of cytoplasmic signaling domains include, but are not limited to, the cytoplasmic domain of CD27, the cytoplasmic domain S of CD28, the cytoplasmic domain of CD137 (also known as 4-1BB and TNFRSF9), the cytoplasmic domain of CD278 (also known as ICOS), the p110α, β, or δ catalytic subunits of PI3 kinases, the human CD3ζ chain, the cytoplasmic domain of CD134 (also known as OX40 and TNFRSF4), the FcεR1γ and β chains, the MB1(Igα) chain, the B29(Igβ) chain, CD3 polypeptides (δ, Δ, ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in transduction into T cells (e.g., CD2, CD5, CD28).

[0228] IL-12(p35 / p40) variants or IL-23(p19 / p40) variants may be administered in combination with first, second, third, or fourth generation CAR-T cells. The term first-generation CAR-T cells refers to cells modified to express a CAR, where the cytoplasmic domain transmits signals from antigen binding only via a single signaling domain, e.g., a signaling domain derived from a high-affinity receptor such as IgE FcεR1 or CD3ζ chain. This domain contains one or three immunoreceptor tyrosine-based activation motifs [ITAM(s)] for antigen-dependent T cell activation. The ITAM-based activation signal gives T cells the ability to lyse target tumor cells and secrete cytokines in response to antigen binding. Second-generation CAR-T cells refer to cells modified to express a CAR that includes a costimulatory signal in addition to the CD3ζ signal. Co-delivery of the costimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T cells. Co-stimulatory domains are typically located proximal to the membrane relative to the CD3ζ domain. Third-generation CAR-T cells refer to cells modified to express CARs containing ternary signaling domains, such as CD28, CD3ζ, OX40, or 4-1BB signaling regions. Fourth-generation or "armored vehicle" CART cells are further modified to express or block immunoenhancing molecules and / or receptors, such as IL-12, IL-18, IL-7, and / or IL-10; 4-1BB ligand, CD-40 ligand. Examples of intracellular signaling domains that can be incorporated into the CARs of the present invention include (amino to carboxy): CD3ζ; CD28-41BB-CD3ζ; CD28-OX40-CD3ζ; CD28-41BB-CD3ζ; 41BB-CD-28-CD3ζ, and 41BB-CD3ζ.

[0229] This term includes, but is not limited to, CAR variants such as split CARs, ON-switched CARs, bispecific or tandem CARs, inhibitory CARs (iCARs), and induced pluripotent stem (iPS) CART cells. The term "split CAR" refers to a CAR in which the extracellular portion, ABD, and cytoplasmic signaling domains of the CAR reside on two separate molecules. CAR variants also include ON-switched CARs, which are conditionally activatable CARs, including split CARs in which the conditional heterodimerization of the two portions of a split CAR is pharmacologically controlled. CAR molecules and their derivatives (i.e., CAR variants) are, for example, PCT application numbers US2014 / 016527, US1996 / 017060, US2013 / 063083; and Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; CARTellieri et al., J Biomed Biotechnol (2010) 956304, which are incorporated herein by reference in their entirety. The term “bispecific or tandem CAR” refers to a CAR containing a secondary CAR-binding domain that can amplify or inhibit the activity of a primary CAR.The terms “inhibitory chimeric antigen receptor” or “iCAR” are used interchangeably herein and refer to a CAR that inhibits primary CAR activation by using dual antigen targeting to shut down the activation of the active CAR through engagement with a secondary inhibitory receptor equipped with an inhibitory signaling domain in the secondary CAR-binding domain. Inhibitory CARs (iCARs) are designed to modulate the activity of CART cells through activation of an inhibitory receptor signaling module. This approach combines the activity of two CARs, one of which generates a dominant-negative signal that limits the response of CART cells activated by the activating receptor. When an iCAR binds to a specific antigen expressed only in normal tissue, it can switch off the response of a counter-activating activator CAR. In this way, iCAR-T cells can distinguish between cancer cells and healthy cells and reversibly block the function of antigen-selectively transduced T cells. The CTLA-4 or PD-1 intracellular domain of an iCAR triggers an inhibitory signal against T lymphocytes, resulting in decreased cytokine production, reduced target cell lysis efficiency, and altered lymphocyte motility. The term "tandem CAR" or "TanCAR" refers to a CAR that mediates bispecific activation of T cells through the binding of two chimeric receptors designed to deliver stimulatory or co-stimulatory signals in response to the independent engagement of two different tumor-associated antigens.

[0230] In general, chimeric antigen receptor T cells (CAR-T cells) are T cells that have been recombinantly modified by transduction with an expression vector encoding a CAR, essentially in accordance with the teachings described above.

[0231] In some embodiments, modified T cells are allogeneic with respect to the individual being treated. Graham et al. (2018) Cell 7(10) E155. In some embodiments, allogeneic modified T cells are HLA-matched. However, not all patients have a perfectly matched donor, and cell products suitable for all patients regardless of HLA type are an alternative.

[0232] If the T cells used in carrying out the methods of this disclosure are allogeneic T cells, such cells may be modified to mitigate graft-versus-host disease. For example, the modified cells of the present invention may be TCRαβ receptor knockouts achieved by gene editing techniques. TCRαβ is a heterodimer, and both the alpha and beta chains must be present for it to be expressed. There is one gene encoding the alpha chain (TRAC), but there are two genes encoding the beta chain, so the TRAC locus KO is deleted for this purpose. Various approaches have been used to achieve this deletion, such as CRISPR / Cas9; meganucleases; and modified I-CreI homing endonucleases. See, for example, Eyquem et al. (2017) Nature 543:113-117 (where the TRAC coding sequence is replaced with the CAR coding sequence); and Georgiadis et al. (2018) Mol. Ther. 26:1215-1227 (where CAR expression and TRAC disruption are linked by clustered, regularly spaced short palindromic repeats (CRISPR) / Cas9, without directly incorporating CAR into the TRAC locus). Another strategy to prevent GVHD is to modify T cells to express inhibitors of TCRαβ signaling, for example, by using terminally cleaved CD3ζ as a TCR inhibitor molecule.

[0233] In some embodiments, the IL-12(p35 / p40) variant or the IL-23(p19 / p40) variant is administered in combination with additional cytokines, including, but not limited to, IL-2, IL-7, IL-15, and IL-18 (including their respective analogs and variants).

[0234] In some embodiments, IL-12(p35 / p40) variants or IL-23(p19 / p40) variants are administered in combination with one or more adjunct agents that inhibit activation-induced cell death (AICD). AICD is a type of programmed cell death resulting from the interaction of Fas receptors (e.g., Fas, CD95) and Fas ligands (e.g., FasL, CD95 ligand), and plays a role in maintaining peripheral immune tolerance. AICD effector cells express FasL, and apoptosis is induced in cells expressing the Fas receptor. Activation-induced cell death is a negative regulator of activated T lymphocytes, resulting from repeated stimulation of T cell receptors. Examples of AICD inhibitors that can be used in combination with the IL-12 (p35 / p40) variants and IL-23 (p19 / p40) variants described herein include, but are not limited to, cyclosporine A (Shih, et al., (1989) Nature 339:625-626), IL-16 and its analogues (including rhIL-16, Idziorek, et al., (1998) Clinical and Experimental Immunology 112:84-91), TGFb1 (Genesteir, et al., (1999) J Exp Med189(2): 231-239), and vitamin E (Li-Weber, et al., (2002) J Clin Investigation 110(5):681-690).

[0235] In some embodiments, the adjunct agents are antineoplastic methods, including but not limited to radiotherapy, cryotherapy, hyperthermia, surgery, laser ablation, and proton beam therapy.

[0236] kit Various kits for carrying out the methods described herein are also provided herein. In particular, some embodiments of the present disclosure relate to kits for methods of modulating IL-12p40-mediated signaling in subjects. Some other embodiments relate to kits for methods of treating conditions in subjects requiring such treatment. In some embodiments, a kit may include one or more recombinant IL-12p40 polypeptides, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein, and instructions for use thereof. For example, in some embodiments herein, a kit is provided comprising one or more recombinant polypeptides, IL-12p40 polypeptide variants, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions of the present disclosure; and instructions for use thereof. In some embodiments, a kit of the present disclosure may further include IL-12p35 polypeptide, or nucleic acids encoding IL-12p35 polypeptide. In some embodiments, a kit of the present disclosure may further include IL-23p19 polypeptide, or nucleic acids encoding IL-23p19 polypeptide.

[0237] In some embodiments, the kits of the present disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided recombinant polypeptides, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. In some embodiments, the kits may have one or more additional therapeutic agents, which may be administered simultaneously with or in succession to other kit components for a desired purpose, for example, to modulate cell activity, to inhibit target cancer cells, or to treat a disease in an individual requiring it.

[0238] Any of the above kits may further include one or more additional reagents, such additional reagents may be selected from: dilution buffer, reconstitution solution, wash buffer, control reagent, control expression vector, negative control polypeptide, positive control polypeptide, and reagent for in vitro production of recombinant polypeptide.

[0239] In some embodiments, the components of the kit may be placed in separate containers. In some other embodiments, the components of the kit may be placed together in a single container. For example, in some embodiments of the present disclosure, the kit may contain one or more of the recombinant IL-12p40 polypeptide, recombinant nucleic acid, recombinant cell, or pharmaceutical composition described herein in one container (e.g., a sterile glass or plastic vial), and further therapeutic agents in another container (e.g., a sterile glass or plastic vial).

[0240] In some embodiments, the kit may further include instructions for use of the kit's components to carry out the methods described herein. For example, the kit may include a package insert containing information about the pharmaceutical compositions and dosage forms contained in the kit. Generally, such information helps patients and physicians to use the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the package insert may provide the following information relating to the combination of the disclosure: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and uses, contraindications, warnings, precautions, adverse reactions, overdose, appropriate doses and methods of administration, supply methods, appropriate storage conditions, references, manufacturer / distributor information, and intellectual property information.

[0241] In some embodiments, the kit may further include instructions for using the kit's components to carry out the methods disclosed herein. Instructions for carrying out the methods are generally recorded on a suitable recording medium. For example, instructions for use may be printed on a substrate such as paper or plastic. Instructions for use may be present as an accompanying document within the kit, such as on the label of the kit or its component containers (e.g., related to the package or partial package). Instructions may exist as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, disk, or flash drive. In some cases, the actual instructions may not be included in the kit, but a means of obtaining the instructions from a remote source (such as via the internet) may be provided. An example of this embodiment is a kit that includes a web address from which the instructions can be viewed and / or downloaded. Similar to the instructions, this means of obtaining the instructions may be recorded on a suitable substrate.

[0242] All publications and patent applications referenced in this disclosure are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated as being incorporated by reference.

[0243] The inclusion of references in this specification does not constitute prior art. The discussions in the references represent the claims of their authors, and the applicant reserves the right to challenge the accuracy and validity of the cited documents. While many sources of information are referenced in this specification, including articles in scientific journals, patent documents, and textbooks, these references do not constitute any recognition that any of these documents constitute common general knowledge in the art.

[0244] The general methods discussed herein are for illustrative purposes only. Other alternative methods and substitutes will be apparent to those skilled in the art upon consideration of this disclosure and are included in the spirit and scope of this application. [Examples]

[0245] The implementation of this invention will utilize the prior art of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, unless otherwise specified. Such techniques are described in detail in the following literature: Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, and Sambrook, J., & Russel, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to as “Sambrook”); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements up to 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998).Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferree, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V. (these disclosures are incorporated herein by reference).

[0246] Additional embodiments are disclosed in more detail in the following examples, which are provided by way of illustration and are not intended to limit the present disclosure or the claims in any way.

[0247] Example 1 General Experimental Procedures Human T Cell Signaling For the production of recombinant human IL-12 and IL-23, IL-12p40 (23-328) was cloned into a pD649 vector having an N-terminal HA signal peptide, a C-terminal AviTag (GLNDIFEAQKIEWHE, SEQ ID NO: 12), and 6xHis. Human IL-12p35 (23-219) and IL-23p19 (28-189) were cloned into pD649 having an N-terminal HA signal peptide, a Flag tag, and a TEV protease site. IL-12 (IL-12p35 and IL-12p40), IL-23 (IL-23p19 and IL-12p40), and IL-12p40 alone were expressed by transient transfection of Expi293F cells (ThermoFisher, #A14527) according to the manufacturer's protocol. The supernatant was purified using Ni-NTA and then subjected to size exclusion chromatography (SEC).

[0248] For human T cell signaling, IL-12 and IL-23 variants were produced in Expi293 cells as described above. IL-12p35 and IL-23p19 were co-transfected with IL-12p40 variants (wild-type, E81A, F82A, or P39A D40A E81A F82A) and purified with Ni-NTA followed by SEC. Human peripheral mononuclear cells (PBMCs) were isolated from Stanford Blood Bank samples using a SepMate-50 column (STEMCELL Technologies, #85450) and Ficoll-PaquePLUS (GEHealthcare, catalog no. GE17-1440-02). Cells were diluted in sterile PBS containing 2% fetal bovine serum (FBS) (Gibco, #20012-050) and added to a SepMate-50 column pre-loaded with 15 ml of Ficoll. Red blood cells were lysed in ACK lysis buffer (Gibco, #A10492-01) for 5 minutes, quenched in PBS containing 2% FBS, and then frozen in 50x10°C medium containing 90% FBS and 10% DMSO. 6The cells were resuspended in 1 / mL. The cells were frozen overnight at -80°C in a Mr.Frosty freezing container (ThermoFisher, #5100-0001) and then transferred to a storage box at -80°C for long-term storage. Human PBMCs were coated with 2.5 μg / ml αCD3 (OKT-3, BioLegend, #317326) in RPMI1640-glutaMax (Gibco, #61870-127), which contains 10% FBS, non-essential amino acids (Gibco, #11140050), sodium pyruvate (Gibco, catalog number 11360-070), 15 mM hepes (Gibco, #15630-080), and 5 μg / mL αCD28 (CD28.2, BioLegend, #302943) and 100 IU / mL recombinant human IL-2 supplemented penicillin-streptomycin (Gibco, catalog number 15140163). Cells were cultured in 5% CO2 at 37°C for 48 hours, washed once, and rested overnight in complete RPMI. Cells were stained with αCD4 PacBlue (RPA-T4, BD, #558116), stimulated with IL-12 and IL-23 variants at 37°C for 20 minutes, fixed with 1.6% paraformaldehyde at room temperature for 10 minutes, and permeabilized with methanol at -20°C. Cells were washed with PBS containing 2% FBS and 2mMEDTA, and stained at room temperature for 1 hour with antibodies against STAT4pY693AF488 (38 / p-Stat4, BD, #558136) and STAT3pY705AF647 (4 / P-STAT3, BD, #557815). Fluorescence intensity was analyzed using a CytoFlex flow cytometer (Beckman Coulter).

[0249] For the analysis of IL-12Rβ1 in human PBMCs, cells were stained directly (ex vivo) or activated as described above to generate T cell blasts. To identify T cells and NK cells, the Fc receptor was blocked with TruStain FcX (BioLegend), and cells were stained using the αCD3 Pacific Blue (UCHT1, BioLegend), αCD4 FITC (OKT4, BioLegend), αCD8 AF750 (R&D systems), and αCD56BV605 (HCD56, BioLegend) phenotyping panel. Human p40 tetramers were prepared by mixing 200 nM streptavidin-AF647 with 4x molar excess biotinylated p40 expressed as described in the surface plasmon resonance section. After staining cells at 4°C for 2 hours, viable cells were detected using propidium iodide (PI, Invitrogen). The samples were analyzed using a CytoFlex flow cytometer (Beckman Coulter) and then analyzed with FlowJo (BD). CD8 + T cells were defined as living CD3+CD8+ cells, and NK cells were defined as living CD3-CD56+ cells. See Figure 7B for details on gating.

[0250] Human CD8 + For T cell IFNγ induction assay, CD8 + CD8 cells were isolated using MACS with the T cell isolation kit (Milteny) and LS magnetic column (Miltenyi). + T cells were isolated from PBMCs. Purified CD8 +T cells were stimulated at 80,000 cells / well in 96-well round-bottom plates coated with 2 μg / mL αCD3 (OKT3, BioLegend) in the presence of 0.5 μg / mL αCD28 (CD28.2, BioLegend) and 5 ng / mL human IL-2. After 48 hours, the cells were pelleted and the supernatant was analyzed using the Human IFNγ ELISA MAX Deluxe (BioLegend) on a Nunc MaxiSorp ELISA plate (BioLegend). For the human NK cell IFNγ induction assay, NK cells were isolated from PBMCs by MACS using the EasySep Human NK Cell Isolation Kit (StemCell) equipped with an EasySep magnet (StemCell). Purified NK cells were stimulated at 40,000 cells / well in 96-well round-bottom plates in the presence of 100 ng / mL IL-18 (R&Dsystems). After 48 hours, the supernatant was collected and analyzed for CD8 + The T cell IFNγ induction assay was performed as described.

[0251] IL-12p40 surface staining For surface staining of mIL-12p40, mouse IL-12p40 (23-335) was cloned into pAcGP67a, which has an N-terminal GP64 signal peptide and a C-terminal AviTag and 6×His tag. Since mouse IL-12p40 is secreted as a disulfide-bonded homodimer, to obtain monomeric IL-12p40, Ni-NTA purified protein was reduced with 20 mM cysteine, alkylated with 40 mM iodoacetamide in hepes-buffered saline (HBS) pH 8.2, and then subjected to SEC. Monomeric IL-12p40 was biotinylated with recombinant BirA and purified by a second SEC.

[0252] Spleens and lymph nodes were isolated from C57 / BL6 mice, and single-cell suspensions were prepared. T cell blasts were activated on plates coated at 37°C for 48 hours using 5 μg / mL αCD28 (37.51, Bio XCell, catalog no. BE0015-1) and 2.5 μg / mL αCD3 (145-2c11, BioLegend, catalog no. 100340) in a complete RMPI containing 100 IU / mL recombinant mouse IL-2. For cell staining, ex vivo cells and T cell blasts were incubated with TruStain FcX (93, BioLegend, 101320) and stained with a phenotypic panel of αCD3 FITC (17A2, eBiosciences, #11-0032-82), αCD4 PerCP-Cy5.5 (GK1.5, BioLegend, #100433), αCD8 BV785 (53-6.7, BioLegend, #100749), and αNK1.1e450 (PK136, eBioscience, #48-5941-82). IL-12p40 tetramers were prepared by mixing biotinylated IL-12p40 in a 4-fold molar excess of 200 nM streptavidin-AF647. Cells were stained at 4°C for 2 hours, and then live cell staining was performed with propidium iodide (PI, ThermoFisher #P3566). Samples were analyzed using a CytoFlex flow cytometer and then analyzed with FlowJo. CD8+ T cells were defined as live CD3+CD8+ cells, and NK cells were defined as live CD3-NK1.1+ cells.

[0253] Mouse IL-12 signaling For IL-12 signaling and functional assays, mouse IL-12 was expressed as a single chain, similar to the previously described approach (Anderson et al., 1997). The 3×GGGS linker, 3C protease site, and mouse IL-12p40(23-335) followed by mouse IL-12p35(23-215) were cloned into pAcGP67a having an N-terminal GP64 signal peptide and a C-terminal 6×His tag. Mouse IL-12 variants were expressed in T.ni cells and purified with Ni-NTA and SEC. For cell signaling, mouse T cell blasts were prepared as described above, rested overnight in complete RPMI, stained with αCD8 BV785 (53-6.7, Biolegend, #100749), stimulated with the IL-12 variant at 37°C for 20 minutes, then fixed, permeabilized, and stained for pSTAT4 as described for human T cell signaling.

[0254] NK cell INFγ induction For the NK cell IFNγ induction assay, NK cells were isolated from the spleen and lymph nodes of C57 / BL6 mice using a mouse NK cell isolation kit (Miltenyi, #130-115-818) and an LS magnetic column (Miltenyi, #130-042-401). The NK cells were stimulated at 37°C for 48 hours in 96-well round-bottom plates containing 50 ng / mL recombinant mouse IL-18 (R&Dsystems, #9139-IL-010) and 1 μM IL-12 variant at a concentration of 25,000 cells / well. GolgiStop (BD#554724) was added during the last 4 hours of culture to prevent further cytokine secretion. Cells were fixed and permeabilized using a Cytofix / Cytoperm kit (BD, #554714) and stained with αIFNγ AF647 (XMG1.2, BD, #557735). Fluorescence intensity was recorded using a CytoFlex flow cytometer and analyzed with FlowJo.

[0255] IFNγ induction in CD8+ T cells For the CD8+ T cell effector assay, OT-I TCR transgenic mice (C57BL / 6-Tg(TcraTcrb)1100Mjb / j) (Hogquist et al., 1994) were obtained from Jackson Labs and maintained at the Stanford Animal Facility according to a protocol approved by the Stanford University Institutional Animal Care and Use Committee. OT-I splenocytes were stimulated in medium containing 1 μg / mL ovalbumin (aa257~264, GenScript, #RP10611), 100 IU / mL rmIL-2, and 1 μM IL-12 variant. For the IFNγ induction assay, cells were stimulated in 96-well round-bottom plates at 80,000 cells / well for 48 hours. GolgiStop was added during the last 4 hours to prevent further cytokine secretion. Cells were stained with αCD3 e450 (17A2, eBioscience, 48-0032-82) and αCD8 BV785 (53-6.7, Biolegend, #100749), then fixed / permeabilized using a Cytofix / Cytoperm kit and stained with αIFNγ AF647. Samples were gated with CD3+CD8+ cells, and αIFNγ AF647 staining was evaluated using a CytoFlex flow cytometer, followed by analysis with FlowJo.

[0256] MHC-I Upgrade Regulation For MHC-I upregulation, 25,000 B16F10 melanoma cells (ATCC, #CRL-6475) were seeded in 96-well flat-bottom plates at 37°C for 4 hours. The supernatant from OT-1 effectors, generated with or without the IL-12 variant as described above, was diluted in culture medium and added to the B16F10 cells at 37°C for 16 hours. After overnight incubation, the medium was removed and the B16F10 cells were isolated using TrypLE (ThermoFisher, #12604013). Cells were then αH-2K b Viable cells were identified by staining with APC (AF6-88.5.5.3, BioLegend, #116512) and PI. Data were collected using a CytoFlex flow cytometer and analyzed with FlowJo.

[0257] Antigen-specific tumor cell death To achieve antigen-specific tumor cell death, B16F10 cells were transduced with pCDH-EF1-cOVA-T2A-copGFP (Tseng et al., 2013), and sorted to obtain a pure population of OVA-GFP-expressing cells. B16F10 wild-type and OVA-GFP were mixed in a 1:1 ratio, and 25,000 cells were seeded in a 96-well flat-bottom plate. After 4 hours at 37°C, the medium was removed, and OT-I effectors, generated with or without the IL-12 variant as described above, were added to complete RPMI at 37°C for 36 hours. After removing the medium, B16F10 was isolated using TrypLe, stained with PI and αCD45.2APC (104, eBioscience, #17-0454-82), and then subjected to CytoFlex. B16F10 was identified as a live CD45.2-, and %GFP+ was quantified compared to conditions without the effector.

[0258] Example 2 Crystal structure of IL-12Rβ1 and quaternary IL-23 receptor complex This example describes the results of experiments conducted to determine the crystal structures of the IL-12Rβ1 and quaternary IL-23 receptor complexes, which will help elucidate the chemistry driving the cytokine-receptor interactions of each of the heteromeric receptor complexes.

[0259] As described above, IL-23 (IL-23p19 / IL-12p40) signals via a receptor complex composed of IL-23R and IL-12Rβ1 (Figure 1A). The ECD of IL-12Rβ1 consists of five fibronectin type III (FNIII) domains, and its N-terminal D1-D2 domain mediates binding to IL-23. Experiments were planned and carried out to crystallize the IL-12Rβ1 D1-D2 complex containing IL-23 and the IL-23R external domain. Table 3 below summarizes the crystallographic data and detailed statistics of the quaternary complex diffracted at a resolution of 3.4 Å.

[0260] The structure of a portion of the complex was determined by molecular substitution using the already published IL-23R ternary (IL-23p19 / IL-12p40 / IL-23R) complex. However, the structure of IL-12Rβ1 was still needed. Therefore, additional experiments were performed using single isomorphic substitution with anomalous scattering (SIRAS) to determine the structure of the human IL-12Rβ 1D1-D2 domain at a resolution of 2.0 Å. Subsequently, this newly established structure was used as a search model that allowed the IL-12Rβ1 D1 domain to be placed in the electron density of the quaternary complex. The D2 domain was not visible, which is thought to be due to the flexibility of the crystal lattice.

[0261] The quaternary IL-23 receptor complex was observed to exhibit a modular structure in which IL-23 functions as a bridge to combine IL-23R and IL-12Rβ1, initiating JAK1 / Tyk2 transphosphorylation within the cell (Figures 1B-1E). Table 3 below shows an overview of the contact site between IL-12p40 and IL-12Rβ1. [Table 3]

[0262] The covalent receptor IL-12Rβ1 binds to the "posterior" portion of IL-12p40 at the intersection of the N-terminal Ig domain of D1 and the fibronectin domain of D2 (Figure 1D). The D1 domain of IL-12p40 is tilted forward relative to the D2 domain, exposing a cleavage between the bottom of D1 and the top of D2 to form the docking site for IL-12Rβ1. The D1 domain of IL-12Rβ1 is a single 1425 Å domain characterized by high charge complementarity between interacting proteins. 2It binds to IL-12p40 at the interface. The bottom of the interface is formed by a continuous positively charged loop (His216, Lys217, and Lys219) of IL-12p40 that interacts with a negatively charged patch of IL-12Rβ1 composed of Glu28, Asp58, and Asp101. Above these charge-charge interaction sites, there is a hydrophobic strip on IL-12p40 formed by aromatic residues (Tryp37 and Phe82) surrounded by polar residues (Glu102, Ser106, Tyr109, Gln132, and Tyr134) of IL-12Rβ1 that form hydrogen bond interaction sites between the side chains and main chain atoms of IL-12p40.

[0263] Example 3 IL-12p40 functions as a common regulator of IL-12 and IL-23 signaling This example describes experiments conducted to demonstrate that IL-12p40 acts as a common regulator of IL-12 and IL-23 signaling.

[0264] The crystal structure of the IL-23 receptor complex described in Example 2 above revealed that IL-12p40 directly engages with IL-12Rβ1, indicating that IL-12p40 can play a conserved role in IL-12 and IL-23 signaling. This was confirmed by surface plasmon resonance (SPR) binding measurements showing that IL-12Rβ1 binds to IL-12p40 with an affinity of 1.7 μM (Figure 2A). To investigate the differences between IL-12 signaling and IL-23 signaling, several experiments were planned and conducted in which human CD4+ T cells were stimulated with IL-12 or IL-23, and phosphorylation of STAT3 and STAT4 was measured by phospho-flow cytometry. It was observed that IL-12 stimulation preferentially resulted in phosphorylation of STAT4, while IL-23 more potently promoted STAT3 phosphorylation (Figures 2B - 2C).

[0265] As discussed above, based on the shared role of IL-12p40 / IL-12Rβ1 interaction in both IL-12 and IL-23 receptor complexes, we planned and conducted additional experiments targeting this interface to modulate the levels of IL-12-related STAT4 signaling and IL-23-related STAT3 signaling by "adjusting" the efficiency of IL-12Rβ1 recruitment. In particular, we created a panel of IL-12 and IL-23 partial agonists by introducing alanine substitutions into the two loops of IL-12p40D1 that mediate interaction with IL-12Rβ1 (Figure 2D). In these experiments, we found that individual alanine mutations (E81A and F82A) reduced the potency of IL-12 and IL-23, as indicated by the rightward shift in the dose-response curves of pSTAT4 and pSTAT3 (Figures 2E-2F). These experiments showed that by combining multiple alanine mutations (4×Ala:P39A / D30A / E81A / F82A), cytokine EC 50 A significant increase and a decrease in maximum STAT phosphorylation were observed.

[0266] A complete list of IL-12p40 amino acid positions that engage IL-12Rβ1 is shown in Figure 2G, and IL-12 signaling with additional alanine mutations is shown in Figure 2H.

[0267] Example 4 IL-12 partial agonists induce cell-type specific activity based on differential IL-12Rβ1 expression. This example describes the results of experiments conducted in mouse IL-12 to demonstrate that an IL-12 partial agonist induces cell-type specific activity based on differential IL-12Rβ1 expression.

[0268] As mentioned above, systemic administration of IL-12 often leads to toxicity due to NK cell-mediated IFNγ production. Therefore, while IL-12 signaling is biased to preferentially activate T cells, reduced IFNγ induction by NK cells may reduce toxicity. A key difference in IL-12 signaling between T cells and NK cells is that antigen stimulation via the T cell receptor enhances IL-12 sensitivity through the upregulation of its receptor subunits. Using IL-12p40 as a FACS staining reagent to evaluate IL-12Rβ1 surface expression, mouse CD8+ T cell blasts were found to have higher IL-12Rβ1 expression than NK cells or ex vivo CD8+ T cells (Figure 3A).

[0269] As its structure indicates, IL-12p40 mediates the recruitment of IL-12Rβ1. Therefore, although not bound by any particular theory, it was hypothesized that reducing the affinity of IL-12p40 for IL-12Rβ1 might more severely impair signaling in NK cells with reduced IL-12Rβ1 expression levels compared to antigen-experiencing T cells. Based on sequence homology with human IL-12p40, additional experiments were planned and performed to design a series of partial agonist alanin mutations in mouse IL-12p40 that were predicted to disrupt binding to IL-12Rβ1 (Figure 3B). Experiments were conducted to test signaling in CD8+ T cell blasts in order to characterize the mouse IL-12 variants. As predicted, mutations in IL-12p40 at the IL-12Rβ1 binding interface increased EC50 and reduced maximal STAT4 phosphorylation by (3x alanine) and (4x alanine) mutants, preventing the induction of measurable STAT4 phosphorylation in this acute signaling assay.

[0270] A well-established harvest of IL-12 signaling in both T cells and NK cells is the induction of IFNγ. To determine the ability of IL-12 partial agonists to promote IFNγ production in antigen-specific CD8+ T cells, ovalbumin-specific OT-I T cells (Hogquist et al., 1994) were stimulated with OVA peptide and IL-12 variants for 48 hours in additional experiments, after which IFNγ production was assessed by intracellular cytokine staining. Despite the fact that 3×Ala and 4×Ala variants did not produce measurable STAT4 phosphorylation upon acute stimulation, IL-12, along with 2×, 3×, and 4× alanine variants, resulted in upregulation of IFNγ (Figure 4A). This discrepancy may be due to differences in sensitivity or longer signal integration times between assays.

[0271] To evaluate the ability of IL-12 variants to stimulate IFNγ production in NK cells, additional experiments were performed in which cells were stimulated with IL-12 variants in the presence of IL-18 for 48 hours, followed by analysis of IFNγ induction by intracellular cytokine staining. Both IL-12 and IL-18 stimulation induced robust IFNγ expression, and this response was attenuated in the (2×Ala) variant and suppressed in the 3×Ala and 4×Ala variants, as measured by intracellular cytokine staining and supernatant ELISA (see, for example, Figure 4B). Thus, IL-12 induces robust IFNγ expression in both CD8+ T cells and NK cells, but the (3×Ala) and (4×Ala) partial agonists preferentially supported IFNγ induction in antigen-experienced CD8+ T cells, while their activity towards NK cells was reduced (Figures 4C and 6A). These results suggest that increased IL-12Rβ1 surface expression leads to greater resistance of activated CD8+ T cells to IL-12p40 mutations, potentially representing a novel mechanism that alters the cell type specificity of IL-12 signaling and reduces NK cell-mediated toxicity. Unlike T cells, which require TCR-mediated stimulation to respond to IL-12, NK cells produce IFNγ in response to IL-12 when combined with the IL-1 family cytokine IL-18 (Figure 6B). IL-12 and IL-18 stimulation induced robust IFNγ expression, and this response was attenuated by 3×Ala and 4×Ala variants, as measured by intracellular cytokine staining (Figures 4B, 6C, and 6D) and supernatant ELISA (Figure 6E). These results were confirmed and expanded by a larger panel of IL-12 partial agonists (Figures 4D-4G).

[0272] IL-12 and IL-18 also promoted upregulation of lfng at the transcript level after 8 hours of stimulation, and this effect was reduced with 3×Ala / IL-18 stimulation (Figure 6F). However, under these conditions, no induction of Tigit by IL-12 was observed (Figure 6G). It has already been shown that the γc family cytokines IL-2 and IL-15 modulate NK cell activity and cause upregulation of IL-12 receptor components. Consistent with these reports, additional experiments were conducted to demonstrate that pre-activation of NK cells with IL-2 results in a slight upregulation of IL-12Rβ1 (Figure 6H). Addition of IL-2 to NK cell cultures increased IFNγ production more than IL-18 alone. However, it was observed that IL-2 did not synergistically enhance IFNγ induction beyond IL-2 / IL-18 in conjunction with 3×Ala and 4×Ala (Figure 6I).

[0273] Additional experiments were conducted to evaluate IL-12Rβ1 expression and IFNγ production in human peripheral blood mononuclear cells (PBMCs), which helped determine whether human IL-12 partial agonists could induce cell-type-specific responses. As summarized in Figures 7A–7D, similar to the findings in mice, TCR stimulation was observed to enhance IL-12Rβ1 expression in CD8+ T cells more than in inactivated T cells and NK cells (Figures 7A and 7B). In these experiments, similar IL-12 mutains were created and tested for pSTAT4 signaling in CD8+ T cell blasts (Figures 7C–7D and 7E). Human IL-12 partial agonists were observed to preferentially support IFNγ induction by CD8+ T cells compared to NK cells (Figures 7C–7D, 7F–7G). These findings indicate that IL-12Rβ1 upregulation is a conserved mechanism used by T cells to increase their sensitivity to IL-12 signaling, and that IL-12 partial agonists can bias signaling towards T cells in both humans and mice.

[0274] Example 5 IL-12 partial agonists promote antigen-specific tumor death. This example describes the results of an experiment conducted to demonstrate that an IL-12 partial agonist promotes antigen-specific tumor death.

[0275] In CD8+ T cells, IL-12 acts to enhance antigen-specific death of tumor and virus-infected cells (Schurich et al., 2013). The effects of IL-12 are mediated by the upregulation of cytotoxic factors such as granzyme B and the secretion of inflammatory cytokines including IFNγ (Aste-Amezaga et al., 1994). A well-explained role of IFNγ in tumor cell death is the upregulation of MHC-I, which can make transformed cells sensitive to T cell surveillance (Zhou, 2009). To determine whether IL-12-induced IFNγ causes MHC-I upregulation in tumor cell lines, supernatants were collected from OT-I effectors generated with or without an IL-12 partial agonist and added to B16F10 mouse melanoma cell lines. MHC-I surface expression was evaluated by antibody staining after overnight incubation. Consistent with the elevation of IFNγ levels measured by intracellular cytokine staining, supernatants from IL-12 and partial agonist cultures induced MHC-I expression more potently than supernatants generated in the absence of IL-12 (Figure 5A).

[0276] The findings described herein that IL-12 partial agonists promote IFNγ production and subsequent MHC-1 upregulation in tumor cell lines led to further testing of the ability of IL-12 partial agonists to enhance tumor cell death. To measure antigen-specific CD8+ T cell death, B16F10 cells were transfected with a plasmid containing ovalbumin along with a GFP marker (OVA-GFP) and mixed with wild-type B16F10 cells. This mixture was incubated with OT-I effectors, and antigen-specific tumor cell death was measured using the frequency of OVA-GFP-expressing cells (Figure 5B). OT-I effectors generated in the presence of IL-12 or a partial agonist were able to kill OVA-expressing tumor cells at a lower effector-to-target cell ratio, demonstrating increased efficacy of the antitumor response (Figure 5C). In summary, these data indicate that IL-12 partial agonists with reduced affinity for IL-12Rβ1 promote IFNγ production and tumor cell death by antigen-specific CD8+ T cells with reduced activity against NK cells.

[0277] Example 6 IL-12 partial agonists support antigen-specific T cell responses with reduced in vivo NK cell activation. To test whether IL-12 partial agonists induce cell-type specific responses in vivo, OT-1 CD8+ T cells were adoptively transferred to Thy1.1 congenic recipients and immunized with OVA (257-264) in an incomplete Freudian adjuvant (OVA-IFA), followed by daily cytokine administration for 5 days (Figure 9A). In the in vivo study, IL-12 and the partial agonist were expressed in mammalian cells (Expi293F). Subsequently, it was confirmed that the mammalian-expressed IL-12 partial agonist maintained cell-type bias in vitro, as had been observed with previously used baculovirus-expressing materials (Figures 8A-8E).

[0278] Treatment with IL-12, rather than 2×Ala or 3×Ala, was observed to induce weight loss and elevated serum IFNg levels (Figure 9B-9C). To evaluate the effect of immunization on T cell activation, the expression of the inhibitory receptor PD-1 on OT-I T cells was tracked. Immuneation increased the frequency of PD-1+ OT-I T cells independently of cytokine treatment and showed activation of adoptive transfer cells (Figure 9D-9E). The effect of immunization was enhanced by IL-12, which increased the frequency of OT-I T cells in the influx region lymph nodes; this effect was not observed with partial agonists (Figure 9F). In NK cells, as measured by the expression of the inhibitory receptor LAG-3, IL-12, rather than partial agonists, increased the population of activated NK cells (Figure 9G).

[0279] CD25, an IL-2Rα chain, has already been described as a marker for activated T cells and NK cells. IL-12 potently upregulated CD25 expression in both OT-I T cells and NK cells, while 2×Ala and 3×Ala partial agonists resulted in intermediate upregulation of CD25 on OT-I T cells without increasing expression in NK cells (Figures 9H-9J). Interestingly, the 2×Ala variant did not exhibit as pronounced a T / NK cell bias in vitro as the 3×Ala variant (Figures 8E-8F), although it was observed to exhibit an equally strong T / NK cell bias in vivo compared to 3×Ala, highlighting the possibility of quantitatively different therapeutic windows in vitro versus in vivo. These results suggest that IL-12 partial agonists support intermediate levels of T cell activation while exhibiting reduced in vivo NK cell stimulation and toxicity.

[0280] Example 7 IL-12 partial agonists support antitumor immunity with lower toxicity compared to IL-12. Based on in vitro characterization and in vivo cell profiling, it was concluded that IL-12 partial agonists can support antitumor T cell immunity without systemic toxicity by biasing IL-12 activity towards antigen-specific T cells and away from NK cells. To determine the ability of IL-12 partial agonists to produce therapeutic effects in vivo, additional experiments were performed on tumors using colon adenocarcinoma MC-38, which has been shown to respond to IL-12. In these experiments, mice were transplanted with MC-38 for one week, followed by daily cytokine therapy for seven days (Figure 10A). Daily administration of 1 μg or 30 μg of IL-12 resulted in severe toxicity, as measured by weight loss (Figure 10B), elevated serum IFNγ (Figure 10C), and decreased activity (Figure 10D). All mice administered 30 μg of IL-12 were observed to die from lethal toxicity between days 13 and 15. As a result, the behavioral capacity of these mice was not measured on day 16. In contrast, 2×Ala and 3×Ala partial agonists were well-tolerated and did not induce toxicity in tumor-bearing mice.

[0281] Both IL-12 and its partial agonists were further observed to attenuate tumor growth and prolong survival compared to treatment with PBS (Figures 10E-10H). However, the 2×Ala and 3×Ala partial agonists exhibited the above effects without inducing the systemic toxicity observed with IL-12 administration. These results provide additional in vivo support for the hypothesis that biased agonists, designed based on the structure of the IL-12Rβ1 covalent interface, possess the ability to isolate T cells from NK cell activation and significantly reduce IL-12 pleiotropy.

[0282] While specific alternatives to those described herein are disclosed, various modifications and combinations are possible, and it should be understood that they are intended to be in the true spirit and scope of the appended claims. Therefore, there is no intention to limit the exact summary and disclosure presented herein.

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Claims

1. Recombinant polypeptide, The interleukin 12 subunit p40 (IL-12p40) polypeptide having the amino acid sequence of SEQ ID NO: 1 comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with the interleukin 12 subunit p40 (IL-12p40) polypeptide, A recombinant polypeptide further comprising one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO:

1.

2. The recombinant polypeptide according to claim 1, wherein the one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO:

1.

3. The recombinant polypeptide according to claim 1 or 2, wherein the one or more amino acid substitutions are independently selected from the group consisting of alanine (A) substitution, arginine (R) substitution, asparagine (N) substitution, aspartic acid (D) substitution, leucine (L) substitution, lysine (K) substitution, phenylalanine (F) substitution, lysine substitution, glutamine (Q) substitution, glutamic acid (E) substitution, serine (S) substitution, and threonine (T) substitution.

4. The recombinant polypeptide according to any one of claims 1 to 3, wherein the one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and K219 of SEQ ID NO:

1.

5. The recombinant polypeptide according to any one of claims 1 to 4, wherein the one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, E81, F82, K106, K217, and K219 of SEQ ID NO:

1.

6. The amino acid sequence contains at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 1, and includes the following amino acid substitutions: a) W37A; b) P39A; c) D40A; d) E81A; e) F82A; f) K106; g) D109A; h) K217A; i) K219A; j) E81A / F82A; k) W37A / E81A / F82A; l) E81A / F82A / K106A; m) E81A / F82A / K106A / K219A; n) E81A / F82A / K106A / K217A; o) 81A / F82A / K106A / E108A / D115A; p) E81F / F82A; q) E81K / F82A; r) E81L / F82A; s) E81H / F82A; t) E81S / F82A; u) E81A / F82A / K106N; v) E81A / F82A / K106Q; w) E81A / F82A / K106T; x) E81A / F82A / K106R; or y) P39A / D40A / E81A / F82A, A recombinant polypeptide according to any one of claims 1 to 5, further comprising the corresponding amino acid substitution.

7. A recombinant polypeptide according to any one of claims 1 to 6, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 8 and 13 to 16.

8. Recombinant polypeptide, An interleukin 12 subunit p40 (IL-12p40) polypeptide having the amino acid sequence of SEQ ID NO: 2, and an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity. A recombinant polypeptide further comprising one or more amino acid substitutions at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X41, X80, X81, X82, X106, X108, X115, X216, X217, X218, and X219 of SEQ ID NO:

2.

9. The recombinant polypeptide according to claim 8, wherein the one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of X37, X39, X40, X81, X82, X106, X217, and X219 of SEQ ID NO:

2.

10. The recombinant polypeptide according to claim 8 or 9, wherein the one or more amino acid substitutions are independently selected from the group consisting of alanine (A) substitution, arginine (R) substitution, asparagine (N) substitution, aspartic acid (D) substitution, leucine (L) substitution, lysine (K) substitution, phenylalanine (F) substitution, lysine substitution, glutamine (Q) substitution, glutamic acid (E) substitution, serine (S) substitution, and threonine (T) substitution.

11. The recombinant polypeptide according to any one of claims 8 to 10, wherein the one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of W37, P39, D40, A41, K80, E81, F82, K106, E108, D115, H216, K217, L218, and E219 of SEQ ID NO:

2.

12. The recombinant polypeptide according to any one of claims 8 to 11, wherein the one or more amino acid substitutions are located at positions corresponding to amino acid residues selected from the group consisting of P39, D40, E81, F82, K106, K217, and E219 of SEQ ID NO:

2.

13. The amino acid sequence contains an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 2, and includes the following amino acid substitutions: a) W37A; b) P39A; c) D40A; d) E81A; e) F82A; f) K106; g) D109A; h) K217A; i) E219A; j) E81A / F82A; k) W37A / E81A / F82A; l) E81A / F82A / K106A; m) E81A / F82A / K106A / K217A; n) E81F / F82A; o) E81K / F82A; p) E81L / F82A; q) E81H / F82A; r) E81S / F82A; s) E81A / F82A / K106N; t) E81A / F82A / K106Q; u) E81A / F82A / K106T; v) E81A / F82A / K106R; or w) P39A / D40A / E81A / F82A, A recombinant polypeptide according to any one of claims 8 to 12, further comprising the corresponding amino acid substitution.

14. A recombinant polypeptide according to any one of claims 8 to 13, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 11 and 17 to 25.

15. A recombinant polypeptide according to any one of claims 1 to 14, having a modified binding affinity to the interleukin-12 receptor, subunit beta 1 (IL-12Rβ1) compared to the binding affinity of a reference polypeptide lacking one or more amino acid sequences.

16. The recombinant polypeptide according to claim 15, having a reduced binding affinity to IL-12Rβ1 compared to the binding affinity of a reference polypeptide lacking one or more amino acid substitutions.

17. The recombinant polypeptide according to any one of claims 15 to 15, which, when measured by surface plasmon resonance (SPR), has a binding affinity to IL-12Rβ1 that is about 10% to about 100% lower than that of a reference polypeptide lacking one or more amino acid substitutions.

18. The recombinant polypeptide according to any one of claims 15 to 17, wherein, when combined with an interleukin 12 subunit p35 (IL-12p35) polypeptide, it has a reduced ability to stimulate STAT4 signaling compared to a reference polypeptide lacking one or more amino acid substitutions.

19. The recombinant polypeptide according to any one of claims 15 to 18, wherein, when combined with an interleukin 23 subunit p19 (IL-23p19) polypeptide, it has a reduced ability to stimulate STAT3 signaling compared to a reference polypeptide lacking one or more amino acid substitutions.

20. The recombinant polypeptide according to claim 18 or 19, wherein STAT3 signaling and / or STAT4 signaling is determined by an assay selected from the group consisting of a gene expression assay, a phosphoflow signaling assay, and an enzyme-linked immunosorbent assay (ELISA).

21. The recombinant polypeptide according to any one of claims 15 to 20, wherein the one or more amino acid substitutions result in cell-type biased signaling of downstream signaling mediated by interleukin-12 (IL-12) and / or interleukin-23 (IL-23) compared to a reference polypeptide lacking the one or more amino acid substitutions.

22. The recombinant polypeptide according to claim 21, wherein the cell type-biased signaling includes a reduced ability of the recombinant polypeptide to stimulate IL-12-mediated signaling in natural killer (NK) cells.

23. The recombinant polypeptide according to claim 21 or 22, wherein the cell type-biased signaling does not substantially alter the ability of the recombinant polypeptide to stimulate IL-12 signaling in CD8+ T cells.

24. The recombinant polypeptide according to any one of claims 21 to 23, wherein the one or more amino acid substitutions result in a reduction of the recombinant polypeptide's ability to stimulate IL-12 signaling in NK cells, but substantially retains its ability to stimulate IL-12 signaling in CD8+ T cells.

25. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide having at least 90% sequence identity with the amino acid sequence of the polypeptide described in any one of claims 1 to 24.

26. The nucleic acid molecule according to claim 25, wherein the nucleic acid sequence is operably linked to a heterologous nucleic acid sequence.

27. A nucleic acid molecule according to any one of claims 25 to 26, further defined as an expression cassette or expression vector.

28. below: a) Recombinant polypeptide according to any one of claims 1 to 24; and / or b) Recombinant nucleic acid according to any one of claims 25 to 27, Recombinant cells, including those mentioned above.

29. The recombinant cell according to claim 28, wherein the recombinant cell is a eukaryotic cell.

30. The recombinant cell according to claim 29, wherein the eukaryotic cell is a mammalian cell.

31. A cell culture comprising at least one recombinant cell and a culture medium according to any one of claims 28 to 30.

32. below: a) To provide one or more recombinant cells according to any one of claims 28 to 30; and b) Culturing one or more recombinant cells in a culture medium so that the cells produce polypeptides encoded by the recombinant nucleic acid molecules. A method for producing recombinant polypeptides, including [a specific compound].

33. The method according to claim 32, further comprising isolating and / or purifying the generated polypeptide.

34. The method according to claim 32 or 33, further comprising structurally modifying the produced polypeptide to extend its half-life.

35. The method according to claim 34, wherein the modification comprises one or more modifications selected from the group consisting of fusion to a human Fc antibody fragment, fusion to albumin, and PEGylation.

36. Recombinant polypeptide produced by the method described in any one of claims 32 to 35.

37. below: a) Recombinant polypeptide according to any one of claims 1 to 24 and 36; b) Recombinant nucleic acid according to any one of claims 25 to 27; c) Recombinant cells according to any one of claims 28 to 30; and / or c) A pharmaceutically acceptable carrier, A pharmaceutical composition containing the following:

38. The pharmaceutical composition according to claim 37, comprising a recombinant polypeptide according to any one of claims 1 to 24 and 36, and a pharmaceutically acceptable carrier.

39. The pharmaceutical composition according to claim 37, comprising a recombinant nucleic acid according to any one of claims 25 to 27 and a pharmaceutically acceptable carrier.

40. A method for modulating IL-12-mediated signaling in a subject, the following: a) Recombinant IL-12p40 polypeptide according to any one of claims 1 to 24 and 36; b) Recombinant nucleic acid according to any one of claims 25 to 27; c) Recombinant cells according to any one of claims 28 to 30; and / or d) The pharmaceutical composition according to claims 37 to 39, A method comprising administering a composition containing the following to a target.

41. The method according to claim 40, further comprising administering to a subject IL-12p35 polypeptide or a nucleic acid encoding IL-12p35 polypeptide.

42. A method for modulating IL-23-mediated signaling in a subject, the following: a) Recombinant IL-12p40 polypeptide according to any one of claims 1 to 24 and 36; b) Recombinant nucleic acid according to any one of claims 25 to 27; c) Recombinant cells according to any one of claims 28 to 30; and / or d) The pharmaceutical composition according to claims 37 to 39, A method comprising administering a composition containing the following to a target.

43. The method according to claim 42, further comprising administering a nucleic acid encoding IL-12p35 polypeptide or IL-23p19 polypeptide.

44. A method for treating a condition in a person requiring it, as follows: a) Recombinant IL-12p40 polypeptide according to any one of claims 1 to 24 and 36; b) Recombinant nucleic acid according to any one of claims 25 to 27; c) Recombinant cells according to any one of claims 28 to 30; and / or d) The pharmaceutical composition according to claims 37 to 39, A method comprising administering a composition containing the following to a target.

45. below: a) IL-12p35 polypeptide; b) IL-23p19 polypeptide; and / or c) nucleic acids encoding (a) or (b) above, The method according to claim 44, further comprising administering to a target.

46. The method according to any one of claims 40 to 45, wherein the recombinant polypeptide has a modified binding affinity to the interleukin-12 receptor, beta-1 (IL-12Rβ1) compared to the binding affinity of the reference polypeptide lacking one or more amino acid substitutions.

47. The method according to any one of claims 40 to 46, wherein the recombinant polypeptide has a reduced binding affinity to IL-12Rβ1 compared to the binding affinity of the reference polypeptide lacking one or more amino acid substitutions.

48. The method according to any one of claims 40 to 47, wherein, as measured by surface plasmon resonance (SPR), the recombinant polypeptide has a binding affinity to IL-12Rβ1 that is about 10% to about 100% lower than the binding affinity of a reference polypeptide lacking one or more amino acid substitutions.

49. The method according to any one of claims 40 to 48, wherein a decrease in the binding affinity of the recombinant polypeptide to the IL-12Rβ1 receptor results in a decrease in STAT4-mediated signaling compared to a reference polypeptide lacking one or more amino acid substitutions.

50. The method according to any one of claims 40 to 49, wherein a decrease in the binding affinity of the recombinant polypeptide to the IL-12Rβ1 receptor results in a decrease in STAT3-mediated signaling compared to a reference polypeptide lacking one or more amino acid substitutions.

51. The method according to any one of claims 49 to 50, wherein the STAT3 signaling and / or STAT4 signaling is determined by an assay selected from the group consisting of a gene expression assay, a phosphoflow signaling assay, and an enzyme-linked immunosorbent assay (ELISA).

52. The method according to any one of claims 40 to 51, wherein the administered composition results in cell-type biased signaling of downstream signaling mediated by interleukin 12 (IL-12) and / or interleukin 23 (IL-23) compared to a reference polypeptide lacking the one or more amino acid substitutions.

53. The method according to claim 52, wherein the cell type-biased signaling includes a reduced ability of recombinant polypeptides to stimulate IL-12-mediated signaling in NK cells.

54. The method according to any one of claims 52 to 53, wherein the cell type-biased signaling includes the substantially unmodified ability of a recombinant polypeptide to stimulate IL-12 signaling in CD8+ T cells.

55. The method according to any one of claims 40 to 54, wherein the administered composition causes a decrease in the ability of recombinant polypeptides to stimulate IL-12 signaling in NK cells, but substantially retains their ability to stimulate IL-12 signaling in CD8+ T cells.

56. The method according to claim 55, wherein the administered composition substantially retains the ability of a recombinant polypeptide to stimulate the expression of interferon-gamma (INFγ) in CD8+ T cells.

57. The method according to any one of claims 40 to 56, wherein the administered composition enhances antitumor immunity in the tumor microenvironment.

58. The method according to any one of claims 40 to 57, wherein the subject is a mammal.

59. The method according to claim 58, wherein the mammal is a human.

60. The method according to any one of claims 40 to 59, wherein the subject has or is suspected to have a condition related to IL-12p40 mediated signaling.

61. The method according to claim 60, wherein the IL-12p40-mediated signaling is IL-12-mediated signaling or IL-23-mediated signaling.

62. The method according to claim 60, wherein the condition is cancer, an immune disease, or a chronic infection.

63. The method according to claim 62, wherein the immune disease is an autoimmune disease.

64. The method according to claim 63, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemia, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, moderate to severe psoriasis vulgaris, psoriatic arthritis, Crohn's disease, ulcerative colitis, and graft-versus-host disease.

65. The method according to claim 62, wherein the condition is a cancer selected from the group consisting of acute myelomatous leukemia, anaplastic lymphoma, astrocytoma, B-cell carcinoma, breast cancer, colon cancer, ependymoma, esophageal cancer, glioblastoma, glioma, leiomyosarcoma, liposarcoma, liver cancer, lung cancer, mantle cell lymphoma, melanoma, neuroblastoma, non-small cell lung cancer, oligodendroglioma, ovarian cancer, pancreatic cancer, peripheral T-cell lymphoma, renal cancer, sarcoma, gastric cancer, carcinoma, mesothelioma, and sarcoma.

66. The method according to any one of claims 40 to 65, wherein the composition is administered to a subject individually as a first treatment or in combination with a second treatment.

67. The method according to claim 66, wherein the second treatment is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery.

68. The method according to any one of claims 66 to 67, wherein the first treatment and the second treatment are administered in conjunction with each other.

69. The method according to any one of claims 66 to 68, wherein the first treatment is administered simultaneously with the second treatment.

70. The method according to any one of claims 66 to 68, wherein the first treatment and the second treatment are administered in succession.

71. The method according to claim 70, wherein the first treatment is administered before the second treatment.

72. The method according to claim 70, wherein the first treatment is administered after the second treatment.

73. The method according to claim 70, wherein the first treatment is administered before and / or after the second treatment.

74. The method according to any one of claims 66 to 73, wherein the first treatment and the second treatment are administered in sequence.

75. The method according to claim 66 or 67, wherein the first treatment and the second treatment are administered together in a single formulation.

76. A kit for modulating IL-12p40-mediated signaling, for modulating IL-12p40-mediated signaling, or for treating a condition requiring such modification, the following: a) Recombinant polypeptide according to any one of claims 1 to 24 and 36; b) Recombinant nucleic acid according to any one of claims 25 to 27; c) Recombinant cells according to any one of claims 28 to 31; and / or d) The pharmaceutical composition according to any one of claims 37 to 39; and Instructions for carrying out the method described in any one of claims 40 to 75, A kit that includes this.

77. For the manufacture of pharmaceuticals for treating and / or preventing conditions related to health conditions associated with disruption of IL-12-p40 mediated signaling, the following: a) Recombinant polypeptide according to any one of claims 1 to 24 and 36; b) Recombinant nucleic acid according to any one of claims 25 to 27; c) Recombinant cells according to any one of claims 28 to 31; and / or d) The pharmaceutical composition according to any one of claims 37 to 39, Use.

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