WO2023133540A1 - Il-12 affinity variants - Google Patents

Abstract

The present disclosure provides IL-12 affinity variants, pharmaceutical compositions comprising the variants, therapeutic cells expressing the variants, and methods or uses of the same for the treatment or prevention of a disease or disorder.

Description

IL-12 AFFINITY VARIANTS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application 63/297,666, filed January 7, 2022, the disclosure of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 10, 2022, is named 026225_W0020_SL.xml and is 97,666 bytes in size.

BACKGROUND OF THE INVENTION

[0003] Interleukin- 12 (IL-12) is a proinflammatory cytokine that can stimulate the proliferation of natural killer (NK) and T cells and drive secretion of IFN-y and TNF-a (Tugues et al., Cell Death Differ. (2015) 22(2):237-46). IL-12 is a heterodimer composed of two subunits, p35 (a.k.a. IL-12A) and p40 (aka. IL-12B). The heterodimer is also called p70. IL-12 binds to IL-12 receptor (IL-12R), which in turn is composed of two subunits, IL-12RP1 and IL-12RP2. IL-12RP1 primarily binds the IL-12 p40 subunit, while IL-12RP2 primarily binds the IL-12 p35 subunit (Presky et al., J Immunol. (1998) 160(5):2174-9). There is no complete co-crystal structure of IL- 12 bound to IL-12R. Simultaneous binding of IL- 12 to both IL-12RP1 and IL-12RP2 may be required to drive intracellular signaling (Presky et al., PNAS (1996) 93(24): 14002-7; Presky et al., Ann NY Acad Sci. (1996) 795:390-3; Robinson, Cytokine (2015) 71(2):348-59). Recent cryogenic electron microscopy studies produced a structure of IL- 12 in complex with IL-12R, but they were unable to solve the detailed interactions between p35 and IL-12RP2 (Glassman et al., Cell (2021) 184:983-99).

[0004] IL-12 is a key cytokine in the initiation of a Thl response and has been explored as a potential therapy to treat cancer (Lasek et al., Cancer Immunol Immunother. (2014) 63(5):419-35). But due to significant systemic toxicity, the approaches to IL-12-based immunotherapy have been focused on direct injections of IL- 12 to tumor sites and on fusions of IL-12 to tumor-targeting moieties. Some researchers have attempted to use a cell-based approach to deliver IL-12 in which cells engineered to express IL-12 are administered in vivo (Wei et al., J Cell Mol Med. (2013) 17(11): 1465-74; Zhang et al., Clin Cancer Res. (2015) 21(10):2278-88). However, this approach is also challenging because IL-12 activates proinflammatory signaling even at very low concentrations in circulation. Furthermore, efficacy is still a critical consideration as the immunosuppressive TME can restrain cell activity even in the presence of IL- 12 (Lasek, supra).

[0005] Thus, there is a long-felt and unmet need for a safe and effective form of IL-12 for stimulation of the immune system, either as a monotherapy or as a combination therapy.

SUMMARY OF THE INVENTION

[0006] The present disclosure provides a composition comprising an IL-12 affinity variant, wherein the IL-12 affinity variant comprises a mutation at one or more positions of SEQ ID NO: 1, wherein the mutation reduces the binding affinity of the IL- 12 affinity variant when compared to IL- 12 not having the mutation.

[0007] In one aspect, the present disclosure provides a human IL-12 p35 subunit mutein, wherein the mutein comprises one or more non-naturally occurring mutations, optionally a substitution, at one or more positions that correspond to position 37, 38, 39, 40, 41, 46, 47, 123, 124, 125, 126, 127, 128, 129, 130, 131, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, and 175 of SEQ ID NO: 1. In some embodiments, the mutein comprises one or more mutations at positions 37, 38, 39, 40, 41, 128, 167, 171, and 175 of SEQ ID NO: 1.

[0008] In some embodiments, the mutein comprises a mutation at position 40 of SEQ ID NO: 1, optionally wherein the mutation is Y40A, Y40G, or Y40S.

[0009] In some embodiments, the mutein comprises a mutation at position 128 of SEQ ID NO: 1, optionally wherein the mutation is K128D.

[0010] In some embodiments, the mutein comprises a mutation at position 167 of SEQ ID NO: 1, optionally wherein the mutation is Y167A.

[0011] In some embodiments, the mutein comprises a mutation at position 171 of SEQ ID NO: 1, optionally wherein the mutation is 1171 A.

[0012] In some embodiments, the mutein comprises a mutation at position 175 of SEQ ID NO: 1, optionally wherein the mutation is I175A.

[0013] In some embodiments, the mutein comprises mutations at positions 167, 171, and 175 of SEQ ID NO: 1, optionally wherein the mutations are Y167A, 1171 A, and 1175 A.

[0014] In some embodiments, the mutein comprises mutations at positions 40 and 128 of SEQ ID NO: 1, optionally wherein the mutations are Y40A and K128D. [0015] In some embodiments, the mutein comprises a mutation at position 37, 38, 39, 40, or 41 of SEQ ID NO: 1, optionally wherein the mutation is a substitution by a glycine (G) or a serine (S).

[0016] In some embodiments, the mutein comprises mutations at positions 37-41 of SEQ ID NO: 1, optionally wherein the mutations are substitutions of LEFYP (SEQ ID NO:7) by GGSGS (SEQ ID NO:8).

[0017] In particular embodiments, the mutein comprises a non-naturally occurring substitution selected from Table 1 or 2.

[0018] In another aspect, the present disclosure provides a human IL-12 variant, comprising the human IL- 12 p35 mutein, and a human IL- 12 p40 subunit. In some embodiments, the IL- 12 variant has reduced binding affinity for IL- 12 receptor P (IL-12RP), optionally for IL-12R P2 subunit, compared to a human IL-12 without the mutation(s).

[0019] In some embodiments, the IL-12 p40 subunit comprises a mutation at a position that corresponds position 39, 40, 81, or 82 of SEQ ID NO:5, optionally wherein the mutation is P39A, D40A, E81 A or F82A. In further embodiments, the IL-12 p40 subunit comprises two or more of P39A, D40A, E81A and F82A.

[0020] In some embodiments, the IL-12 variant is a heterodimer comprising the p35 subunit variant and the p40 subunit. In other embodiments, the IL-12 variant is a singlechain fusion protein (scIL-12) comprising the p35 subunit variant and the p40 subunit, optionally wherein the two subunits are linked by a peptide linker, optionally a flexible peptide linker.

[0021] In some embodiments, the IL-12 variant comprises a membrane anchor or a transmembrane domain.

[0022] In one aspect, the present disclosure also provides a pharmaceutical composition comprising the human IL-12 variant herein and a pharmaceutically acceptable carrier.

[0023] In another aspect, the present disclosure a mouse IL-12 p35 mutein, comprising one or more mutations selected from Table 3 or 4; and a mouse IL-12 variant comprising the mouse IL-12 p35 mutein and a mouse IL-12 p40 subunit, wherein the p40 is wildtype or contains one or more mutations optionally selected from E81A and F82A. In some embodiments, the mouse IL-12 variant is a heterodimer comprising the p35 mutein and the p40 subunit; or an scIL-12 comprising the p35 mutein and the p40 subunit, optionally wherein the two subunits are linked by a peptide linker, optionally a flexible peptide.

[0024] In other aspects, the present disclosure provides an isolated nucleic acid molecule or isolated nucleic acid molecules encoding the present IL-12 p35 mutein or IL-12 variant; and an expression vector or expression vectors comprising the isolated nucleic acid molecule(s), optionally wherein the expression vector(s) are viral vectors, further optionally wherein the viral vectors are lentiviral vectors, adenoviral vectors, or adeno-associated viral (AAV) vectors.

[0025] Provided also are mammalian cells comprising the expression vector(s) and expressing the IL-12 variant of the present disclosure. In some embodiments, the mammalian cells are CHO cells. In other embodiments, the mammalian cells are human immune cells, optionally T cells or natural killer (NK) cells. In further embodiments, the human immune cells are T cells or NK cells engineered to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR), optionally wherein the CAR or engineered TCR targets a tumor antigen.

[0026] In one aspect, the present disclosure provides a method of stimulating the immune system, or treating cancer, in a human subject in need thereof, comprising administering the present pharmaceutical composition, or the present engineered immune cells, to the human subject. Also provided are use of the present human IL-12 variant, expression vector(s) therefor, or engineered immune cells, for the manufacture of a medicament for stimulating the immune system or treating cancer in a human subject in need thereof; as well as the present pharmaceutical composition, expression vector(s), or engineered immune cells for use in stimulating the immune system or treating cancer in a human subject in need thereof.

[0027] In another aspect, the present disclosure provides a method of producing a human IL-12 variant, comprising: culturing the present mammalian host cell (e.g., CHO) under conditions that allow expression of the human IL-12 variant, and isolating the human IL-12 variant from the culture.

[0028] In another aspect, the present disclosure provides a mouse IL-12 p35 mutein, comprising one or more mutations selected from Table 3 or 4.

[0029] In another aspect, the present disclosure provides a mouse IL-12 variant comprising the mouse IL-12 p35 mutein comprising one or more mutations selected from Table 3 or 4, and a mouse IL-12 p40 subunit. In some embodiments, the p40 is wildtype or contains one or more mutations optionally selected from E81A and F82A. In some embodiments, the mouse IL-12 variant is a heterodimer comprising the p35 mutein and the p40 subunit; or a single-chain fusion protein (scIL-12) comprising the p35 mutein and the p40 subunit. In some embodiments, the two subunits are linked by a peptide linker, such as a flexible peptide. [0030] Other features, objectives, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a pair of graphs depicting an exemplary gating strategy for identifying IL-12 p35* muteins after site- saturation mutagenesis (SSM). A library of p35* mutants was transduced into Jurkat cells in the context of membrane-anchored, surface-expressed scIL-12 with an N-terminal strep tag II (STII), and cells were sorted into four populations binned based on FITC (STII) and Alexa Fluor 647 (IL-12Rp2) staining. “LL,” “ML,” “MM,” and “MH”: see Materials and Methods used in the Examples for details. Top graph: 5’ p35 library. Bottom graph: 3’ p35 library.

[0032] FIGs. 2A-2D are heatmaps from SSM of IL- 12 p35 identifying positions that are important for binding to IL-12RP2 (FIGs. 2A and 2B) and for expression of single-chain IL- 12 (scIL-12; FIGs. 2C and 2D). Black boxes indicate mutations with enrichment ratios above the 95th percentile for the ML bin with mutants that disrupt protein expression subtracted (ML-LL bins) (FIGs. 2 A and 2B); and for the LL bin (FIGs. 2C and 2D). Values < or = 0 converted to 0 (white). Values greater than 0 converted to 1 (black).

[0033] FIG. 3A is a pair of bar plots depicting the sum of the subtracted ML enrichment ratios at each residue in IL-12 p35 for the top 5th percentile. Top bar plot: 5’ library. Bottom bar plot: 3’ library. See Materials and Methods used in the Examples for details.

[0034] FIG. 3B is a schematic diagram depicting that enriched positions are mapped onto the IL-12 p35 structure PDB ID 3HMX (Luo et al., J Mol Biol. (2010) 402(5):797-812) (corresponding to GenBank Accession No. 3HMX B; also known as IL-12A or p35; SEQ ID NO: 1). Residues in the top 50% of summed enrichment ratios within the selected set of sequences are shown in dark gray, whereas residues in the lower 50% are shown in medium gray.

[0035] FIG. 4 is a series of schematic diagrams depicting subunits of IL-23 and IL-12. Top and bottom-left panels (PDB ID 5MZV) depict positions on IL-23 pl9 subunit that may be involved in receptor binding. The bottom-right panel identifies positions Y167, 1171, and 1175 on IL-12 p35 subunit that may be involved in binding to IL-12RP2. [0036] FIG. 5 is a graph demonstrating that p35 and p40 mutations reduce activity of purified human scIL-12 proteins in an HEK-Blue™ IL-12R reporter cell line assay (InvivoGen). Purified proteins were serially diluted and added to HEK-Blue™ IL-12R reporter cells at the indicated final concentrations. IL-12R activation was measured using a colorimetric assay that monitored secreted alkaline phosphatase activity in the supernatant of the reporter cells at 20 hours.

[0037] FIG. 6 is a pair of graphs demonstrating that IL- 12 p35 mutations at homologous positions in human (top) and mouse (bottom) scIL-12 proteins reduced IL- 12 activity in an HEK-Blue™ IL-12R reporter cell line assay. Purified proteins were serially diluted and added to HEK-Blue™ reporter cells at the indicated final concentrations. IL-12R activation was measured using a colorimetric assay that monitored secreted alkaline phosphatase activity in the supernatant of the reporter cells at 20 hours.

[0038] FIG. 7 is a graph depicting the results of an IFN-y secretion assay, which shows reduced activity of p35 mutants in primary T cells. Dilution series of purified scIL-12 proteins containing the indicated mutations in p35 were added to expanded T cells and stimulated with TransACT™ (Miltenyi Biotec). T cell supernatants were harvested at 20 hours and IFN-y was measured by Meso Scale Discovery (MSD). The last mutein, E81A, was a p40 mutein.

[0039] FIG. 8 is a schematic diagram depicting the structure of IL- 12 p35 PDB ID 3HMX (white) with IL-12R binding residues shown in dark gray, sticks representation.

[0040] FIG. 9 is a graph demonstrating that scIL-12 affinity variants enhanced tumor killing of an antigen-specific TCR-expressing T cells against A375 target cells proportionally to the variants’ affinity for IL-12R. The antigen-specific TCR was expressed in all conditions. 25% of each co-culture was carried over to new target cells every 3 days, until differences in cytolysis between conditions were observed. Each data point indicates a biological replicate. “Triple Alanine”: p35 mutein having the Y167A I171A I175A mutations. 37-41GS: p35 mutein where residues 37-41 LEFYP (SEQ ID NO:7) by GGSGS (SEQ ID NO:8).

[0041] FIG. 10 is a graph demonstrating that IL- 12 affinity variants enhanced IFN-y secretion from antigen-specific TCR-expressing T cells following co-culture with A375 target cells proportionally to the variants’ affinity for IL-12R. The antigen-specific TCR was expressed in all conditions.

[0042] FIG. 11 is a graph demonstrating that scIL-12 affinity variants can enhance IFN-y secretion of antigen-specific TCR-expressing T cells in an in vivo xenograft model using A375 target cells. The increase in IFN-y secretion was directly proportional to the variants’ affinity for IL-12R. The antigen-specific TCR was expressed in all conditions except PBS, and IFN-y was detected in the serum of mice by MSD. The “4X NF AT WT IL-12” condition includes an inducible promoter consisting of a 4X NF AT sequence and a wildtype human beta globin sequence that drives expression of a wildtype IL- 12 sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0043] The present disclosure describes studies that elucidate interface residues on IL-12 p35 that interact with IL-12R. Through these studies, the present inventors have discovered that mutations (e.g., substitutions) at certain positions of IL-12 p35 can modulate IL-12’s biological activity. Without being bound by theory, it is believed that such mutations impact p35’s interaction with IL-12R, particularly with IL-12RP2, causing a change of the IL- 12 variants’ affinity for IL-12R.

[0044] The present inventors have translated the discovery into a rational design for IL-12 affinity variants with a wide range of controlled signaling activity. In some embodiments, the mutations at the positions weaken the IL- 12 variants’ binding affinity for IL-12R such as IL- 12RP and thereby weaken the variants’ biological activity. IL-12 affinity variants with weakened activity can still be efficacious in immunotherapy while causing fewer adverse side effects as compared to IL-12 without the mutations such as wildtype IL-12.

[0045] Existing approaches to using IL-12 as a therapeutic agent pose substantial risk because the potent activity of IL-12 even at very low concentrations. Even for local administration, small amounts of IL-12 that diffuse from sites of interest can drive unwanted activity and cause toxicity. The IL-12 affinity variants of the present disclosure circumvent this problem because they are capable of driving IL-12R signaling at high concentrations, particularly when localized to sites of interest (e.g., tumor microenvironment (TME)), but are far less potent than wildtype IL-12 at low concentrations due to reduced binding affinity. Thus, the present disclosure provides a new approach to regulating IL-12 activity that balances safety with efficacy.

I. IL-12 Affinity Variants

[0046] The IL-12 variants of the present disclosure encompass only non-naturally occurring IL-12 molecules. It is believed that the IL-12 variants disclosed herein are non- naturally occurring. In some embodiments, the IL-12 variant is a heterodimer comprising human IL- 12 p35 and p40 subunits, with one or more mutations in the p35 subunit, the p40 subunit, or both subunits. In other embodiments, the IL-12 variant is a fusion protein comprising amino acid sequences of human p35 and p40, with mutations in the p35 sequence, the p40 sequence, or both sequences; the amino acid sequences of p35 and p40 may be linked via a peptide linker, such as a flexible peptide linker. In further embodiments, the heterodimeric or single-chain IL-12 (scIL-12) variants comprise a membrane anchor or a transmembrane domain such that they are tethered to the cell surface when expressed.

[0047] In some preferred embodiments, the IL-12 variants are variants of human IL-12. By “variants of human IL-12” or “human IL-12 variants’ is meant an IL-12 molecule comprising sequences of human IL-12 p35 and p40 subunits where one or both of the p35 and p40 sequences contain one or more amino acid mutations.

[0048] In other preferred embodiments, the IL-12 variants are variants of mouse IL-12.

By “variants of mouse IL-12” or “mouse IL-12 variants” is meant an IL-12 molecule comprising sequences of mouse IL-12 p35 and p40 subunits where one or both of the p35 and p40 sequences contain one or more amino acid mutations.

[0049] The IL- 12 variants herein have altered binding affinity for IL-12R, and thus are also called “IL-12 affinity variants.” In preferred embodiments, the IL-12 affinity variants herein have reduced affinity for IL-12R.

A. p35 Muteins

[0050] The IL-12 affinity variants herein comprise a p35 mutein, i.e., a p35 polypeptide containing mutations relative to a wildtype p35 sequence. An exemplary mature wildtype human p35 sequence is shown as SEQ ID NO: 1 below:

RNLPVATPDP GMFPCLHHSQ NLLRAVSNML QKARQTLEFY PCTSEE IDHE

DITKDKTSTV EACLPLELTK NESCLNSRET SFITNGSCLA SRKTSFMMAL

CLSS IYEDLK MYQVE FKTMN AKLLMDPKRQ I FLDQNMLAV IDELMQALNF

NSETVPQKSS LEEPDFYKTK IKLCILLHAF RIRAVTIDRV MSYLNAS

( SEQ ID NO : 1 )

See also IL- 12 p35 structure PDB ID 3HMX in Luo, supra, and GenBank Accession No.

3HMX B. An unprocessed wildtype human p35 sequence (UniProt ID No. P29459) is shown below, where the signal sequence (residues 1-22) is boxed:

|MCPARSLLLV ATLVLLDHLS LA]RNLPVATP DPGMFPCLHH SQNLLRAVSN MLQKARQTLE FYPCTSEEID HEDITKDKTS TVEACLPLEL TKNESCLNSR ETSFITNGSC LASRKTS FMM ALCLSS IYED LKMYQVE FKT MNAKLLMDPK RQI FLDQNML AVIDELMQAL NFNSETVPQK SSLEEPDFYK TKIKLCILLH AFRIRAVT ID RVMSYLNAS ( SEQ ID NO : 2 )

[0051] In some embodiments of the disclosure, an IL-12 p35 subunit mutein may comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between identity to SEQ ID NO: 1 or to a sequence comprising SEQ ID NO: 1.

[0052] The percent identity of two amino acid sequences (or of two nucleic acid sequences) may be obtained by, e.g., BLAST® using default parameters (available at the U.S. National Library of Medicine’s National Center for Biotechnology Information website). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the reference sequence.

[0053] In some embodiments, the p35 mutein may be identical to SEQ ID NO: 1 but for a mutation (e.g., a substitution) at one to five positions selected from L37, E38, F39, Y40, P41, K128, Y167, 1171, and 1175 of SEQ ID NO: 1. For example, the p35 mutein may comprise one or more mutations at positions Y40, K128, Y167, 1171, and 1175 of SEQ ID NO: 1 (FIG.

8)

[0054] The discovery of these positions was unexpected. As shown in the Working Examples below, the inventors have found that even though IL-23 and IL- 12 have structural homology, the interface structure between IL-23 and its receptor cannot be extrapolated to predict the interface structure between IL-12 and IL-12R. Mutagenesis studies of IL-23 cannot predict the impact of mutations at homologous positions in IL- 12 p35 on the IL- 12 variant’s binding affinity for IL-12R. Without performing the SSM studies described below, identification of the IL-12 muteins of the present disclosure and the degree to which they modulate IL-12R signaling could not have been accomplished based on homology with IL-23 alone. For example, the amino acid positions 40 and 128 of the p35 subunit had not been identified previously, by either structural or functional means, as positions capable of modifying IL- 12 affinity for IL-12R; however, the present studies show that these two positions play a role in modulating IL-12 activity.

[0055] Accordingly, the present disclosure provides human IL-12 affinity variants comprising heretofore unknown p35 mutations. In some embodiments, the p35 mutein comprises a mutation at position 37 of SEQ ID NO: 1. In further embodiments, the mutein comprises an amino acid substitution at position 37 of SEQ ID NO: 1, wherein leucine (L) is substituted with an amino acid other than leucine (L). In certain embodiments, the substitute amino acid is glycine (G) or serine (S).

[0056] In some embodiments, the p35 mutein comprises a mutation at position 38 of SEQ ID NO: 1. In further embodiments, the mutein comprises an amino acid substitution at position 38 of SEQ ID NO: 1, wherein glutamate (E) is substituted with an amino acid other than glutamate (E). In certain embodiments, the substitute amino acid is glycine (G) or serine (S).

[0057] In some embodiments, the p35 mutein comprises a mutation at position 39 of SEQ ID NO: 1. In further embodiments, the mutein comprises an amino acid substitution at position 39 of SEQ ID NO:1, wherein phenylalanine (F) is substituted with an amino acid other than phenylalanine (F). In certain embodiments, the substitute amino acid is glycine (G) or serine (S).

[0058] In some embodiments, the p35 mutein comprises a mutation at position 40 of SEQ ID NO: 1. In further embodiments, the mutein comprises an amino acid substitution at position 40 of SEQ ID NO: 1, wherein tyrosine (Y) is substituted with an amino acid other than tyrosine (Y). In certain embodiments, the substitute amino acid is alanine (A). In other embodiments, the substitute amino acid is glycine (G) or serine (S).

[0059] In some embodiments, the p35 mutein comprises a mutation at position 41 of SEQ ID NO: 1. In further embodiments, the mutein comprises an amino acid substitution at position 41 of SEQ ID NO:1, wherein proline (P) is substituted with an amino acid other than proline (P). In certain embodiments, the substitute amino acid is glycine (G) or serine (S).

[0060] In some embodiments, the p35 mutein comprises a mutation at position 128 of SEQ ID NO:1. In further embodiments, the mutein comprises an amino acid substitution at position 128 of SEQ ID NO: 1, wherein lysine (K) is substituted with an amino acid other than lysine (K). In certain embodiments, the substitute amino acid is asparagine (D).

[0061] In some embodiments, the p35 mutein comprises a mutation at position 167 of SEQ ID NO:1. In further embodiments, the mutein comprises an amino acid substitution at position 167 of SEQ ID NO:1, wherein tyrosine (Y) is substituted with an amino acid other than tyrosine (Y). In certain embodiments, the substitute amino acid is alanine (A).

[0062] In some embodiments, the p35 mutein comprises a mutation at position 171 of SEQ ID NO:1. In further embodiments, the mutein comprises an amino acid substitution at position 171 of SEQ ID NO:1, wherein isoleucine (I) is substituted with an amino acid other than isoleucine (I). In certain embodiments, the substitute amino acid is alanine (A).

[0063] In some embodiments, the p35 mutein comprises a mutation at position 175 of SEQ ID NO:1. In further embodiments, the mutein comprises an amino acid substitution at position 175 of SEQ ID NO:1, wherein isoleucine (I) is substituted with an amino acid other than isoleucine (I). In certain embodiments, the substitute amino acid is alanine (A).

[0064] In some embodiments, the p35 mutein comprises one or more mutations at positions within SEQ ID NO: 1, as shown in the table below. Boxes in the diagonal are blackened as each variant can be substituted once per position; however, the present disclosure provides p35 muteins comprising two or more mutations as described by Table 1 below. Boxes with hatched lines are redundant to white boxes as both contain the same mutations.

Table 1

[0065] The present disclosure provides additional p35 muteins as shown in Table 2 below. This table shows a list of mutations that were shown to reduce the affinity of human IL- 12 p35 to IL-12R in SSM experiments.

Table 2

[0066] The present disclosure also provides IL-12 affinity variants that are not derived from SEQ ID NO: 1 but contain mutations relative to their cognate wildtype sequences at positions that correspond to the numbered positions disclosed herein in relation to SEQ ID NO: 1. By “corresponding” amino acid residue is meant an amino acid residue that aligns with (though not necessarily identical to) the reference residue, when the subject sequence and the reference sequence containing the residues are aligned to achieve maximum homology (allowing gaps that are recognized in the art).

[0067] In some embodiments, the IL-12 affinity variants of the present disclosure are mouse IL-12 affinity variants. An exemplary wildtype mouse IL-12 p35 amino acid sequence (UniProt ID No. P43431) is shown below, where the signal peptide (residues 1-22) is boxed:

|MCQSRYLLFL ATLALLNHLS L |RVIPVSGP ARCLSQSRNL LKTTDDMVKT

AREKLKHYSC TAEDIDHEDI CLPLELHKNE SCLATRETSS

TTRGSCLPPQ KTSLMMTLCL

QTE FQAINAA LQNHNHQQI I

LDKGMLVAID ELMQSJ LNHNG

EADPYRVKMK LCILLHAFST

RVVT INRVMG YLSSA ( SEQ ID NO : 3 )

In some embodiments, a mouse IL-12 p35 mutein of the present disclosure may comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between identity to SEQ ID NO: 3 or to a sequence comprising SEQ ID NO:3.

[0068] In some embodiments, the mouse p35 mutein may comprise one or more mutations at positions 33, 34, 35, 36, 124, and 163 of the mature mouse IL-12 p35 sequence (SEQ ID NO:3 without the signal peptide). See also FIG. 6. Table 3 below shows exemplary mouse p35 muteins and the corresponding human p35 muteins containing mutations at orthologous positions and having reduced affinity to IL-12R.

Table 3

B. p40 Muteins

[0069] In some embodiments, the IL-12 affinity variants herein comprise a p35 mutein and a wildtype p40 subunit. In other embodiments, the IL-12 affinity variants comprise a mutated p40 subunit. An exemplary mature wildtype human p40 has a sequence shown in

SEQ ID NO:4 below:

IWELKKDVYV VELDWYPDAP GEMVVLTCDT PEEDGITWTL DQSSEVLGSG

KTLTIQVKEF GDAGQYTCHK GGEVLSHSLL LLHKKEDGIW STDILKDQKE PKNKTFLRCE AKNYSGRFTC WWLTTISTDL TFSVKSSRGS SDPQGVTCGA ATLSAERVRG DNKEYEYSVE CQEDSACPAA EESLPIEVMV DAVHKLKYEN YTSSFFIRDI IKPDPPKNLQ LKPLKNSRQV EVSWEYPDTW STPHSYFSLT

FCVQVQGKSK REKKDRVFTD KTSATVICRK NASISVRAQD RYYSSSWSEW ASVPCS ( SE( > ID NO : 4 )

See also GenBank Accession No. 3HMX A. An unprocessed wildtype human p40 sequence (UniProt ID No. P29460) is shown below, where the signal sequence (residues 1-22) is boxed:

|MCHQQLVISW FSLVFLASPL VA]I ELKKDV YVVELDWYPD APGEMVVLTC DTPEEDGITW TLDQSSEVLG SGKTLTIQVK EFGDAGQYTC HKGGEVLSHS LLLLHKKEDG IWSTDILKDQ KEPKNKTFLR CEAKNYSGRF TCWWLTTIST DLTFSVKSSR GSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACP AAEESLPIEV MVDAVHKLKY ENYTSSFFIR DIIKPDPPKN LQLKPLKNSR QVEVSWEYPD TWSTPHSYFS LTFCVQVQGK SKREKKDRVF TDKTSATVIC RKNASISVRA QDRYYSSSWS EWASVPCS ( SEQ ID NO : 5 )

[0070] In some embodiments, an IL-12 p40 subunit may comprise an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between identity to SEQ ID NO:4 or to a sequence comprising SEQ ID NO:4. The mutations in p40 may further reduce the affinity of the IL- 12 variants for IL-12R, such as IL-12Rpl. [0071] For example, the p40 subunit may contain mutations, such as substitutions, at the interface with IL-2RP1. In some embodiments, the p40 subunit contains mutations at, e.g., positions that correspond to residues P39, D40, E81, and F82 of SEQ ID NO:5. In further embodiments, the p40 mutein is derived from human p40 and has one or more mutations selected from P39A, D40A, E81A, and F82A in SEQ ID NO:5. See, e.g., Glassman et al., supra.

[0072] In some embodiments, the IL-12 variants are mouse IL-12 variants and comprise a mouse p40 subunit, with or without mutations. An exemplary wildtype mouse p40 amino acid sequence (UniProt ID No. P43432) is shown below, with the signal sequence (amino acids 1-22) in box:

|MCPQKLTISW FAIVLLVSPL MA|MWELEKDV YVVEVDWTPD APGETVNLTC DTPEEDDITW TSDQRHGVIG SGKTLTITVK EFLDAGQYTC HKGGETLSHS HLLLHKKENG IWSTEILKNF KNKTFLKCEA PNYSGRFTCS WLVQRNMDLK FNIKSSSSSP DSRAVTCGMA SLSAEKVTLD QRDYEKYSVS CQEDVTCPTA EETLPIELAL EARQQNKYEN YSTSFFIRDI IKPDPPKNLQ MKPLKNSQVE VSWEYPDSWS TPHSYFSLKF FVRIQRKKEK MKETEEGCNQ KGAFLVEKTS TEVQCKGGNV CVQAQDRYYN SSCSKWACVP CRVRS ( SEQ ID NO : 6 )

[0073] In some embodiments, the mouse p40 contains mutations that correspond to those described above for human p40 (e.g., E81A and F82A mutations in SEQ ID NO:6).

C. Single-Chain IL-12

[0074] In some embodiments, the present IL- 12 affinity variants are single-chain IL- 12 (scIL-12) fusion proteins comprising a p35 mutein sequence fused to a p40 sequence (with or without mutations). In further embodiments, the scIL-12 variants comprise a membrane anchor or a transmembrane domain such that they are expressed at the cell surface. Examples of membrane anchors are glycolipid anchors such as a glycosylphosphatidylinositol (GPI) anchor, covalent lipid modifications, membrane anchor peptides, and a fusion protein (e.g., derived from a mammalian sequence, a viral sequence, and/or a bacterial sequence).

Examples of transmembrane domains include any transmembrane domain derived from a cell surface protein and an artificial transmembrane domain. The p35 sequence may be either N- terminal, or C-terminal, to the p40 sequence. The p35 sequence and the p40 sequence may be linked by a peptide linker, such as a glycine-serine (GS) linker (e.g., a linker comprising G4S or GeS, or two, three, four or more repeats of G4S or GeS) or a peptide linker of any sequence.

[0075] In some embodiments, the scIL-12 variant comprises a CLS transmembrane domain, and optionally an N-terminal tag such as a strep tag II (STII). In certain embodiments, the scIL-12 has the structure STII-pdO^-GeS-pSS^-CLS, where (*) denotes the possibilities of having or not having mutations, and GeS denotes a linker having the sequence GGGGGGS (SEQ ID NO: 17). In some embodiments, scIL-12 comprises a wildtype sequence of the p35 subunit of IL-12.

[0076] Table 4 below shows positions important for expression of mouse IL- 12 p35 in the context of scIL-12, as identified by SSM. Position numbering is based off of SEQ ID NO:1).

Table 4

[0077] In some embodiments, the scIL-12 variants herein do not contain the mutations listed in Table 4.

D. Biological Activities of IL-12 Variants

[0078] In preferred embodiments, the present IL- 12 variants have reduced signaling activity as compared to parent IL-12 that has no mutations. In some embodiments, the IL-12 variants have reduced activity by 10, 20, 30, 40, 50, 60, 70, 80, or 90%, or by one, two, three, four, five, or more fold when measured at 0.1 to 10 ng/mL in an IL-12R report assay (e.g., the HEK-Blue™ assay, infra).

[0079] The signaling activities of exemplary human scIL-12 variants are summarized in Table 5 below. Unless otherwise indicated, the IL-12 variants exemplified in the table contain a wildtype human p40 sequence. The signaling activities were evaluated using IL- 12R reporter cell lines (see Examples, infra). “Y” indicates mutants that exhibited reduced signaling activity compared to wildtype human IL-12 and scIL-12 comprising wildtype human p35 and p40 sequences (SEQ ID NOs: 1 and 4) linked by a GeS linker. Position numbering is based off of SEQ ID NO: 1.

Table 5

[0080] In preferred embodiments, the present IL- 12 variants have reduced stimulatory effects on T cells (e.g. CD4⁺ and/or CD8⁺ T cells) when at low concentrations, and comparable effects when at high concentrations, as compared to parent IL- 12 without mutations. In some embodiments, the IL- 12 variants have reduced activity by 10, 20, 30, 40, 50, 60, 70, 80, or 90%, or by one, two, three, four, five, or more fold when measured at 1 to 100,000 pg/mL, as indicated by interferon-y secretion from stimulated primary T cells (using, e.g., the TransACT™ assay, and the sequential killing assay, infra),- and the variants have activity within 80-100% of wildtype IL-12 activity when measured at 100,000 pg/mL or higher.

[0081] Table 6 below summarizes the effects of exemplary human scIL-12 affinity variants in a TransAct™ assay (FIG. 7) or sequential killing assays (FIGs. 9 and 10). Position numbering is based off of SEQ ID NO: 1. Unless otherwise indicated, the IL-12 variants contain a wildtype p40 sequence. Table 6

[0082] The present disclosure provides IL-12 variants comprising the p35 muteins shown in Tables 5 and 6.

III. Expression of IL-12 Variants

[0083] The present disclosure provides nucleic acid molecules encoding the IL-12 affinity variants herein. If the variant is a heterodimer, the coding sequences for the two IL-12 subunits may be placed on separate expression vectors; alternatively, the two coding sequences may be placed on the same vector, with one polycistronic expression cassette, or with two separate expression cassettes. The IL-12 affinity variants (e.g., dimeric IL-12 or scIL-12 proteins having the mutations described herein) can be expressed in cells of interest, such as eukaryotic host cells where purified proteins are desired, or immune cells where cellbased therapies are desired.

[0084] The nucleic acid molecules (e.g., DNA or RNA vectors containing them) may be introduced into the cells by well-known techniques, including without limitation, electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, colloidal dispersion systems (e.g., as macromolecule complexes, nanocapsules, microspheres, and beads), and lipid-based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, and liposomes). Alternatively, the nucleic acid molecules may be introduced into the cells by transduction of recombinant viruses whose genomes comprise the nucleic acid molecules. Examples of viral vectors include, without limitation, vectors derived from lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, and vaccinia virus. In certain embodiments, the recombinant virus is pseudotyped with a heterologous envelope protein. In one embodiment, the recombinant virus is a lentivirus pseudotyped with an envelope glycoprotein derived from vesicular stomatitis virus (VSV), measles virus, or another virus (see e.g., Cronin et al., Curr Gene Ther. (2005) 5(4):387-98; Gutierrez-Guerrero et al., Viruses (2020) 12(9): 1016).

[0085] Mammalian host cells for producing IL-12 affinity variants include, without limitations, CHO cells, NS0 cells, HEK cells, 293 cells, and the like. Methods of producing therapeutic proteins in mammalian host cells are well known in the art. Insect cells such as Sf9 cells and Sf21 cells may also be used.

[0086] In the context of cell-based therapy, the IL- 12 variant herein may be expressed in immune cells such as T cells (e.g., CD4⁺ T cells and CD8⁺ T cells) and natural killer (NK) cells. For therapy of human patients, human immune cells are preferred. In some embodiments, the T cells are engineered T cells. In some embodiments, the T cells are tumor-infiltrating T cells (TILs). The combination of an IL-12 affinity variant with control strategies that localize the variant to a site of interest (e.g., a tumor or a TME) maintains IL- 12R signaling by increased localized concentrations at the site of interest, while avoiding unwanted signaling activity at lower concentrations away from the site of interest. Thus, expression of the IL-12 affinity variant in therapeutic immune cells homed to a target site provides an unprecedented method of tightly regulating IL- 12 activity and balancing safety with efficacy.

[0087] In some embodiments, the IL- 12 affinity variants are expressed under the control of a constitutive promoter. In alternative embodiments, the IL- 12 affinity variants are expressed under the control of an inducible promoter. In some embodiments, the inducible promoter responds to an extracellular signal, e.g., a stimulatory or inhibitory immunomodulatory signal. In some embodiments, the inducible promoter responds to an intracellular signal. In some embodiments, the promoter herein responds directly or indirectly to a CD3-mediated signal, e.g., a CD3-mediated signal triggered by CAR or TCR stimulation. Examples of promoters useful herein include, without limitation, an immediate early cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) early promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an elongation factor- la (EF-la) promoter, an MND promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Core or minimal promoters derived from the aforementioned promoters also are contemplated. Exemplary inducible promoter systems include, without limitation, hormone-regulated elements, synthetic ligand-regulated elements, ionizing radiation-regulated elements, tetracycline (Tet) systems (e.g., “Tet-Off” and “Tet-On” systems), and NF AT systems (see, e.g., Kallunki et al., Cells (2019) 8(8):796; Uchibori et al., Mol Ther Oncolytics. (2018) 12: 16-25). In some embodiments, the inducible promoter systems comprise a human beta globin promoter sequence, for example, one or more NF AT binding motifs (e.g., 4 X NF AT) in combination with a human beta globin promoter sequence (see, e.g., Na et al., Blood (2010) 116(11):el8-e25).

[0088] In some embodiments, the expression cassettes also include Kozak sequences, polyadenylation sites, and other elements that facilitate transcription and/or translation of the coding sequences. For example, a woodchuck hepatitis virus post-transcriptional response element (WPRE) or variants thereof may be included at the 3 ’ untranslated region of the expression cassette.

[0089] In the expression cassettes, the transcription/translation regulatory elements such as the promoters, any enhancers, and the like are operably linked to the coding sequences so as to allow efficient expression of the coding sequences and efficient translation of the RNA transcripts.

IV. Use of the IL-12 Affinity Variants

[0090] The IL- 12 affinity variants herein can be used in a cytokine therapy and delivered in a pharmaceutical composition systemically or locally to subjects in need of immune stimulation, such as subjects with cancer or a compromised immune system. For example, the pharmaceutical composition may be injected directly to a tumor site. Pharmaceutical compositions herein may comprise a pharmaceutically acceptable carrier or excipients. Examples of such carriers and excipients include water, saline, phosphate-buffered saline, sodium chloride, sodium phosphate, polyols (e.g., sucrose, mannitol, and trehalose), methionine, albumin, chelating agents, and the like.

[0091] The IL-12 affinity variants herein may also be used in a cell-based therapy. As described above, immune cells such as T cells may be engineered to express the variants. In some embodiments, the T cells also express recombinant antigen receptor. As used herein, a “recombinant antigen receptor” refers to an antigen receptor that is not natively expressed by the T cells. A recombinant antigen receptor may be a cell surface molecule that binds to an antigen of interest on another cell (e.g., a tumor cell), and may, for example, be derived from a T cell receptor or an antibody. The recombinant antigen receptor may be, for example, an antibody, an engineered antibody such as an scFv, a CAR, an engineered TCR, a TCR mimic (e.g., an antibody-T cell receptor (abTCR) or a chimeric antibody-T cell receptor (caTCR)), a chimeric signaling receptor (CSR), TCR mimics (e.g., antibodies that recognize epitopes similar to those recognized by TCRs), TCR fusion constructs (TRuCs). See, e.g., EP340793B1, WO 2017/070608, WO 2018/200582, WO 2018/200583, WO 2018/200585, Xu et al., Cell Discovery (2018) 4:62, Baeuerle et al., Nat Comm. (2019) 10:2087.

[0092] By way of example, a CAR may comprise an extracellular antigen-binding domain (e.g., a scFv domain), a transmembrane domain, and intracellular signaling domains, optionally peptide stretches linking the domains (e.g., a hinge region linking the antigenbinding domain and the transmembrane domain). In some embodiments, the transmembrane domain may be derived from a natural source, for example, the TCR alpha, beta, gamma, or delta chain, CD3 epsilon, CD4, CD5, CD8, CD9, CD 16, CD 19, CD20, CD21, CD22, CD25, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or 4-1BB. Alternatively, the transmembrane domain may be synthetic and may comprise predominantly hydrophobic residues (e.g., alanine, leucine, valine, glycine, isoleucine, proline, phenylalanine, and tryptophan). In some embodiments, the intracellular signaling domains are those that provide a signal similar to that from a natural antigen receptor and may comprise, for example, a costimulatory domain (e.g., one derived from CD28, 4-1BB, 0X40, DAP 10, or ICOS) and a primary signaling domain (e.g., one derived from CD3 zeta chain).

[0093] In some embodiments, an abTCR may comprise an engineered TCR in which the antigen-binding domain of a TCR (e.g., an alpha/beta TCR or a gamma/delta TCR) has been replaced by that of an antibody (with or without the antibody’s constant domains); the engineered TCR then becomes specific for the antibody’s antigen while retaining the TCR’s signaling functions. [0094] In some embodiments, a CSR may comprise (1) an extracellular binding domain (e.g., natural/modified receptor extracellular domain, natural/modified ligand extracellular domain, scFv, nanobody, Fab, DARPin, and affibody), (2) a transmembrane domain, and (3) an intracellular signaling domain (e.g., a domain that activates transcription factors, or recruits and/or activates JAK/STAT, kinases, phosphatases, and ubiquitin; SH3; SH2; and PDZ).

[0095] The recombinant antigen receptor may target an antigen of interest (e.g., a tumor antigen or an antigen of a pathogen). The antigens may include, without limitation, AFP (alpha-fetoprotein), avP6 or another integrin, BCMA, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (C-C motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7/8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD 123, CD 138, CD171, CD352, CEA (carcinoembryonic antigen), Claudin 18.2, Claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrinB2, EPHa2 (ephrine receptor A2), ERBB dimers, estrogen receptor, ETBR (endothelin B receptor), FAP-a (fibroblast activation protein a), fetal AchR (fetal acetylcholine receptor), FBP (a folate binding protein), FCRL5, FR-a (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican- 2), GPC3, gplOO (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G Protein Coupled Receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen Al), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight-melanoma-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Ig kappa, Ig lambda, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL- 13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI -CAM), Liv-1, LRRC8A (leucine rich repeat containing 8 Family member A), Lewis Y, melanoma- associated antigen (MAGE)-Al, MAGE-A3, MAGE-A6, MART-1 (melan A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC/peptide complexes (e.g., HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE- A, and WT1), NC M (neural cell adhesion molecule), Nectin-4, NKG2D (natural killer group 2 member D) ligands, NY-ESO, oncofetal antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen ), PSMA (prostate specific membrane antigen), ROR1, ROR2, SIRPa (signal -regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (six transmembrane epithelial antigen of the prostate 1), survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV, and other pathogens.

[0096] In some embodiments, the antigen receptor may be bispecific and target two different antigens, such as two of the antigens listed above. For example, the antigen receptor, such as a CAR, targets CD 19 and CD20, or CD 19 and CD22.

[0097] The present pharmaceutical compositions and engineered immune cells may be used to prevent a disease or disorder by being administered in a therapeutically effective amount, wherein an onset, progression, or a relapse of a sign or symptom of the disease or disorder is delayed or inhibited, thereby preventing the disease or disorder. In some embodiments, the disease or disorder is a cancer.

[0098] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of immunology, medicine, medicinal and pharmaceutical chemistry, and cell biology described herein are those well-known and commonly used in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art. As used herein, the term “approximately” or “about” as applied to one or more values of interest refers to a value that is similar to a stated reference value. In certain embodiments, the term refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context. [0099] According to the present disclosure, back-references in the dependent claims are meant as short-hand writing for a direct and unambiguous disclosure of each and every combination of claims that is indicated by the back-reference. Any molecule disclosed herein can be used in any of the treatment method here, wherein the individual to be treated is as defined anywhere herein. Further, headers herein are created for ease of organization and are not intended to limit the scope of the claimed invention in any manner.

[0100] In order that this invention may be better understood, the following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the invention in any manner.

EXAMPLES

[0101] Materials and methods used in the following Examples are as follows. p35 SSM Library Generation

[0102] The p35 SSM libraries were tested in the following format: a membrane-anchored, surface-expressed scIL-12 with an N-terminal strep tag II (STII) and a CLS transmembrane domain (STII-p40-G6S-p35*-CLS). To generate the libraries, synthetic DNA for the 5’ and 3’ p35 SSM libraries were ordered as oligo pools from Integrated DNA Technologies (IDT). A single codon in each oligo was substituted with all possible amino acids including stop codons using either NNK or NNN codons. Since p35 is 591bp or 197 amino acids in length, we made 197 constructs with NNK or NNN at each amino acid position. 98 degenerate oligos were in 5’ pool and 99 degenerate oligos were in 3’ pool. First, we amplified the oligo pools via PCR. Second, we cloned purified PCR products into our destination vector using Gibson assembly. Third, we purified the Gibson assembly reactions using Solid Phase Reversible Immobilization beads. Fourth, we transformed the purified DNA into NEB 10- beta electrocompetent E. coli. Fifth, we plated 0.0005% of transformations and counted single colonies to measure transformation efficiency. Sixth, we inoculated the rest of the transformations into 50 mL of Terrific Broth (Thermo Fisher). Seventh, we midi-prepped the bacterial cultures. Eighth, we conducted amplicon-Next Generation Sequencing (NGS) to determine the quality of the SSM plasmid libraries.

[0103] To clone a 5’ SSM library, we cut the first round 5’ SSM library with Nhel and SexAI and ligated it into a new cloning backbone. To clone a 3’ SSM library, we cut the first round 3’ SSM library with BstXI and MslI and Gibson-assembled into a new cloning backbone. We also added a mouse CD90.1 (mCD90.1) tag to the SSM libraries for use as a transduction marker. p35 SSM Library Screening

[0104] For each library (5’ and 3’) containing about 6000 unique sequences, 18xl0⁶ Jurkat cells were transduced at a low MOI to obtain a transduction rate of 25% in 6-well G-Rex plates to achieve about 750-fold coverage of each library at the time of transduction. Transduced cells were diluted with 25 mL of RPMI 1640 and GlutaMAX™ and 10% FBS (R10), and allowed to expand for 72 hours after transduction before enrichment using the EasySep™ mouse CD90.1 (mCD90.1) positive selection kit (StemCell Technologies) according to the manufacturer’s instructions. Transduced CD90.1 -positive cells obtained from positive selection were diluted in 25 mL R10 and allowed to grow overnight in a 6-well G-Rex plate.

[0105] The next day, 7xl0⁶ cells were spun down for each library and resuspended in PBS with 1 mM EDTA, 25 mM HEPES, and 2% FBS (Sorting Buffer) with anti-STII-FITC (GenScript) at 1 : 100 and IL-12Rp2-Fc-AF647 at 250 nM. Cells were washed in Sorting Buffer, and resuspended at a density of 5xlO⁶/mL. The viability dye 7-AAD (BioLegend) was added to the cells (5 pL/10⁶ cells) 10 min prior to sorting on a Sony MA900. Cells were sorted into four populations based on FITC (STII) and Alexa Fluor 647 (IL-12RP2) staining (FIG. 1), labeled as follows: “LL” is FITC^LoAF647^Lo, “ML” is FITC⁺AF647^Lo, “MM” is FITC⁺AF647⁺, and “MH” is FITC⁺AF647⁺⁺. Approximately 2xl0⁶ cells were recovered among the four bins for each library after sorting, providing about 300-fold coverage. Cells from each sorted population and 10⁶ unsorted cells were washed with PBS and pelleted, before lysis using the ZYMO genomic extraction kit (Zymo Research). Lysed pellets were frozen at -20°C until processing with the ZYMO Quick-DNA™ Microprep Kit. p35 SSM Library Sequencing

[0106] To sequence p35 SSM libraries, we used the amplicon-NGS protocol. For PCR1, we added 25 pL of 2xQ5 master mix, 1 pL of Ing/pL plasmids pool, 2 pL of 10 pM primers mix, and 22 pL of water. We ran 10 cycles of Q5 PCR at 63°C annealing temperature. We purified PCR1 using SPRI beads at 1 : 1 ratio and eluted in 50 pL water. For PCR2, we added 5 pL of SPRIed PCR1, 10 pL of 0.25 pM Nextera™ indices mix, 1 pL of 10 pM PPC primers mix, 25 pL of 2xQ5 master mix, and 9 pL of water. We ran 7 cycles of Q5 PCR at 63°C annealing temperature. We purified PCR2 using SPRI beads at 0.6: 1 SPRI to PCR ratio and eluted in 40 pL of water. We conducted SYBR™-Green qPCR to quantify the Illumina libraries. We ran sequencing on Illumina MiSeq™ System. For genomic DNA PCR templates, we use 200-500 ng of genomic DNA templates and conducted 28 cycles of PCR1. p35 SSM Data Analysis

[0107] Forward and reverse reads were merged using Geneious BBMerge with default setting. The merged reads were then filtered based on adapter sequences flanking the SSM library using the Cutadapt tool and a custom script was used to enumerate the number of sequences in each run that code for an amino acid sequence that was expected in each SSM library. These counts were used to calculate the frequency of each sequence (the number of merged reads coding for a given sequence divided by the total number of merged reads with the correct adapter sequences) in each of the sorted bins, “LL,” “ML,” “MM,” and “MH” as described above. The enrichment ratio for each sequence in each bin was calculated by comparing the frequency of a given sequence to the frequency of that sequence in the unsorted cell population. To identify mutations that reduce binding to IL-12RP2, we focused on sequences that were enriched in the “ML” bin relative to the unsorted population and the other bins. Because some sequences may have shown apparent weak binding to IL-12RP2 because of low levels of membrane-anchored IL-12 expression, we subtracted the enrichment ratios of the “LL” bin from those of the “ML” bin in order to remove sequences that were enriched in the “ML” bin because of a mutation’s impact on expression. These subtracted enrichment ratios were used to generate the heatmaps in FIGs. 2A-2D. The bar plots in FIG. 3A reflect the sum of the enrichment ratios at a given position in the p35 primary sequence in order to identify which regions of the p35 contribute the most to IL-12RP2 binding.

Protein Production and Purification

[0108] Expi293 cells of passage less than 30 were transfected with plasmid DNA encoding CMV promoter and scIL-12 variant at a concentration of 1 pg/mL of culture in 25 or 50 mL cultures. Generally, all complexation reaction amounts are as described in the Expi293 system manual. Feed/Enhancers were added 18-22 hours post transfection, and harvest of cells by centrifugation at 4,000 x g was done 5 or 6 days post transfection. Purification was done by subjecting supernatants to end over end mixing in the presence of Nickel Sepharose excel (GE) using 0.5 mL of resin (1 mL 50% slurry) that had been exchanged into PBS before addition. After binding, the supernatant/resin mixture was spun at 1000 x g to pellet the resin. Supernatant/FT was removed such that about 9 mL remained, which was then resuspended and transferred to an Amicon® Purification device. All subsequent spins except for the elution step were done at 1000 x g. Buffer A was PBS with 500 mM NaCl and Buffer B was the same with 500 mM imidazole. Washing was done with 20 column volume (CV) of PBS with 500 mM NaCl and 20-25 mM imidazole (4 or 5% Buffer B). Elution was typically about 3 CV of Buffer B. All flow-through (FT), wash and elution steps were collected in Falcon tubes and analyzed by SDS page prior to pooling fractions with the target protein. These fractions were then buffer exchanged into PBS pH7.2 and concentrated to between 1 and 4 mg/Ml. Final material was analyzed by size exclusion chromatography (SEC)-HPLC.

IL-12Rp2-Fc Conjugation to Alexa Fluor 647

[0109] IL-12RP2-Fc protein from R&D Biosystems/Bio-Techne was resuspended at 0.2 mg/mL and buffer-exchanged into PBS using Zeba™ desalting columns (Thermo Fisher). The pH of the protein solution was adjusted with 20X borate buffer (Pierce) just prior to the addition of Alexa Fluor 647-NHS (Thermo Fisher) in DMSO, using a 10-fold molar ratio of dye to protein. Labeling was carried out at room temperature for 2 hours before dye removal and buffer-exchange into PBS using Zeba™ columns (Thermo Fisher).

IL-12R Reporter Cell Line

[0110] HEK-Blue IL-12R cells (InvivoGen, Inc.) cells, a IL-12 receptor (IL-12R) reporter cell line containing a STAT4-inducible Secreted Embryonic Alkaline Phosphatase (SEAP) reporter gene, were used to monitor IL-12 activation of the IL-12R. The cells were passaged according to the manufacturer’s recommendations. On the day of the activity experiment, cells at about 80% confluency were resuspended using PBS (without Ca2⁺ or Mg2⁺), spun down at 300xg for 5 min, and resuspended at 0.28xl0⁶ live cells/mL in DMEM with 10% FBS. 180 pL of the HEK-Blue suspension (50,000 cells/well) was added to each well of a flat-bottom tissue-culture treated 96-well plate and allowed to attach for about 1 hour before the addition of purified scIL-12 proteins. The IL- 12 proteins were diluted to a starting concentration of 500 pg/mL in PBS and diluted 3-fold down into a total 12 concentrations per construct. These scIL-12 proteins were diluted 10-fold further upon the final addition of 20 pL of serially-diluted protein to 180 pL of cells in the 96-well plate. Cells were cultured in the presence of the IL-12 proteins for 16 hours at 37°C, 5% CO2, 95% humidity. The measurement of IL- 12 activity was performed by adding 20 pL of cell supernatant to 180 pL of QUANTI-Blue™ reagent, incubating at 37°C for about 1 hour to monitor the color-change reaction driven by secreted alkaline phosphatase, and measuring absorbance at 640 nm on a Synergy Hl plate reader.

IFN-y Secretion Assay to Monitor IL-12 Activity in Primary T Cells

[0111] CD4+ and CD8+ T cells from matching donors were mixed at a 1 : 1 : ratio, and stimulated using TransACT™ (Miltenyi Biotec) for 24 hours prior to expansion for 7 days in media containing IL-2, IL-7 and IL- 15 (R&D). Expanded T cells were washed and cryopreserved in CyroStor® CS10 (StemCell Technologies) at 50xl0⁶ cells/mL for storage. [0112] T cells were thawed into fresh, warm cytokine free media and rested in 37°C for 30 minutes prior to plating. 50,000 T cells were stimulated with TransACT™ at a 1 :500 dilution and added to flat bottom 96-well plates with 180 pL per well. 20 pL of serially-diluted IL- 12 proteins in PBS were added to each well, and the T cells + IL-12 were cultured at 37°C, 5% CO2, 95% humidity. After 16 hours, cell culture supernatants were harvested and frozen at - 80°C until processing or analyzed for IFN-y by MSD.

[0113] T cell supernatants were thawed at 4°C, then diluted 20X. IFN-y and IL-12 p70 in T cell supernatants were measured using V-PLEX Proinflammatory Panel 1 Human Kit (Meso Scale Discovery) according to the manufacturer's protocol. Data were analyzed using MSD Discovery Workbench 4.0.

Functional Testing of IL-12 Affinity Variants in vitro

[0114] T cell production was carried out in TCM media. At day 0, CD4+ and CD8+ T cells were thawed into OpTmizer™ CTS media, mixed 1 : 1, and activated for 24-28 hours with 1 : 100 dilution of TransACT™. On day 1, T cells were counted and co-transduced with two lentiviral vectors. The first vector encodes constitutive expression of an antigen-specific TCR, the second vector encodes constitutive expression of human LNGFR and activation- induced expression of an IL-12 mutein. On day 2, at least 24 hours post-transduction, T cells were scaled up to the appropriately sized G-Rex® plate. On day 7, cells were harvested and assessed phenotypically by flow for activation markers, memory markers, and transduction efficiency of both TCR and IL-12 (LNGFR). The cells were also counted and assessed for viability by AOPI dye, then transferred directly into sequential kill assays.

[0115] Sequential kill assays were carried out in RPMI 1640 + 10% FBS media. On the first day of stimulation (Day 7 post-production), A375-NLR or H1703-NLR cells were counted, and seeded into 24-well plates, and incubated at 37°C for 2 hours to allow for adherence. Effector T cells from above were seeded at between 1 : 1 to 1 : 10 E:T (depending on the assay). E:T ratios were based on number of Live, TCR+ cells. Effector-target coculture plates were placed in an IncuCyte®, with readings every 2 hours, to measure killing. Supernatant was collected at 24 hours post seeding of effector cells, and frozen for later analysis of cytokine levels. Three days after seeding effector and target cells (Day 10 postproduction) new 24-well plates of A375-NLR or H1703-NLR cells were seeded as above, and 25% of the previous culture was transferred to this new plate, to initiate the second stimulation of the sequential kill assay. A fraction of the cells were also transferred to 96- well plates and assessed phenotypically by flow for cell expansion, activation markers, memory markers, and transduction efficiency of both TCR and IL- 12 (LNGFR). Supernatant was again collected at 24 hours post seeding of effector cells, and frozen for later analysis of cytokine levels. Cells were re-plated and analyzed by flow cytometry as on Day 10 at Days 13 and 16 post-production until stimulations provided the required resolution to differentiate between various constructs. Supernatants collected 24 hours after each seeding of cells were analyzed by MSD at 1 :5 and 1 :20 dilutions due to concentration differences between IL-12 and IFN-y, according to the manufacturer’s instructions.

Structural Visualization and Images for Figures

[0116] All structural images shown in figures were generated using PyMOL software (The PyMOL Molecular Graphics System, Schrodinger, LLC.)

Example 1: Identification of IL-12 Affinity Variants

[0117] To identify mutations on p35 that reduce the affinity of IL-12 for IL-12RP2, we performed site- saturation mutagenesis (SSM) to introduce all possible amino acids at each position along the primary sequence of p35. This library contains p35* mutants that were transduced into Jurkat cells and displayed on the cell surface in the following format: a membrane-anchored, surface-expressed scIL-12 with an N-terminal strep tag II (STII) and a CLS transmembrane domain (STII-p40-G6S-p35*-CLS). The transduced Jurkat cells were stained with an anti-STII-FITC antibody to measure surface expression of IL-12 (STII-p40- G6S-p35*-CLS) and stained with IL-12Rp2-Fc-AF647 to measure binding of IL-12RP2.

[0118] The cells were then sorted into four bins (FIG. 1; see Materials and Methods above for details). To identify mutations that reduce binding to IL-12RP2, we focused on sequences that were enriched in the FITC⁺AF647^Lo bin (hereafter labeled as “ML”) relative to the unsorted library. To calculate this relative enrichment, we first counted the number of reads for a given sequence in the SSM library, and then calculated the frequency of that sequence in the pool of reads for that sample. The frequency of a mutation in each bin (in this case the “ML” bin) was then divided by the frequency of that mutation in the unsorted library to calculate an “enrichment ratio.” Because some sequences may have been enriched in the “ML” bin because of stochastic sampling of low abundance sequences, we excluded sequences that were absent in any of the bins.

[0119] It is important to distinguish mutations that directly impact binding to IL-12R from mutations that show less binding due to decreased levels of membrane-presented IL-12, i.e. mutations that alter the expression, secretion, or stability of IL-12. Some sequences may have been enriched in the “ML” bin as a result of low expression of IL- 12 on the cell surface. To identify mutations that were enriched in the “ML” bin independent of IL- 12 expression on the cell surface, we subtracted the enrichment ratios from the “ML” bin from the low expressing FITC^LoAF647^Lo bin (hereafter labeled as “LL”). After performing this subtraction, we selected sequences that were the most highly enriched, with enrichment ratios in the top 5th percentile (FIGs. 2A and 2B). All mutations identified as capable of reducing the binding affinity to IL-12RP2 are summarized in Table 1. To identify mutations that affect expression levels of IL-12 p35, we divided the “LL” frequency for each mutation by its frequency in the unsorted library, selecting the top 5th percentile for enrichment ratios in the “LL” bin (FIGs. 2C and 2D). The complete list of mutations that affect expression of IL-12 p35 based on the SSM results are listed in Table 2. Throughout, we use amino acid position numbering consistent with the crystal structures of human IL- 12: PDB ID 3HMX (Luo, supra) (corresponding to GenBank Accession No. 3HMX B (also known as IL-12A or p35; SEQ ID NO: 1)) and PDB ID 1F45 (Yoon et al., EMBO J. (2000) 19(14):3530-41), the contents of each of which is incorporated herein by reference in its entirety.

[0120] Taking the sum of the subtracted “ML” enrichment ratios at each residue provides a metric to assess which positions on p35 contribute to binding IL-12RP2 (FIG. 3, top). The positions identified as contributing to binding IL-12RP2 in these experiments are near each other in three-dimensional space when mapped onto the structure of p35 (FIG. 3, bottom) at a location that is consistent with the receptor binding interface of the homologous cytokine IL-23 (FIG. 4). Bloch et al. solved a co-complex crystal structure of the interface between IL-23 pl9 and IL-23R (Bloch et al., Immunity (2018) 48(l):45-58), and showed that W157, L161, and KI 65 on IL-23 pl9 are critical residues for IL-23R binding. Homologous positions on IL-12 are Y167, 1171, and 1175, respectively (FIG. 4), indicating that these residues might be involved p35 binding to IL-12R; however, it was unclear from the homology alone how much these residues would contribute to binding, and also unclear how mutating these residues would affect activity. All three of these positions were identified as IL-12R binding residues in our SSM studies (FIGs. 1 and 3). To address a potential structure-function relationship in view of the results of our SSM, we designed IL- 12 variants with different combinations of mutations with the goal of achieving a wide range of altered binding affinities and concentration-dependent signaling activity.

Example 2: Functional Evaluation of IL-12 Affinity Variants

[0121] We produced and purified 42 different variants of human scIL-12 (Table 3) and evaluated their functional impact using an IL-12R reporter cell line assay (see Materials and Methods above for details). The results of these reporter cell line assays are summarized in FIG. 5. The Y40A single mutant showed small decreases in activity compared to wildtype scIL-12. The Y167A-I171A-I175A triple mutant and Y40A-K128D double mutant show substantial decreases in signaling activity at lower concentrations but only modest decreases in signaling activity at higher concentrations (greater than 100 nM). Combinations of mutations enabled a wide range of different relative signaling strengths, and generally the more mutations that were combined, the greater the decrease in signaling activity was observed. Y40A-Y167A and 37-41GS-K128D dramatically decreased activity even at high concentrations, and Y40A-Y167A-I171A, 37-41GS-K128D-Y167A, and Y40A-K128D- Y167A nearly eliminated activity in the reporter assay (FIG. 5).

Example 3: Mouse Orthologs of IL-12 Affinity Variants

[0122] To enable syngeneic mouse studies, we produced mouse orthologs for IL- 12 p35 mutations that exhibited altered IL-12R signaling activity (Table 4). Even though human IL- 12 does not drive signaling through mouse IL-12R, mouse IL-12 is able to activate human IL- 12R and drive signaling in the reporter cell lines, enabling the reporter cell line assay to be used to compare relative activity levels of the mouse orthologs.

[0123] Using the IL-12R reporter line assay, we demonstrated that the mouse orthologs of the IL- 12 affinity variants exhibited a similar trend of altered signaling (FIG. 6). These data suggest that mouse orthologs of human IL-12 affinity variants could be effectively used to facilitate syngeneic mouse studies.

Example 4: Impact of IL-12 Affinity Variants on Primary Human T Cells

[0124] We next tested the functional impact of these IL-12 affinity variants on primary human T cells in vitro by measuring IFN-y production in CD3/CD28-stimulated T cells in response to the addition of purified scIL-12 proteins. The addition of wildtype scIL-12 drove the T cells to secrete large amounts of IFN-y, whereas the relative levels of IFN-y secretion driven by the IL-12 affinity variants showed a range of decreased activities (FIG. 7) that is consistent with the reporter cell line assays (FIG. 5). The full set of IL-12 affinity variants tested against primary human T cells is summarized in Table 5.

[0125] The data show that the Y40A-K128D and Y167A-I171A-I175A variants have substantially reduced activity at low concentrations but their activity was comparable to wildtype scIL-12 at higher concentrations (FIGs. 5-7). These results indicate that these IL- 12 variants can drive enhanced proinflammatory activity when localized to higher concentrations at sites of interest. The results also show that IL-12 affinity variants having one or more mutations at the five positions Y40, K128, Y167, 1171, and 1175, which group together in three-dimensional space on the surface of the IL-12 p35 structure (FIG. 8).

Example 5: Effects of IL-12 Affinity Variants on T-Cell Mediated Cytolysis of Tumor Cells

[0126] To test the ability of these mutants to drive T cell-mediated cytolysis of tumor cell lines in in vitro tumor challenge assays, T cells were transduced with lentiviral constructs that constitutively express an antigen-specific TCR and inducibly express scIL-12 (or designed variants thereof) under the control of an inducible 4 X NF AT human beta-globin promoter, and the transduced T cells were sequentially co-cultured with A375 target cells (see Materials and Methods above for details). The Y40A variant resulted in enhanced cytolysis approaching the levels observed for WT scIL-12, and the Y40A-K128D and 37-41GS variants resulted in enhanced cytolysis compared to TCR alone (no IL-12) but at levels less than WT scIL-12 or exogenously added WT IL-12 (FIG. 9). We also measured IFN-y production during the co-culture assays (FIG. 10); similarly, these variants drove at levels between that of TCR alone and WT scIL-12.

Example 6: Effects of IL-12 Affinity Variants on In Vivo T-Cell Activation

[0127] Additionally, we interrogated whether the IL-12 affinity variants could activate T cell activity in vivo. Specifically, we measured IFN-y production in a xenograft model (FIG. 11). A375 cell productions were carried out in RPMI1640 supplemented with 10% FBS. Cells were thawed, counted, and plated, then passaged/expanded every 2-3 days afterwards. Once the required number of cells were reached, cells were harvested. First, the media was aspirated, then the cells were washed with PBS, and then treated with TrypLE for 5 minutes at 37°C. The detached cells were washed with RPMI1640, and then counted and resuspended at the proper concentration in HBSS. Cells were then mixed 1 : 1 with Matrigel before being injected into the mice. T cell productions were carried out as described above.

[0128] Once the tumors reached 100 mm³, mice were injected with antigen-specific TCR- expressing T cells, and IFN-y secretion levels in the serum were monitored via blood draws on Days 1, 7, 14, 21, 28, and 42 post-T cell infusion. Blood draws were diluted 1 :5 in PBS+0.1% BSA solution, and then centrifuged at 2,000 x g for 15 minutes at 4°C to separate the serum and red blood cells. Serum was collected from the supernatant for further analysis via MSD at 1 : 10 dilutions, according to the manufacturer’s instructions. Similar to the in vitro assays illustrated in FIG. 10, these variants drove expression of IFN-y at levels between that of TCR alone and WT scIL-12 (FIG. 11).

Claims

1. A human IL-12 p35 subunit mutein, wherein the mutein comprises a non-naturally occurring mutation, optionally a substitution, at a position that corresponds to position 37, 38, 39, 40, 41, 46, 47, 123, 124, 125, 126, 127, 128, 129, 130, 131, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, or 175 of SEQ ID NO: L

2. The human IL-12 p35 mutein of claim 1, wherein the mutein comprises a mutation at position 37, 38, 39, 40, 41, 128, 167, 171, or 175 of SEQ ID NO: l.

3. The human IL- 12 p35 mutein of claim 1 or 2, wherein the mutein comprises a mutation at position 40 of SEQ ID NO: 1, optionally wherein the mutation is Y40A, Y40G, or Y40S.

4. The human IL-12 p35 mutein of any one of claims 1-3, wherein the mutein comprises a mutation at position 128 of SEQ ID NO: 1, optionally wherein the mutation is K128D.

5. The human IL-12 p35 mutein of any one of claims 1-4, wherein the mutein comprises a mutation at position 167 of SEQ ID NO: 1, optionally wherein the mutation is Y167A.

6. The human IL-12 p35 mutein of any one of claims 1-5, wherein the mutein comprises a mutation at position 171 of SEQ ID NO: 1, optionally wherein the mutation is 1171 A.

7. The human IL-12 p35 mutein of any one of claims 1-6, wherein the mutein comprises a mutation at position 175 of SEQ ID NO: 1, optionally wherein the mutation is I175A.

8. The human IL-12 p35 mutein of claim 1-7, wherein the mutein comprises mutations at positions 167, 171, and 175 of SEQ ID NO: 1, optionally wherein the mutations are Y167A, I171A, and I175A.

9. The human IL-12 p35 mutein of any one of claims 1-8, wherein the mutein comprises mutations at positions 40 and 128 of SEQ ID NO: 1, optionally wherein the mutations are Y40A and K128D.

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10. The human IL-12 p35 mutein of any one of claims 1-8, wherein the mutein comprises a mutation at position 37, 38, 39, 40, or 41 of SEQ ID NO: 1, optionally wherein the mutation is a substitution by a glycine (G) or a serine (S).

11. The human IL-12 p35 mutein of claim 10, wherein the mutein comprises mutations at positions 37-41 of SEQ ID NO: 1, optionally wherein the mutations are substitutions of LEFYP (SEQ ID NO:7) by GGSGS (SEQ ID NO:8).

12. A human IL- 12 p35 mutein, wherein the mutein comprises a non-naturally occurring substitution selected from Table 1 or 2.

13. A human IL-12 variant, comprising the human IL-12 p35 mutein of any one of claims 1-12, and a human IL- 12 p40 subunit.

14. The human IL-12 variant of claim 13, wherein the IL-12 variant has reduced binding affinity for IL- 12 receptor P (IL-12RP), optionally for IL-12R P2 subunit, compared to a human IL-12 without the mutation(s).

15. The human IL-12 variant of claim 13 or 14, wherein the IL-12 p40 subunit comprises a mutation at a position that corresponds position 39, 40, 81, or 82 of SEQ ID NO:5, optionally wherein the mutation is P39A, D40A, E81 A or F82A.

16. The human IL-12 variant of claim 15, wherein the IL-12 p40 subunit comprises two or more of P39A, D40A, E81A and F82A.

17. The human IL-12 variant of any one of claims 13-16, wherein the IL-12 variant is a heterodimer comprising the p35 subunit variant and the p40 subunit.

18. The human IL-12 variant of any one of claims 13-16, wherein the IL-12 variant is a single-chain fusion protein (scIL-12) comprising the p35 subunit variant and the p40 subunit, optionally wherein the two subunits are linked by a peptide linker, optionally a flexible peptide linker.

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19. The human IL-12 variant of claim 17 or 18, wherein the IL-12 variant comprises a membrane anchor or a transmembrane domain.

20. A pharmaceutical composition comprising the IL-12 variant of any one of claims 13- 19 and a pharmaceutically acceptable carrier.

21. An isolated nucleic acid molecule or isolated nucleic acid molecules encoding the IL- 12 p35 mutein of any one of claims 1-12 or the IL-12 variant of any one of claims 13-19.

22. An expression vector or expression vectors comprising the isolated nucleic acid molecule(s) of claim 21, optionally wherein the expression vector(s) are viral vectors, further optionally wherein the viral vectors are lentiviral vectors, adenoviral vectors, or adeno- associated viral (AAV) vectors.

23. A mammalian cell comprising the expression vector(s) of claim 22 and expressing the human IL-12 variant of any one of claims 13-19.

24. The mammalian cell of claim 23, wherein the mammalian cell is a CHO cell.

25. The mammalian cell of claim 23, wherein the mammalian cell is a human immune cell, optionally a T cell or a natural killer (NK) cell.

26. The mammalian cell of claim 25, wherein the human immune cell is a T cell or a NK cell engineered to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR), optionally wherein the CAR or engineered TCR targets a tumor antigen.

27. A method of stimulating the immune system, or treating cancer, in a human subject in need thereof, comprising administering the pharmaceutical composition of claim 20, or the cell of claim 25 or 26, to the human subject.

28. Use of the human IL-12 variant of any one of claims 13-19, the expression vector(s) of claim 22, or the cell of claim 25 or 26, for the manufacture of a medicament for stimulating the immune system or treating cancer in a human subject in need thereof.

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29. The human IL-12 variant of any one of claims 13-19, the pharmaceutical composition of claim 20, the expression vector(s) of claim 22, or the cell of claim 25 or 26, for use in stimulating the immune system or treating cancer in a human subject in need thereof.

30. A method of producing a human IL-12 variant, comprising: culturing a mammalian cell of claim 23 or 24 under conditions that allow expression of the human IL- 12 variant, and isolating the human IL-12 variant from the culture.

31. A mouse IL- 12 p35 mutein, comprising one or more mutations selected from Table 3 or 4.

32. A mouse IL-12 variant comprising the mouse IL-12 p35 mutein of claim 31 and a mouse IL-12 p40 subunit, wherein the p40 subunit is wildtype or contains one or more mutations optionally selected from E81A and F82A.

33. The mouse IL-12 variant of claim 32, wherein the mouse IL-12 variant is a heterodimer comprising the p35 mutein and the p40 subunit; or a single-chain fusion protein (scIL-12) comprising the p35 mutein and the p40 subunit, optionally wherein the two subunits are linked by a peptide linker, optionally a flexible peptide.