CAIX-TARGETED IL-12 FUSION PROTEINS AND METHODS OF USE THEREOF

Abstract

Provided herein are IL-12p40 and IL-12p35 polypeptides and compositions (e.g., pharmaceutical compositions) comprising them; and methods for making IL-12p40 and IL-12p35 polypeptides and compositions. Further provided herein are fusion proteins (e.g., antibody fusion proteins) comprising IL-12p40 polypeptides (e.g., IL-12p40 polypeptides described herein) and / or IL-12p35 polypeptides (e.g., IL-12p35 polypeptides described herein). The IL-12p40 polypeptides, IL-12p35 polypeptides, and fusion proteins provided herein are useful in pharmaceutical compositions and methods for treating diseases (e.g., cancer).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 245,523, filed September 17, 2021, the entire contents of which are incorporated herein by reference.

[0002] 1. Field The present disclosure relates to IL-12p40 variants, IL-12p35 variants, IL-12 fusion proteins, and methods of using them. The present disclosure further relates to multispecific IL-12 fusion proteins that target IL-12 to cells expressing a target protein (e.g., hCAIX) on their surface, and methods of using them. [Background technology]

[0003] 2, Background Human IL-12 (hIL-12) is a pleiotropic secreted cytokine composed of the α subunit, human IL-12p35 (hIL-12p35), and the β subunit, human IL-12p40 (hIL-12p40). The native hIL-12p35 and hIL-12p40 subunits are linked via disulfide bonds to form the bioactive hIL12-p70 cytokine. hIL-12 is, among other things, proinflammatory and mediates its function through binding to the hIL-12 receptor (hIL-12R). The high-affinity hIL-12R is a heterodimer containing the hIL-12Rβ1 and hIL-12Rβ2 subunits. hIL-12R is constitutively or inducibly expressed in various immune cells, including natural killer (NK) cells, T cells, and B cells. For example, when hIL-12 binds to hIL-12R expressed on activated T cells, NK cells, and dendritic cells, it activates the TYK2, JAK2, and STAT signaling pathways. One of the major roles of hIL-12 is to activate T cells and NK cells, leading to increased production of INF-γ, proliferation, and cytotoxicity. Summary of the Invention

[0004] 3. Overview Provided herein, inter alia, are IL-12p40 variant and IL-12p35 polypeptides and polynucleotides encoding same; IL-12 fusion proteins and conjugates; methods of manufacture; pharmaceutical compositions; and methods of use, including, for example, methods of treating disease (e.g., cancer).

[0005] In one aspect, provided herein is a nucleic acid sequence encoding a nucleotide sequence (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) at amino acid positions (i) W37, F82, and K219; (ii) W37, F82, and K217; (iii) K106, K217, and K219; (iv) W37 and F82; (v) W37 and K217; (vi) W37 and K219; (xv) F82 and K219; (xvi) K217 and K219; (xvii) K106 and K217; or (xviii) K106 and K219, (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0006] In some embodiments, the hIL-12p40 polypeptide has the following amino acid substitutions: (i) W37A, F82A, and K219A; (ii) W37A, F82A, and K217A; (iii) K106A, K217A, and K219A; (iv) W37A and F82A; (v) W37A and K217A; (vi) W37A and K219A; (vii) W37A and K (xiv) F82A and K217A; (xv) F82A and K219A; (xvi) K217A and K219A; (xvii) K106A and K217A; or (xviii) K106A and K219A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0007] In some embodiments, the hIL-12p40 polypeptide comprises or consists of an amino acid substitution at each of amino acid positions W37, F82, and K219, (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0008] In some embodiments, the hIL-12p40 polypeptide comprises or consists of each of the following amino acid substitutions: W37A, F82A, and K219A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0009] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of any one of the polypeptides shown in Table 5 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide shown in Table 5.

[0010] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of a set of amino acid substitutions set forth in the amino acid sequence of any one of SEQ ID NOs: 38-65 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-65.

[0011] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of any one of SEQ ID NOs: 38-51 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 38-51.

[0012] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of a set of amino acid substitutions set forth in the amino acid sequence of any one of SEQ ID NOs: 52-65 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 52-65.

[0013] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of SEQ ID NO: 38 or 52 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 38 or 52.

[0014] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of SEQ ID NO:38 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO:33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:38.

[0015] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide is 100% identical to the amino acid sequence of SEQ ID NO:38.

[0016] In some embodiments, the hIL-12p40 polypeptide specifically binds to the hIL-12 receptor (hIL-12R).

[0017] In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates an increase in phosphorylated STAT4 (pSTAT4) levels in cells expressing hIL-12R on their surface that is less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0018] In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates an increase in phosphorylated STAT4 (pSTAT4) levels in cells expressing hIL-12R on their surface that is about 0.5-1000 fold, 0.5-100 fold, 0.5-10 fold, 0.5-5 fold, 0.5-2 fold, 1-1000 fold, 1-100 fold, 1-10 fold, 1-5 fold, 1-2 fold, 10-1000 fold, or 100-1000 fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0019] In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates an increase in the level of interferon gamma (IFN-γ) produced by expressing hIL-12R on its surface that is less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0020] In some embodiments, the hIL-12p40 protein, when combined with the hIL-12p35 protein, mediates an increase in the level of IFN-γ produced by cells expressing hIL-12R on their surface that is about 0.5-1000-fold, 0.5-100-fold, 0.5-10-fold, 0.5-5-fold, 0.5-2-fold, 1-1000-fold, 1-100-fold, 1-10-fold, 1-5-fold, 1-2-fold, 10-1000-fold, or 100-1000-fold less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0021] In one aspect, provided herein is a human interleukin-12p35 (hIL-12p35) polypeptide comprising: (a) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31; and (b) an amino acid modification (e.g., substitution, addition, deletion (e.g., substitution)) at one or more of the following amino acid positions: E60, F61, P63, K150, F188, Y189A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0022] In some embodiments, the hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., a substitution, addition, deletion (e.g., substitution)) at each of the following amino acid positions: (i) F188; (ii) Y189; (iii) F188 and Y189; or (iv) E60, F61, P63, K150, and F188 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0023] In some embodiments, the hIL-12p35 polypeptide comprises or consists of one or more of the following amino acid substitutions: E60K, F61H, P63S, K150H, F188P, F188A, and / or Y189A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0024] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the set of amino acid substitutions (relative to the amino acid sequence of SEQ ID NO: 31) set forth in the amino acid sequence of any one of SEQ ID NOs: 111-114; and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 111-114.

[0025] In some embodiments, the hIL-12p35 polypeptide comprises or consists of each of the following amino acid substitutions: (i) F188A; (ii) Y189A; (iii) F188A and Y189A; or (iv) E60K, F61H, P63S, K150H, and F188P (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0026] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is selected from the group consisting of amino acids A55 to K92, N50 to K92, M51 to K92, L52 to K92, Q53 to K92, K54 to K92, N50 to N93, M51 to N93, L52 to N93, Q53 to N93, K54 to N93, N50 to E94, M51 to E94, L52 to E94, Q53 to E94, K54 to E94, N50 to S95, M51 to S95, L52 to S95, Q53 to S95, K54 to S95, N50 to C96, M51 to C96, L52 to C96, Q53 to C96, K54 to C96, N50 to L97, M51~L97, L52~L97, Q53~L97, K54~L97, N50~P87, M51~P87, L52~P87, Q53~P87, K 54~P87, N50~L88, M51~L88, L52~L88, Q53~L88, K54~L88, N50~E89, M51~E89, L52~E8 9, Q53~E89, K54~E89, N50~L90, M51~L90, L52~L90, Q53~L90, K54~L90, N50~T91, M51 ~T91, L52~~T91, Q53~T91, K54~T91, R56~K92, Q57~K92, T58~K92, L59~K92, E60~K92 A55~N93, R56~N93, Q57~N93, T58~N93, L59~N93, E60~N93, A55~E94, R56~E94, Q57~E94, T58~E94, L59~E94, E60~E94, A55~S95, R56~S95, Q57~S95, T58~S95, L59~S95, E60~S95, A55~C96, R56~C96, Q57~C96, T58~C96, L59~C96, E60~C96, A55~L97, R56~L97, Q57~L97, T58~L97, L59~L97, E60~L97, A55~P87, R56~P87, Q57~P87, T58~P87, L59~P87, E60~P87, A55~L88, R56~L88, Q57~L88, T58~L88, L59~L88, E60~L88, A55~E89, R56~E89, Q57~E89, T58~E89, L59~E89, E60~E89, A55~L90, R56~L90, Q57~L90, T58~L90, L59~L90, E60~L90, A55~T91, R56~T91,comprising or consisting of a deletion of Q57 to T91, T58 to T91, L59 to T91, or E60 to T91 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30), and 51~N93, L52~N93, Q53~N93, K54~N93, N50~E94, M51~E94, L52~E94, Q53~E94, K54~E94, N50~S95, M51~S95, L52~S95, Q53~S95, K54~S95, N50~C96, M51~C96, L52~C96, Q53~C96, K54~C96, N50~L97, M51~L97, L52~L97, Q53~L97, K54~L97, N50~P87, M51~P87, L52~P87 , Q53~P87, K54~P87, N50~L88, M51~L88, L52~L88, Q53~L88, K54~L88, N50~E89, M51~E89 , L52~E89, Q53~E89, K54~E89, N50~L90, M51~L90, L52~L90, Q53~L90, K54~L90, N50~T91 , M51~T91, L52~T91, Q53~T91, K54~T91, R56~K92, Q57~K92, T58~K92, L59~K92, E60~K92 A55~N93, R56~N93, Q57~N93, T58~N93, L59~N93, E60~N93, A55~E94, R56~E94, Q57~E94, T58~E94, L59~E94, E60~E94, A55~S95, R5 6~S95, Q57~S95, T58~S95, L59~S95, E60~S95, A55~C96, R56~C96, Q57~C96, T58~C96, L59~C96, E60~C96, A55~L97, R56~L97, Q57~ L97, T58~L97, L59~L97, E60~L97, A55~P87, R56~P87, Q57~P87, T58~P87, L59~P87, E60~P87, A55~L88, R56~L88, Q57~L88, T58~L8 8, L59~L88, E60~L88, A55~E89, R56~E89, Q57~E89, T58~E89, L59~E89, E60~E89, A55~L90, R56~L90, Q57~L90, T58~L90, L59~L90,Other than the deletion of E60 to L90, A55 to T91, R56 to T91, Q57 to T91, T58 to T91, L59 to T91, or E60 to T91, the amino acid sequence of the polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31.

[0027] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of a deletion of amino acids A55-K92 (amino acid numbering relative to the amino acid sequence of SEQ ID NO:30), and other than the deletion of amino acids A55-K92, the amino acid sequence of the polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:31.

[0028] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the set of amino acid deletions (relative to the amino acid sequence of SEQ ID NO: 31) set forth in the amino acid sequence of any one of SEQ ID NO: 110; and other than said set of amino acid deletions, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 110.

[0029] In one aspect, provided herein is a single-chain hIL-12 (schIL-12) polypeptide comprising a hIL-12p40 polypeptide described herein operably linked to a hIL-12p35 polypeptide. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 31 or 110-114. In some embodiments, the hIL-12p35 polypeptide is a hIL-12p35 polypeptide described herein. In some embodiments, the hIL-12p40 polypeptide is operably linked to the hIL-12p35 polypeptide via a peptide linker. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a hIL-12p40 polypeptide, a peptide linker, and a hIL-12p35 polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a hIL-12p35 polypeptide, a peptide linker, and a hIL-12p40 polypeptide.

[0030] In one aspect, provided herein is a schIL-12 polypeptide comprising a hIL-12p35 polypeptide described herein operably linked to a hIL-12p40 polypeptide. In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 10. In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 38-51 or 90-109. In some embodiments, the hIL-12p40 polypeptide is a hIL-12p40 polypeptide described herein. In some embodiments, the hIL-12p40 polypeptide is operably linked to the hIL-12p35 polypeptide via a peptide linker. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a hIL-12p40 polypeptide, a peptide linker, and a hIL-12p35 polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a hIL-12p35 polypeptide, a peptide linker, and a hIL-12p40 polypeptide.

[0031] In one aspect, provided herein is a fusion protein comprising a hIL-12p40 polypeptide described herein (eg, a mutant hIL-12p40 polypeptide), a hIL-12p35 polypeptide; and a heterologous moiety.

[0032] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide shown in Table 6.

[0033] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 31 or 110-114. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide shown in Table 10. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33, or 38-51, or 90-109. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33 or 38.

[0034] In some embodiments, the hIL-12p40 polypeptide and the hIL-12p35 polypeptide are operably linked as a schIL-12 polypeptide. In some embodiments, the hIL-12p40 polypeptide is operably linked to the hIL-12p35 polypeptide via a peptide linker. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a hIL-12p40 polypeptide, a peptide linker, and a hIL-12p35 polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a hIL-12p35 polypeptide, a peptide linker, and a hIL-12p40 polypeptide.

[0035] In some embodiments, the fusion protein mediates an increase in phosphorylated STAT4 (pSTAT4) levels in cells expressing hIL-12R on their surface that is less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the fusion protein mediates an increase in phosphorylated STAT4 (pSTAT4) levels in cells expressing hIL-12R on their surface that is about 0.5-1000 fold, 0.5-100 fold, 0.5-10 fold, 0.5-5 fold, 0.5-2 fold, 1-1000 fold, 1-100 fold, 1-10 fold, 1-5 fold, 1-2 fold, 10-1000 fold, or 100-1000 fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the fusion protein mediates an increase in the level of interferon gamma (IFN-γ) produced by expressing hIL-12R on its surface that is less than the increase in the level of IFN-γ produced in the presence of a suitable control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the fusion protein mediates an increase in the level of IFN-γ produced by expressing hIL-12R on its surface that is about 0.5-1000 fold, 0.5-100 fold, 0.5-10 fold, 0.5-5 fold, 0.5-2 fold, 1-1000 fold, 1-100 fold, 1-10 fold, 1-5 fold, 1-2 fold, 10-1000 fold, or 100-1000 fold less than the increase in the level of IFN-γ produced in the presence of a suitable control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)).

[0036] In some embodiments, the heterologous moiety comprises or consists of an antibody (or antigen-binding domain thereof) and / or one or more Fc regions. In some embodiments, the heterologous moiety comprises or consists of an antibody (or antigen-binding domain thereof). In some embodiments, the heterologous moiety comprises or consists of a full-length antibody, scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2, Fab-Fc, a single-domain antibody (e.g., VHH), or a single-domain antibody-Fc (e.g., VHH-Fc). In some embodiments, the antibody (or antigen-binding domain thereof) comprises a first variable heavy chain region (VH) comprising three VH complementarity-determining regions (VH CDRs): VH CDR1, VH CDR2, and VH CDR3; and a first variable light chain region (VL) comprising three VL CDRs: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the heterologous moiety comprises or consists of a full-length antibody.

[0037] In some embodiments, the antibody (or antigen-binding domain thereof) specifically binds to a human tumor-associated antigen (hTAA).

[0038] In some embodiments, the antibody (or antigen-binding domain thereof) specifically binds to human carbonic anhydrase IX (hCAIX).

[0039] In some embodiments, the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise or consist of the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively, of the antibodies shown in Table 17.

[0040] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of SEQ ID NO: 237 or the amino acid sequence of SEQ ID NO: 237 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of SEQ ID NO: 238 or the amino acid sequence of SEQ ID NO: 238 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises the amino acid sequence of SEQ ID NO: 239 or the amino acid sequence of SEQ ID NO: 239 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises the amino acid sequence of SEQ ID NO: 240 or the amino acid sequence of SEQ ID NO: 240 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 comprises the amino acid sequence of SEQ ID NO: 241 or the amino acid sequence of SEQ ID NO: 242 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises the amino acid sequence of SEQ ID NO: 243 or the amino acid sequence of SEQ ID NO: 243 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0041] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any VH polypeptide shown in Table 17; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any VL polypeptide shown in Table 17.

[0042] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 7, 246, 256, 264, 274, or 284; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 12, 247, 257, 265, 275, or 285.

[0043] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:12.

[0044] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15.

[0045] In some embodiments, the fusion protein comprises a first Fc region comprising a CH2 region and a CH3 region; and a second Fc region comprising a CH2 region and a CH3 region. In some embodiments, (a) the first Fc region comprises a CH2 region and a CH3 region; and the second Fc region comprises a CH2 region and a CH3 region; or (b) the first Fc region comprises a hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region and the second Fc region are hIgG1 or hIgG4 Fc regions, respectively, or functional variants thereof. In some embodiments, the first Fc region and the second Fc region are portions of full-length antibodies.

[0046] In some embodiments, the CH3 region of the first Fc region and the CH3 region of the second Fc region each comprise at least one amino acid modification that promotes heterodimerization of the first Fc region and the second Fc region.

[0047] In some embodiments, the first Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitutions T366S, L368A, and Y407V (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises an amino acid substitution at amino acid position Y349 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitution Y349C (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises amino acid substitutions at amino acid positions T366, L368, Y407, and Y349 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitutions T366S, L368A, Y407V, and Y349C (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises an amino acid substitution at amino acid position T366 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitution T366W (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises an amino acid substitution at amino acid position S354 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitution S354C (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises amino acid substitutions at amino acid positions T366 and S354 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitutions T366W and S354C (numbering according to the EU index of Kabat): In some embodiments, the first Fc region comprises the following amino acid substitutions T366S, L368A, Y407V, and Y349C, and the second Fc region comprises the following amino acid substitutions T366W and S354C (numbering according to the EU index of Kabat).

[0048] In some embodiments, the second Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitutions T366S, L368A, and Y407V (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises an amino acid substitution at amino acid position Y349 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitution Y349C (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises amino acid substitutions at amino acid positions T366, L368, Y407, and Y349 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitutions T366S, L368A, Y407V, and Y349C (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises an amino acid substitution at amino acid position T366 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitution T366W (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises an amino acid substitution at amino acid position S354 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitution S354C (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises amino acid substitutions at amino acid positions T366 and S354 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitutions T366W and S354C (numbering according to the EU index of Kabat): In some embodiments, the first Fc region comprises the following amino acid substitutions T366S, L368A, Y407V, and Y349C, and the first Fc region comprises the following amino acid substitutions T366W and S354C (numbering according to the EU index of Kabat).

[0049] In some embodiments, the first Fc region and the second Fc region each comprise at least one amino acid modification (e.g., substitution, deletion, addition) that reduces or eliminates an effector function of the Fc region relative to a reference Fc region that does not comprise the at least one amino acid modification (e.g., substitution, deletion, addition). In some embodiments, the at least one effector function comprises the ability of the Fc region to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC), bind to an Fc receptor (e.g., an Fcγ receptor), or any combination thereof.

[0050] In some embodiments, the first Fc region and the second Fc region each comprise an amino acid substitution at one, two, or three of amino acid positions L234, L235, and / or P329 (numbering according to EU index of Kabat). In some embodiments, the first Fc region and the second Fc region each comprise one, two, or three of the following amino acid substitutions: L234A, L235A, and / or P329G or P329A (numbering according to EU index of Kabat). In some embodiments, the first Fc region and the second Fc region each comprise an L234A and an L235A amino acid substitution (numbering according to EU index of Kabat). In some embodiments, the first Fc region and the second Fc region each comprise an L234A, L235A, and P329A amino acid substitution (numbering according to EU index of Kabat). In some embodiments, the first Fc region and the second Fc region each comprise L234A, L235A, and P329G amino acid substitutions (numbering according to the EU index of Kabat).

[0051] In some embodiments, the N-terminus of the hIL-12p40 polypeptide is operably linked to the C-terminus of a first Fc region; and the N-terminus of the hIL-12p35 polypeptide is operably linked to the C-terminus of a second Fc region. In some embodiments, the hIL-12p40 polypeptide is operably linked to the first Fc region via a first peptide linker; and the hIL-12p40 polypeptide is operably linked to the second Fc region via a second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 88-303, or 369; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 288-303, or 369. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 72; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 72.

[0052] In some embodiments, the N-terminus of the hIL-12p35 polypeptide is operably linked to the C-terminus of a first Fc region; and the N-terminus of the hIL-12p40 polypeptide is operably linked to the C-terminus of a second Fc region. In some embodiments, the hIL-12p40 polypeptide is operably linked to the second Fc region via a first peptide linker; and the hIL-12p40 polypeptide is operably linked to the first Fc region via a second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 288-303, or 369; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 288-303, or 369. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 72; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 72.

[0053] In some embodiments, the hIL-12p40 polypeptide and the hIL-12p35 polypeptide are operably linked as a schIL-12 polypeptide, and the N-terminus of the schIL-12 polypeptide is operably linked to the C-terminus of a first Fc region or the C-terminus of a second Fc region. In some embodiments, the schIL-12 polypeptide is operably linked to the first Fc region or the second Fc region via a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 288-303, or 369. In some embodiments, the amino acid sequence of the first peptide comprises or consists of the amino acid sequence of SEQ ID NO: 72.

[0054] In one aspect, provided herein is a fusion protein comprising a hIL-12p40 polypeptide; a hIL-12p35 polypeptide described herein (eg, a mutant hIL-12p35 polypeptide); and a heterologous moiety.

[0055] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide shown in Table 6.

[0056] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 31 or 110-114. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 31. In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide shown in Table 10. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 33, or 38-51, or 90-109. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 33 or 38.

[0057] In some embodiments, the hIL-12p40 polypeptide and the hIL-12p35 polypeptide are operably linked as a schIL-12 polypeptide. In some embodiments, the hIL-12p40 polypeptide is operably linked to the hIL-12p35 polypeptide via a peptide linker. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a hIL-12p40 polypeptide, a peptide linker, and a hIL-12p35 polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a hIL-12p35 polypeptide, a peptide linker, and a hIL-12p40 polypeptide.

[0058] In some embodiments, the fusion protein mediates an increase in phosphorylated STAT4 (pSTAT4) levels in cells expressing hIL-12R on their surface that is less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the fusion protein mediates an increase in phosphorylated STAT4 (pSTAT4) levels in cells expressing hIL-12R on their surface that is about 0.5-1000 fold, 0.5-100 fold, 0.5-10 fold, 0.5-5 fold, 0.5-2 fold, 1-1000 fold, 1-100 fold, 1-10 fold, 1-5 fold, 1-2 fold, 10-1000 fold, or 100-1000 fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the fusion protein mediates an increase in the level of interferon gamma (IFN-γ) produced by expressing hIL-12R on its surface that is less than the increase in the level of IFN-γ produced in the presence of a suitable control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the fusion protein mediates an increase in the level of IFN-γ produced by expressing hIL-12R on its surface that is about 0.5-1000-fold, 0.5-100-fold, 0.5-10-fold, 0.5-5-fold, 0.5-2-fold, 1-1000-fold, 1-100-fold, 1-10-fold, 1-5-fold, 1-2-fold, 10-1000-fold, or 100-1000-fold less than the increase in the level of IFN-γ produced in the presence of a suitable control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)).

[0059] In some embodiments, the heterologous moiety comprises or consists of an antibody (or antigen-binding domain thereof) and / or one or more Fc regions. In some embodiments, the heterologous moiety comprises or consists of an antibody (or antigen-binding domain thereof). In some embodiments, the heterologous moiety comprises or consists of a full-length antibody, scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2, Fab-Fc, a single-domain antibody (e.g., VHH), or a single-domain antibody-Fc (e.g., VHH-Fc). In some embodiments, the antibody (or antigen-binding domain thereof) comprises a first variable heavy chain region (VH) comprising three VH complementarity-determining regions (VH CDRs): VH CDR1, VH CDR2, and VH CDR3; and a first variable light chain region (VL) comprising three VL CDRs: VL CDR1, VL CDR2, and VL CDR3. In some embodiments, the heterologous moiety comprises or consists of a full-length antibody.

[0060] In some embodiments, the antibody (or antigen-binding domain thereof) specifically binds to a human tumor-associated antigen (hTAA).

[0061] In some embodiments, the antibody (or antigen-binding domain thereof) specifically binds to human carbonic anhydrase IX (hCAIX).

[0062] In some embodiments, the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 comprise or consist of the amino acid sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively, of the antibodies shown in Table 17.

[0063] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of SEQ ID NO: 237 or the amino acid sequence of SEQ ID NO: 237 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of SEQ ID NO: 238 or the amino acid sequence of SEQ ID NO: 238 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises the amino acid sequence of SEQ ID NO: 239 or the amino acid sequence of SEQ ID NO: 239 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises the amino acid sequence of SEQ ID NO: 240 or the amino acid sequence of SEQ ID NO: 240 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 comprises the amino acid sequence of SEQ ID NO: 241 or the amino acid sequence of SEQ ID NO: 242 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises the amino acid sequence of SEQ ID NO: 243 or the amino acid sequence of SEQ ID NO: 243 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0064] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any VH polypeptide shown in Table 17; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any VL polypeptide shown in Table 17.

[0065] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 7, 246, 256, 264, 274, or 284; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 12, 247, 257, 265, 275, or 285.

[0066] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:12.

[0067] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15.

[0068] In some embodiments, the fusion protein comprises a first Fc region comprising a CH2 region and a CH3 region; and a second Fc region comprising a CH2 region and a CH3 region. In some embodiments, (a) the first Fc region comprises a CH2 region and a CH3 region; and the second Fc region comprises a CH2 region and a CH3 region; or (b) the first Fc region comprises a hinge region, a CH2 region, and a CH3 region; and the second Fc region comprises a hinge region, a CH2 region, and a CH3 region. In some embodiments, the first Fc region and the second Fc region are hIgG1 or hIgG4 Fc regions, respectively, or functional variants thereof. In some embodiments, the first Fc region and the second Fc region are portions of full-length antibodies.

[0069] In some embodiments, the CH3 region of the first Fc region and the CH3 region of the second Fc region each comprise at least one amino acid modification that promotes heterodimerization of the first Fc region and the second Fc region.

[0070] In some embodiments, the first Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitutions T366S, L368A, and Y407V (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises an amino acid substitution at amino acid position Y349 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitution Y349C (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises amino acid substitutions at amino acid positions T366, L368, Y407, and Y349 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitutions T366S, L368A, Y407V, and Y349C (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises an amino acid substitution at amino acid position T366 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitution T366W (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises an amino acid substitution at amino acid position S354 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitution S354C (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises amino acid substitutions at amino acid positions T366 and S354 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitutions T366W and S354C (numbering according to the EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitutions T366S, L368A, Y407V, and Y349C, and the second Fc region comprises the following amino acid substitutions T366W and S354C (numbering according to the EU index of Kabat).

[0071] In some embodiments, the second Fc region comprises amino acid substitutions at amino acid positions T366, L368, and Y407 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitutions T366S, L368A, and Y407V (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises an amino acid substitution at amino acid position Y349 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitution Y349C (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises amino acid substitutions at amino acid positions T366, L368, Y407, and Y349 (numbering according to EU index of Kabat). In some embodiments, the second Fc region comprises the following amino acid substitutions T366S, L368A, Y407V, and Y349C (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises an amino acid substitution at amino acid position T366 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitution T366W (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises an amino acid substitution at amino acid position S354 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitution S354C (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises amino acid substitutions at amino acid positions T366 and S354 (numbering according to EU index of Kabat). In some embodiments, the first Fc region comprises the following amino acid substitutions T366W and S354C (numbering according to the EU index of Kabat): In some embodiments, the first Fc region comprises the following amino acid substitutions T366S, L368A, Y407V, and Y349C, and the first Fc region comprises the following amino acid substitutions T366W and S354C (numbering according to the EU index of Kabat).

[0072] In some embodiments, the first Fc region and the second Fc region each comprise at least one amino acid modification (e.g., substitution, deletion, addition) that reduces or eliminates an effector function of the Fc region relative to a reference Fc region that does not comprise the at least one amino acid modification (e.g., substitution, deletion, addition). In some embodiments, the at least one effector function comprises the ability of the Fc region to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC), bind to an Fc receptor (e.g., an Fcγ receptor), or any combination thereof.

[0073] In some embodiments, the first Fc region and the second Fc region each comprise an amino acid substitution at one, two, or three of amino acid positions L234, L235, and / or P329 (numbering according to EU index of Kabat). In some embodiments, the first Fc region and the second Fc region each comprise one, two, or three of the following amino acid substitutions: L234A, L235A, and / or P329G or P329A (numbering according to EU index of Kabat). In some embodiments, the first Fc region and the second Fc region each comprise an L234A and an L235A amino acid substitution (numbering according to EU index of Kabat). In some embodiments, the first Fc region and the second Fc region each comprise an L234A, L235A, and P329A amino acid substitution (numbering according to EU index of Kabat). In some embodiments, the first Fc region and the second Fc region each comprise L234A, L235A, and P329G amino acid substitutions (numbering according to the EU index of Kabat).

[0074] In some embodiments, the N-terminus of the hIL-12p40 polypeptide is operably linked to the C-terminus of a first Fc region; and the N-terminus of the hIL-12p35 polypeptide is operably linked to the C-terminus of a second Fc region. In some embodiments, the hIL-12p40 polypeptide is operably linked to the first Fc region via a first peptide linker; and the hIL-12p40 polypeptide is operably linked to the second Fc region via a second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 88-303, or 369; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 288-303, or 369. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 72; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 72.

[0075] In some embodiments, the N-terminus of the hIL-12p35 polypeptide is operably linked to the C-terminus of a first Fc region; and the N-terminus of the hIL-12p40 polypeptide is operably linked to the C-terminus of a second Fc region. In some embodiments, the hIL-12p40 polypeptide is operably linked to the second Fc region via a first peptide linker; and the hIL-12p40 polypeptide is operably linked to the first Fc region via a second peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 288-303, or 369; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 288-303, or 369. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 72; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 72.

[0076] In some embodiments, the hIL-12p40 polypeptide and the hIL-12p35 polypeptide are operably linked as a schIL-12 polypeptide, and the N-terminus of the schIL-12 polypeptide is operably linked to the C-terminus of a first Fc region or the C-terminus of a second Fc region. In some embodiments, the schIL-12 polypeptide is operably linked to the first Fc region or the second Fc region via a first peptide linker. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of a peptide linker set forth in Table 18. In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 66-81, 288-303, or 369. In some embodiments, the amino acid sequence of the first peptide comprises or consists of the amino acid sequence of SEQ ID NO: 72.

[0077] In one aspect, provided herein is a full-length antibody that specifically binds to an hTAA, comprising: a first light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL) region; a first heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH), a CH1 region, a hinge region, a CH2 region, and a CH3 region; a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; a first light chain and a second light chain comprising a VH region; a first light chain and a first heavy chain that associate to form a first antigen-binding domain; a second light chain and a second heavy chain that associate to form a second antigen-binding domain; and a first heavy chain and a second heavy chain that associate to form a dimer; a full-length antibody comprising a hIL-12p40 polypeptide (e.g., a mutant hIL-12p40), a hIL-12p35 polypeptide described herein; and a full-length antibody wherein the CH3 region of the first heavy chain of the full-length antibody is a reference CH3 region (e.g., a wild-type CH3 region) that does not contain one or more amino acid modifications. the CH3 region of the second heavy chain of the full-length antibody comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 122) that does not contain the one or more amino acid modifications; the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; A fusion protein is provided in which one or more amino acid modifications in the H3 region and one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimerization of the first heavy chain and the second heavy chain of the full-length antibody; the N-terminus of the hIL-12p40 polypeptide is operably linked to the C-terminus of the CH3 region of the first heavy chain via a first peptide linker; and the N-terminus of the hIL-12p35 polypeptide is operably linked to the C-terminus of the CH3 region of the second heavy chain via a second peptide linker.

[0078] In some embodiments, the hIL-12p40 polypeptide comprises an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) comprises, or consists of, an amino acid substitution at each of amino acid positions W37, F82, and K219, (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0079] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of each of the following amino acid substitutions: W37A, F82A, and K219A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0080] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of SEQ ID NO:38 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO:33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:38.

[0081] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:38.

[0082] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 31 or 110-114.

[0083] In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of SEQ ID NO:72; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of SEQ ID NO:72.

[0084] In some embodiments, the first antigen-binding domain specifically binds to hCAIX and the second antigen-binding domain specifically binds to hCAIX.

[0085] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of SEQ ID NO: 237 or the amino acid sequence of SEQ ID NO: 237 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of SEQ ID NO: 238 or the amino acid sequence of SEQ ID NO: 238 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises the amino acid sequence of SEQ ID NO: 239 or the amino acid sequence of SEQ ID NO: 239 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises the amino acid sequence of SEQ ID NO: 240 or the amino acid sequence of SEQ ID NO: 240 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 comprises the amino acid sequence of SEQ ID NO: 241 or the amino acid sequence of SEQ ID NO: 242 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises the amino acid sequence of SEQ ID NO: 243 or the amino acid sequence of SEQ ID NO: 243 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0086] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:12.

[0087] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15.

[0088] In some embodiments, the first heavy chain and the second heavy chain each comprise at least one amino acid modification (e.g., substitution, deletion, addition) that reduces or eliminates an effector function of the Fc region relative to a reference Fc region that does not comprise the at least one amino acid modification (e.g., substitution, deletion, addition). In some embodiments, the at least one effector function comprises the ability to induce ADCC, ADCP, or CDC, bind to an Fc receptor, or any combination thereof.

[0089] In one embodiment, provided herein is a polypeptide comprising, from N-terminus to C-terminus, a first polypeptide comprising a first light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; a second polypeptide comprising, from N-terminus to C-terminus: (i) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) a first peptide linker, and (iii) a hIL-12p40 polypeptide described herein (e.g., a mutant hIL-12p40); a third polypeptide comprising, from N-terminus to C-terminus: (i) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region, (ii) a second peptide linker; and (iii) a hIL-12p35 polypeptide; and a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain comprising, from N-terminus to C-terminus, a VL region and a CL region; The H associates to form a first antigen-binding domain that specifically binds to a first hTAA; the CH3 region of the first heavy chain comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 122) that does not contain the one or more amino acid modifications; the CH3 region of the second heavy chain comprises one or more amino acid modifications (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 122) that does not contain the one or more amino acid modifications; the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; and the one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody and the one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimerization of the first heavy chain and the second heavy chain of the full-length antibody.

[0090] In some embodiments, the hIL-12p40 polypeptide comprises an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) comprises, or consists of, an amino acid substitution at each of amino acid positions W37, F82, and K219 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0091] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of each of the following amino acid substitutions: W37A, F82A, and K219A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0092] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of SEQ ID NO:38 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO:33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:38.

[0093] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:38.

[0094] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 31 or 110-114.

[0095] In some embodiments, the amino acid sequence of the first peptide linker comprises or consists of the amino acid sequence of SEQ ID NO:72; and the amino acid sequence of the second peptide linker comprises or consists of the amino acid sequence of SEQ ID NO:72.

[0096] In some embodiments, the full-length antibody specifically binds to hCAIX.

[0097] In some embodiments, the amino acid sequence of VH CDR1 comprises the amino acid sequence of SEQ ID NO: 237 or the amino acid sequence of SEQ ID NO: 237 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR2 comprises the amino acid sequence of SEQ ID NO: 238 or the amino acid sequence of SEQ ID NO: 238 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VH CDR3 comprises the amino acid sequence of SEQ ID NO: 239 or the amino acid sequence of SEQ ID NO: 239 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); the amino acid sequence of VL CDR1 comprises the amino acid sequence of SEQ ID NO: 240 or the amino acid sequence of SEQ ID NO: 240 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); The amino acid sequence of CDR2 comprises the amino acid sequence of SEQ ID NO: 241 or the amino acid sequence of SEQ ID NO: 242 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.); and the amino acid sequence of VL CDR3 comprises the amino acid sequence of SEQ ID NO: 243 or the amino acid sequence of SEQ ID NO: 243 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions, etc.).

[0098] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:12.

[0099] In some embodiments, the amino acid sequence of the VH comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:7; and the amino acid sequence of the VL comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:15.

[0100] In some embodiments, the first heavy chain and the second heavy chain each comprise at least one amino acid modification (e.g., substitution, deletion, addition) that reduces or eliminates an effector function of the Fc region relative to a reference Fc region that does not comprise the at least one amino acid modification (e.g., substitution, deletion, addition). In some embodiments, the at least one effector function comprises the ability to induce ADCC, ADCP, or CDC, bind to an Fc receptor, or any combination thereof.

[0101] In one aspect, provided herein is an antibody (or antigen-binding domain thereof) that specifically binds to hCAIX and comprises a VH and a VL, wherein the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10 to 17.

[0102] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:15.

[0103] In one aspect, provided herein is a polynucleotide encoding a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, or an antibody (or one or more polypeptides thereof) described herein. In some embodiments, the polynucleotide is RNA (e.g., mRNA) or DNA. In some embodiments, the polynucleotide is codon-optimized.

[0104] In one aspect, provided herein is an expression vector comprising a polynucleotide described herein. In some embodiments, the expression vector is a viral vector or a plasmid.

[0105] In one aspect, provided herein is a host cell comprising a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, an antibody (or one or more polypeptides thereof) described herein, a polynucleotide described herein, or an expression vector described herein.

[0106] In one aspect, provided herein is a carrier comprising a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, an antibody (or one or more polypeptides thereof) described herein, a polynucleotide described herein, or an expression vector described herein. In some embodiments, the carrier is a lipid nanoparticle, liposome, lipoplex, or nanoliposome.

[0107] In one aspect, provided herein is a pharmaceutical composition comprising a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, an antibody (or one or more polypeptides thereof) described herein, a polynucleotide described herein, or an expression vector described herein, a host cell described herein, or a carrier described herein, and a pharmaceutically acceptable excipient.

[0108] In one aspect, provided herein is a kit comprising a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, an antibody (or one or more polypeptides thereof) described herein, a polynucleotide described herein, or an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein.

[0109] In one aspect, provided herein is a method of making a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, or an antibody (or one or more polypeptides thereof) described herein, the method comprising introducing a polynucleotide described herein or an expression vector described herein into a population of in vitro or ex vivo cells, culturing the cell population under conditions sufficient for the cell population to express the multispecific protein, and optionally isolating and / or purifying the hIL-12p40 polypeptide, hIL-12p35 polypeptide, schIL-12 polypeptide, or fusion protein (or one or more polypeptides thereof).

[0110] In one aspect, provided herein is a method of delivering a polypeptide, fusion protein, antibody, polynucleotide, expression vector, host cell, carrier, or pharmaceutical composition to a subject, comprising administering a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, an antibody (or one or more polypeptides thereof) described herein, a polynucleotide described herein, or an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein to a subject in an amount and for a time sufficient to deliver the hIL-12p40 polypeptide, hIL-12p35 polypeptide, schIL-12 polypeptide, fusion protein, polynucleotide, expression vector, host cell, carrier, or pharmaceutical composition to the subject.

[0111] A method of stimulating effector function of T cells or NK cells in a subject, comprising administering to the subject a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, an antibody (or one or more polypeptides thereof) described herein, a polynucleotide described herein, or an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein in an amount and for a time sufficient to stimulate effector function of T cells or NK cells in the subject.

[0112] A method of preventing or treating cancer in a subject, comprising administering to a subject in need thereof a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, an antibody (or one or more polypeptides thereof) described herein, a polynucleotide described herein, or an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein in an amount and for a time sufficient to prevent or treat cancer in the subject.

[0113] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is lung cancer, central nervous system cancer (e.g., brain cancer or spinal cancer, e.g., astrocytoma, glioblastoma), breast cancer, colorectal cancer, colon cancer, rectal cancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, skin cancer, bladder cancer, uterine cancer, brain cancer, endometrial cancer, lip cancer, oral cancer, mesothelioma, sarcoma, thyroid cancer, thymic cancer, renal cancer, anal cancer, head cancer, neck cancer, or head and neck cancer.

[0114] In some embodiments, the cancer is renal cancer (e.g., renal cell carcinoma), bladder cancer, colon cancer, small intestine cancer, esophageal / gastric junction (GEJ) cancer, central nervous system cancer (e.g., brain or spinal cord cancer, e.g., glioblastoma), cervical cancer, gastric cancer, lung cancer (e.g., small cell lung cancer), or gastrointestinal cancer.

[0115] In one aspect, the present specification provides a method for determining the expression of CAIX in cancer (e.g., solid cancer) cells of a subject, the method comprising obtaining a sample from the subject, the sample not containing cancer cells (or not containing a substantial number of cancer cells), and determining the presence or absence of soluble CAIX (or a fragment or mutant thereof) in the sample.

[0116] In some embodiments, the sample is blood, serum, or plasma. In some embodiments, the subject is a human.

[0117] In some embodiments, the cancer is (e.g., a solid tumor). In some embodiments, the solid tumor is renal cancer (e.g., renal cell carcinoma), bladder cancer, colon cancer, small intestine cancer, esophageal / gastric junction (GEJ) cancer, central nervous system cancer (e.g., brain or spinal cord cancer, e.g., glioblastoma), cervical cancer, gastric cancer, lung cancer (e.g., small cell lung cancer), or gastrointestinal cancer.

[0118] In one aspect, provided herein is a method for diagnosing a subject having a cancer (e.g., a solid tumor) comprising cancer cells that express CAIX, the method comprising obtaining a sample from the subject, the sample not containing cancer cells (or not containing a substantial number of cancer cells), determining the presence or absence of soluble CAIX (or a fragment or variant thereof) in the sample, and diagnosing the subject as having a cancer (e.g., a solid tumor) comprising cancer cells that express CAIX if soluble CAIX is determined to be present in the sample.

[0119] In some embodiments, the sample is blood, serum, or plasma. In some embodiments, the subject is a human.

[0120] In some embodiments, the cancer is (e.g., a solid tumor). In some embodiments, the solid tumor is renal cancer (e.g., renal cell carcinoma), bladder cancer, colon cancer, small intestine cancer, esophageal / gastric junction (GEJ) cancer, central nervous system cancer (e.g., brain or spinal cord cancer, e.g., glioblastoma), cervical cancer, gastric cancer, lung cancer (e.g., small cell lung cancer), or gastrointestinal cancer.

[0121] In one aspect, provided herein is a method of treating cancer (e.g., a solid cancer) in a subject, the method comprising receiving test results determining the presence of soluble CAIX in a sample from the subject, wherein the sample does not contain cancer cells (or does not contain a substantial number of cancer cells), diagnosing the subject as having a cancer (e.g., a solid cancer) comprising cancer cells that express CAIX, and administering to a subject in need thereof a hIL-12p40 polypeptide described herein, a hIL-12p35 polypeptide described herein, a schIL-12 polypeptide described herein, a fusion protein (or one or more polypeptides thereof) described herein, an antibody (or one or more polypeptides thereof) described herein, a polynucleotide described herein, or an expression vector described herein, a host cell described herein, a carrier described herein, or a pharmaceutical composition described herein in an amount and for a time sufficient to treat cancer (e.g., a solid cancer) in the subject.

[0122] In some embodiments, the sample is blood, serum, or plasma. In some embodiments, the subject is a human.

[0123] In some embodiments, the cancer is (e.g., a solid tumor). In some embodiments, the solid tumor is renal cancer (e.g., renal cell carcinoma), bladder cancer, colon cancer, small intestine cancer, esophageal / gastric junction (GEJ) cancer, central nervous system cancer (e.g., brain or spinal cord cancer, e.g., glioblastoma), cervical cancer, gastric cancer, lung cancer (e.g., small cell lung cancer), or gastrointestinal cancer. 4. Brief description of the drawings [Brief explanation of the drawings]

[0124] [Figure 1]1 is an illustration of an exemplary antibody (e.g., an anti-CAIX antibody) IL-12 fusion protein described herein. In the specific embodiment shown, the fusion protein comprises a full-length antibody operably linked to the C-terminus of a second Fc region of the full-length antibody, and a full-length antibody, IL-12p35 (e.g., an IL-12p35 polypeptide described herein), operably linked to the C-terminus of a first Fc region of IL-12p40 (e.g., an IL-12p40 polypeptide described herein). In the specific embodiment shown, the first Fc region and the second Fc region are heterodimers, and each Fc region comprises at least one amino acid modification (e.g., a substitution) that promotes heterodimer formation between the first Fc region and the second Fc region. In some embodiments, the first Fc region and the second Fc region each comprise one or more amino acid modifications (e.g., substitutions) that eliminate or reduce one or more Fc effector functions (e.g., antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), Fc receptor binding). [Figure 2] Figure 2 is an illustration of an exemplary antibody (e.g., an anti-CAIX antibody) IL-12 fusion protein described herein. In the specific embodiment shown, the fusion protein comprises a full-length antibody and scIL-12 (e.g., an scIL-12 polypeptide described herein) operably linked to the C-terminus of the first Fc region of the full-length antibody. In the specific embodiment shown, the first Fc region and the second Fc region are heterodimers, and each Fc region comprises at least one amino acid modification (e.g., substitution) that promotes heterodimer formation between the first Fc region and the second Fc region. In some embodiments, the first Fc region and the second Fc region each comprise one or more amino acid modifications (e.g., substitutions) that eliminate or reduce one or more Fc effector functions (e.g., ADCC, ADCP, CDC, Fc receptor binding). [Figure 3]3 is an illustration of an exemplary antibody (e.g., anti-CAIX antibody) IL-12 fusion protein described herein. In the specific embodiment shown, the fusion protein comprises two scFvs (one scFv operably linked to the N-terminus of a first Fc region and a second scFv operably linked to the N-terminus of a second Fc region); IL-12p35 (e.g., an IL-12p35 polypeptide described herein) operably linked to the C-terminus of the first Fc region; and IL-12p40 (e.g., an IL-12p40 polypeptide described herein) operably linked to the C-terminus of the second Fc region. In the specific embodiment shown, the first Fc region and the second Fc region are heterodimers, and each Fc region comprises at least one amino acid modification (e.g., a substitution) that promotes heterodimer formation between the first Fc region and the second Fc region. In some embodiments, the first Fc region and the second Fc region each comprise one or more amino acid modifications (e.g., substitutions) that eliminate or reduce one or more Fc effector functions (e.g., ADCC, ADCP, CDC, Fc receptor binding). [Figure 4] 4 is an illustration of an exemplary antibody (e.g., anti-CAIX antibody) IL-12 fusion protein described herein. In the specific embodiment shown, the fusion protein comprises two scFvs (one scFv operably linked to the N-terminus of a first Fc region and a second scFv operably linked to the N-terminus of a second Fc region); and scIL-12 (e.g., an sc-IL-12 polypeptide described herein) operably linked to the C-terminus of either the first or second Fc region. In the specific embodiment shown, the first and second Fc regions are heterodimers, and each Fc region comprises at least one amino acid modification (e.g., substitution) that promotes heterodimer formation between the first and second Fc regions. In some embodiments, the first Fc region and the second Fc region each comprise one or more amino acid modifications (e.g., substitutions) that eliminate or reduce one or more Fc effector functions (e.g., ADCC, ADCP, CDC, Fc receptor binding). [Figure 5]5 is an illustration of an exemplary antibody (e.g., anti-CAIX antibody) IL-12 fusion protein described herein. In the specific embodiment shown, the fusion protein comprises a full-length antibody and IL-12p35 (e.g., an IL-12p35 polypeptide described herein) operably linked to the C-terminus of one of the first or second Fc regions of the full-length antibody. In the specific embodiment shown, the first Fc region and the second Fc region are heterodimers, and each Fc region comprises at least one amino acid modification (e.g., substitution) that promotes heterodimer formation between the first Fc region and the second Fc region. In some embodiments, the first Fc region and the second Fc region each comprise one or more amino acid modifications (e.g., substitutions) that eliminate or reduce one or more Fc effector functions (e.g., ADCC, ADCP, CDC, Fc receptor binding). [Figure 6] 6 is an illustration of an exemplary antibody (e.g., an anti-CAIX antibody) IL-12 fusion protein described herein. In the specific embodiment shown, the fusion protein comprises a first DART operably linked to a first Fc region via a coil domain (e.g., a coil domain described herein) and a second DART operably linked to a second Fc region (e.g., a coil domain described herein); IL-12p35 (e.g., an IL-12p35 polypeptide described herein) operably linked to the C-terminus of the first Fc region; and IL-12p40 (e.g., an IL-12p40 polypeptide described herein) operably linked to the C-terminus of the second Fc region. In some embodiments, a non-native disulfide bond is introduced into the heavy chains of the first and second DARTs. In certain embodiments shown, the first Fc region and the second Fc region are heterodimeric, and each Fc region comprises at least one amino acid modification (e.g., substitution) that promotes heterodimerization of the first Fc region and the second Fc region. In some embodiments, the first Fc region and the second Fc region each comprise one or more amino acid modifications (e.g., substitutions) that eliminate or reduce one or more Fc effector functions (e.g., ADCC, ADCP, CDC, Fc receptor binding). [Figure 7]7 is an illustration of an exemplary antibody (e.g., anti-CAIX antibody) IL-12 fusion protein described herein. In the specific embodiment shown, the fusion protein comprises two scFvs operably linked in tandem to the N-terminus of a first Fc region; and scIL-12 (e.g., an scIL-12 polypeptide described herein) operably linked to the N-terminus of a second Fc region. In the specific embodiment shown, the first Fc region and the second Fc region are heterodimers, and each Fc region comprises at least one amino acid modification (e.g., substitution) that promotes heterodimer formation between the first Fc region and the second Fc region. In some embodiments, the first Fc region and the second Fc region each comprise one or more amino acid modifications (e.g., substitutions) that eliminate or reduce one or more Fc effector functions (e.g., ADCC, ADCP, CDC, Fc receptor binding). [Figure 8] Figure 8 is an illustration of an exemplary antibody (e.g., anti-CAIX antibody) IL-12 fusion protein described herein. In the specific embodiment shown, the fusion protein comprises two scFvs operably linked in tandem to the N-terminus of a first Fc region; and scIL-12 (e.g., an scIL-12 polypeptide described herein) operably linked to a single-domain antibody operably linked to the N-terminus of a second Fc region. In the specific embodiment shown, the first Fc region and the second Fc region are heterodimers, and each Fc region comprises at least one amino acid modification (e.g., substitution) that promotes heterodimer formation between the first Fc region and the second Fc region. In some embodiments, the first Fc region and the second Fc region each comprise one or more amino acid modifications (e.g., substitutions) that eliminate or reduce one or more Fc effector functions (e.g., ADCC, ADCP, CDC, Fc receptor binding). [Figure 9] Figure 9 is a line graph showing pSTAT4 signaling (measured by SEAP production) from HEK-Blue cells treated with the indicated concentrations of the indicated constructs. Data were analyzed using the 4-PL model and presented as mean ± SD of optical density. [Figure 10A]Figure 10A is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of three replicates from a single experiment. [Figure 10B] Figure 10B is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of three replicates from a single experiment. [Figure 10C] Figure 10C is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of three replicates from a single experiment. [Figure 10D] Figure 10D is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of three replicates from a single experiment. [Figure 10E] Figure 10E is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of three replicates from a single experiment. [Figure 10F] Figure 10F is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of three replicates from a single experiment. [Figure 11A] Figure 11A is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of triplicates from a single experiment. [Figure 11B]Figure 11B is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of triplicates from a single experiment. [Figure 11C] Figure 11C is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of triplicates from a single experiment. [Figure 11D] Figure 11D is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of triplicates from a single experiment. [Figure 11E] Figure 11E is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of three replicates from a single experiment. [Figure 11F] Figure 11F is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of triplicates from a single experiment. [Figure 12] Figure 12 is a line graph showing the level of hIL-12R signaling in vitro (measured by SEAP production) from HEK-Blue cells cultured with the indicated concentrations of the indicated constructs. Reported values ​​(mean ± SD) are the average of three independent replicates. [Figure 13]13 is a line graph showing the levels of IFN-γ produced in vitro by PHA-stimulated human PBMCs (hPBMCs) cultured with the indicated concentrations of the indicated constructs. Each data point is the average (mean ± SD) of triplicate values, and the line graph is representative of data from three independent experiments. [Figure 14] 14 is a line graph showing the levels of IFN-γ produced in vitro by IL-2-primed NK cells cultured with the indicated concentrations of the indicated constructs. Each data point is the average (mean ± SD) of triplicate values, and the line graph is representative of data from three independent experiments. [Figure 15] Figure 15 is a line graph showing the levels of pSTAT4 expressed in vitro by gated CD8+ T cells using anti-CD3 and anti-CD28 stimulated hPBMCs cultured with the indicated concentrations of the indicated constructs. Each point is data from a single well, and the line graph is representative of data from three independent experiments. [Figure 16] 16 is a line graph showing the killing rate of CAIX-expressing SNU16 tumor cells by SEB-stimulated hPBMCs treated with each of the indicated constructs at the indicated concentrations. Each data point is from a single well, and the line graph is representative of data from two independent experiments. [Figure 17A] FIG. 17A is a bar graph showing granzyme B release by SEB-stimulated hPBMCs treated with the indicated concentrations of each of the indicated constructs. [Figure 17B] FIG. 17B is a bar graph showing IFN-γ release by SEB-stimulated hPBMCs treated with the indicated concentrations of each of the indicated constructs. [Figure 17C] FIG. 17C is a bar graph showing TNF-α release by SEB-stimulated hPBMCs treated with the indicated concentrations of each of the indicated constructs. [Figure 17D] FIG. 17D is a bar graph showing IL-10 release by SEB-stimulated hPBMCs treated with the indicated concentrations of each of the indicated constructs. [Figure 17E]FIG. 17E is a bar graph showing MIP-3α release by SEB-stimulated hPBMCs treated with the indicated concentrations of each of the indicated constructs. [Figure 17F] FIG. 17F is a bar graph showing CD40-L release by SEB-stimulated hPBMCs treated with the indicated concentrations of each of the indicated constructs. [Figure 17G] FIG. 17G is a bar graph showing Flt3-L release by SEB-stimulated hPBMCs treated with the indicated concentrations of each of the indicated constructs. [Figure 17H] Figure 17H ​​is a bar graph showing GMCSF release by SEB-stimulated hPBMCs treated with each of the indicated constructs at the indicated concentrations. In Figures 17A-17H, each bar is a value from a single well, and the line graphs are representative data from experiments evaluated at 24, 72, and 120 hours. [Figure 18] Figure 18 shows boxplots depicting the cytotoxicity of each indicated construct against HCT116 CAIX-expressing spheroids by NK cells. Cytotoxicity data are pooled data from three donors in four independent experiments. Statistical analysis was performed using one-way ANOVA and Kruskal-Wallis test, comparing all groups with each other (*p-values ​​<0.05, **p-values ​​<0.01 considered significant). [Figure 19] FIG. 19 is a dot plot (left) and histogram (right) showing the expression of eGFP-CAIX by transfected A549 cells. [Figure 20] Figure 20 shows bright field and fluorescent images of A459 spheroids expressing CAIX-eGFP fusion protein (images taken from Cytation 5). [Figure 21A] Figure 21A is a bar graph showing the intensity density of single CAIX-eGFP fusion protein-expressing A459 spheroids treated with the indicated constructs. Each bar is from a single spheroid, and the bar graph is representative of data from two independent experiments. [Figure 21B]Figure 21B is a bar graph showing the cytotoxicity induced by each of the indicated constructs in a CAIX-eGFP fusion protein-expressing A549 model. Cytotoxicity was calculated by considering IgG-treated spheroids as 100% viable. Each bar represents the value from a single spheroid, and the bar graph is representative of data from two independent experiments. [Figure 22] 22 is a line graph showing the tumor growth profile of HCT116-CAIX tumors in mice (n=6) treated with the indicated constructs. Arrows on the x-axis indicate the day of administration. [Figure 23] Figure 23 is a line graph showing the tumor growth profile of B16F10 allografts expressing human CAIX in hIL-12 and hIL-12 receptor gene knock-in transgenic mice (n=6) treated with the indicated constructs. Arrows on the x-axis indicate the days of administration. [Figure 24A] Figure 24A shows immunohistochemistry images showing CAIX expression in the indicated normal or cancer tissues. Upper panels: CAIX expression in normal bladder (a), colon (b), cervix (c), kidney (d), and brain (e) (10x magnification). Lower panels: Representative images of heterogeneous CAIX expression in various cancer types: low expression (f, j, n, r, v), moderate expression (g, k, o, s, w), and high expression (h, l, p, t, x) (10x magnification). Representative images of heterogeneous CAIX expression in clear cell carcinoma (f, g, h, i), bladder cancer (j, k, l, m), small intestine cancer (n, o, p, q), colon cancer (r, s, t, u), and gastric cancer (v, w, x, y). Membrane staining of CAIX in tumor cells (i, m, q, u, y; 30x magnification) of the corresponding tumor types shown in panels h, l, p, t, and x, respectively. [Figure 24B]Figure 24B is a bar graph showing, for each cancer type (Y-axis), the percentage of cases corresponding to the H-score categories (X-axis): CAIX high (201-300), CAIX moderate (101-200), CAIX low (1-100), and CAIX absent (0). BC(ER+): Breast cancer (ER+); BLC: Bladder cancer; BC(Her2+): Breast cancer (Her2+); CC: Cervical cancer; CRC: Colorectal cancer; DLBLC: Diffuse large B-cell lymphoma; EnC: Endometrial cancer; EsC: Esophageal / GEJ cancer; GBM: Glioblastoma; GC: Gastric cancer; GIST: Gastrointestinal stromal tumor; HCC :Hepatocellular carcinoma; HNC: Head and neck cancer; MEL: Melanoma; NHL: Non-Hodgkin's lymphoma; NSCLC: Non-small cell lung cancer; OC: Ovarian cancer; PC: Pancreatic cancer; PrC: Prostate cancer; RCC: Renal cell carcinoma; SARC: Sarcoma; SBC: Small bowel cancer; SCLC: Small cell lung cancer; TC: Thyroid cancer; TNBC: Triple negative breast cancer. [Figure 25A] Figure 25A shows representative microscopy images showing low (1–199; left panel), moderate (200–499; middle panel), and high (≥500; right panel) lymphocytic infiltration in hematoxylin-eosin-stained tumor tissue cores. Red arrowheads: tumor cells; green arrowheads: lymphocytes. [Figure 25B] Figure 25B is a bar graph showing the percentage of cases (X-axis) corresponding to high, moderate, low, and absent lymphocytic infiltration for each tumor type (Y-axis). BC(ER+): breast cancer (ER+); BLC: bladder cancer; BC(Her2+): breast cancer (Her2+); CC: cervical cancer; CRC: colorectal cancer; DLBLC: diffuse large B-cell lymphoma; EnC: endometrial cancer; EsC: esophageal / GEJ cancer; GBM: glioblastoma; GC: gastric cancer; GIST: gastrointestinal stromal tumor; HCC: hepatocellular carcinoma; HNC: head and neck cancer; MEL: melanoma; NSCLC: non-small cell lung cancer; OC: ovarian cancer; PC: pancreatic cancer; PrC: prostate cancer; RCC: renal cell carcinoma; SARC: sarcoma; SBC: small intestine cancer; SCLC: small cell lung cancer; TC: thyroid cancer; TNBC: triple-negative breast cancer. [Figure 25C]Figure 25C is a graph showing the density of lymphocytic infiltration [mean score (0-3), X-axis] in CAIX-expressing tumors (mean H-score, Y-axis). BC(ER+): breast cancer (ER+); BC(Her2+): breast cancer (Her2+); BLC: bladder cancer; CC: cervical cancer; CRC: colorectal cancer; DLBLC: diffuse large B-cell lymphoma; EnC: endometrial cancer; EsC: esophageal / GEJ cancer; GBM: glioblastoma; GC: gastric cancer; GIST: gastrointestinal stromal tumor; HCC: hepatocellular carcinoma; HNC: head and neck cancer; MEL: melanoma; NSCLC: non-small cell lung cancer; OC: ovarian cancer; PC: pancreatic cancer; PrC: prostate cancer; RCC: renal cell carcinoma; SARC: sarcoma; SBC: small intestine cancer; SCLC: small cell lung cancer; TC: thyroid cancer; TNBC: triple-negative breast cancer. [Figure 26A] FIG. 26A is a pie chart showing the percentage of different cell types (total of 10,468 cells) identified by single-cell RNA sequencing for colon adenocarcinoma tissue. [Figure 26B] Figure 26B is a t-distributed stochastic neighbor embedding (t-SNE) plot showing the clustering of different cell types of colon adenocarcinoma and the expression of CA9, IL12RB1, and IL12RB2 in these cell clusters. [Figure 26C] FIG. 26C is a bar graph showing CA9, IL12RB1, and IL12RB2 gene expression (Y-axis) in cell types (X-axis) identified by single-cell RNA sequencing analysis of colon adenocarcinoma tissue. [Figure 26D] FIG. 26D is a bar graph showing the percentage of cell types (Y-axis) expressing CA9, IL12RB1, and IL12RB2 in various gene combinations (X-axis). [Figure 27] Figure 27 is a line graph showing pSTAT4 signaling (measured by SEAP production) from HEK-Blue cells treated with the indicated concentrations of the indicated constructs. Data were analyzed using the 4-PL model and presented as mean ± SD of the optical density. BCA307.16 could not be tested at 100 nM concentration due to stock concentration limitations. [Figure 28A] Figure 28A is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated hIL-12 constructs. Data are plotted using a 4-PL model, and each point represents the average (mean ± SD) of triplicate values ​​from a single experiment. [Figure 28B] Figure 28B is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated hIL-12 constructs. Data are plotted using a 4-PL model, and each point represents the average (mean ± SD) of triplicate values ​​from a single experiment. [Figure 28C] Figure 28C is a line graph showing the levels of IFN-γ released by activated T cells in the presence of the indicated concentrations of the indicated hIL-12 constructs. Data are plotted using a 4-PL model, and each point represents the average (mean ± SD) of triplicates from a single experiment. [Figure 29A] Figure 29A is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data are plotted using a 4-PL model, and each data point is the average (mean ± SD) of triplicates from a single experiment. [Figure 29B] Figure 29B is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of triplicates from a single experiment. [Figure 29C] Figure 29C is a line graph showing the levels of IFN-γ released by enriched hIL-2-primed NK cells in the presence of the indicated concentrations of the indicated constructs. Data were plotted using a 4-PL model, and each data point represents the average (mean ± SD) of triplicates from a single experiment. [Figure 30]FIG. 30 is a dot plot showing Pearson's correlation coefficient analysis between cellular CAIX quantified as immunohistochemical H-scores in tumor tissue (X-axis) and soluble CAIX (pg / mL) in tumor-matched plasma quantified by ELISA (Y-axis) in cancer patients (n=86). DETAILED DESCRIPTION OF THE INVENTION

[0125] 5. Detailed Description hIL-12 has been evaluated as a therapeutic agent for the treatment of cancer, but clinical success has been limited. To achieve a therapeutic effect, hIL-12 must be administered at relatively high levels, which is highly potent and causes severe and intolerable side effects. In particular, when administered systemically, hIL-12 can activate immune cells expressing hIL-12R (e.g., T cells, e.g., CD8+ T cells) in the bloodstream, resulting in a systemic inflammatory response that can contribute to the severe side effects associated with hIL-12-based therapy. The present inventors have created improved hIL-12 (e.g., hIL-12p40, hIL-12p35) proteins that exhibit reduced efficacy while retaining, inter alia, the ability to mediate tumor cell killing via the activation of immune cells (e.g., T cells and NK cells). Thus, the novel hIL-12 proteins and fusion proteins containing them (e.g., anti-CAIX fusion proteins) described herein are excellent candidates for the treatment of diseases (e.g., cancer). Thus, the present disclosure provides, inter alia, hIL-12 proteins and fusion proteins containing same (eg, anti-CAIX fusion proteins) for use in pharmaceutical compositions for the treatment of disease (eg, cancer).

[0126] 5.1 Definition The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0127] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any claimed subject matter.

[0128] As used herein, the use of the singular includes the plural unless clearly indicated otherwise. For example, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Furthermore, the use of the term "including" and other forms ("include," "includes," and "included") is non-limiting.

[0129] Where an embodiment is described herein using the term "comprising," it is understood that other similar embodiments described using the terms "consisting of" and "consisting essentially of" are also provided.

[0130] The term "and / or," as used herein, means that each of the two specified features or components is considered to be specifically disclosed, regardless of the presence or absence of the other. Thus, when the term "and / or" is used herein in phrases such as "A and / or B," it is intended to include "A and B," "A or B," "A (alone)," and "B (alone)." Similarly, when the term "and / or" is used in phrases such as "A, B, and / or C," it is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0131] As described herein, concentration ranges, percentage ranges, ratio ranges, or integer ranges are understood to include any integer value within the stated range, and fractions thereof, where applicable (such as integer tenths and hundredths), unless otherwise specified.

[0132] The terms "about" or "consisting essentially of" refer to a value or composition that is within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When a particular value or composition is provided in the present application and claims, unless otherwise specified, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0133] Unless otherwise stated or clear from the context, the use of terms such as "first and second," or "(a) and (b)," or "(i) and (ii)," and similar terms herein do not denote any order or direction, but are used to identify multiple components of a composition or method. It will be clear to one of ordinary skill in the art from the context when these terms are intended to denote order or direction.

[0134] Where proteins and / or polypeptides are described herein, it is understood that the polynucleotides (e.g., RNA (e.g., mRNA) or DNA polynucleotides) that encode the proteins or polypeptides are also provided herein.

[0135] Where a protein, polypeptide, polynucleotide, cell, expression vector, etc. is described herein, it is understood that isolated forms of the protein, polypeptide, polynucleotide, cell, expression vector, etc. are also provided herein.

[0136] Where a protein, polypeptide, polynucleotide, etc. is described herein, it is understood that recombinant forms of that protein, polypeptide, polynucleotide, etc. are also provided herein.

[0137] When a polypeptide or set of polypeptides is described herein, it is understood that proteins comprising that polypeptide or set of polypeptides folded into their three-dimensional structures (i.e., tertiary or quaternary structures) are also provided herein, or vice versa.

[0138] As used herein, the term "administering" refers to the physical introduction of an agent, e.g., a therapeutic agent (or a precursor of a therapeutic agent that is metabolized or altered in the subject's body to produce the therapeutic agent in vitro), to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Administration can be, for example, one time, multiple times, and / or over one or more extended periods of time.

[0139] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to an immune mechanism that leads to the lysis of antibody (or Fc-region-containing polypeptide or protein) (e.g., an Ig Fc-containing fusion protein or polypeptide described herein)-coated target cells by immune effector cells (e.g., NK cells). As used herein, terms such as "reduction in ADCC" refer to a decrease in the number of target cells that are lysed in a given time in the medium surrounding the target cells with a given concentration of antibody (or Ig Fc-region-containing polypeptide or protein) (e.g., an Fc-region-containing fusion protein or polypeptide described herein) by the mechanism of ADCC as defined above, and / or an increase in the concentration of antibody (or Fc-region-containing polypeptide or protein) (e.g., an Fc-containing fusion protein or polypeptide described herein) in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time by the mechanism of ADCC as defined above. The reduction in ADCC is relative to the ADCC mediated by the same antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc-containing fusion protein or polypeptide described herein) produced by the same type of host cell using the same standard production, purification, formulation, and storage methods (known to those of skill in the art), but which has not been engineered (e.g., does not contain one or more amino acid modifications, e.g., amino acid substitutions, that mediate reduced ADCC). For example, the reduction in ADCC mediated by an antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc-containing fusion protein or polypeptide described herein) that contains an amino acid substitution in the Fc region that minimizes ADCC is relative to the ADCC mediated by the same antibody (or Fc region-containing polypeptide or protein) (e.g., an Fc-containing fusion protein or polypeptide described herein) that does not have said amino acid substitution in the Fc region.

[0140] As used herein, the term "affinity" refers to the strength of binding between one protein (e.g., an antibody) and another protein (e.g., an antigen). Protein affinity is measured by the dissociation constant, Kd, ​​defined as [antibody] x [antigen] / [antibody-antigen], where [antibody-antigen] is the molar concentration of the antibody-antigen complex, [antibody] is the molar concentration of unbound antibody, and [ligand] is the molar concentration of unbound antigen. The affinity constant, Ka, is defined as 1 / Kd. Standard methods for measuring affinity are known to those of skill in the art. Exemplary methods for measuring affinity are described herein. See, e.g., Sections 5.2.2 and 5.2.3.

[0141] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of immunoglobulin (Ig) (e.g., human Ig (hIg)) structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific (e.g., bispecific, trispecific) antibodies, and antibody fragments (i.e., antigen-binding fragments or variants) that exhibit the desired antigen-binding activity. Thus, the term antibody includes, for example, full-length antibodies; antigen-binding fragments of full-length antibodies; molecules comprising the CDRs, VH regions, and / or VL regions of an antibody; and antibody-like scaffolds (e.g., fibronectin). Examples of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelid antibodies, intrabodies, affibodies, diabodies, tribodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies (e.g., VHH, (VHH)2), single chain antibodies, single chain Fv (scFv; (scFv)2), Fab fragments (e.g., Fab, single chain Fab (scFab), F(ab')2 fragments, disulfide-linked Fv ( Antibodies include Fc fusions (e.g., Fab-Fc, scFv-Fc, VHH-Fc, (scFv)2-Fc, (VHH)2-Fc), and antigen-binding fragments of any of the above, and conjugates or fusion proteins comprising any of the above. The antibody may be of the Ig isotype (e.g., IgG, IgE, IgM, IgD, or IgA), and may be of any class of Ig (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b) In certain embodiments, the antibodies described herein are IgG antibodies, or subclasses thereof (e.g., human IgG1 or IgG4). In some embodiments, the antibodies are human, humanized, or chimeric IgG1 or IgG4 monoclonal antibodies. In some embodiments, the term antibody refers to a monoclonal or polyclonal antibody population. The antibodies described herein can be produced by any standard method known in the art, including, for example, recombinant production in a host cell, e.g., see Section 5.4; or synthetic production.

[0142] "Antibody-like scaffolds" are known in the art; for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen-binding domains; see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008), the entire contents of each of which are incorporated herein by reference for any purpose. Exemplary antibody-like scaffold proteins include, but are not limited to, lipocalins (anticalins), protein A-derived molecules such as the Z-domain (affibody), A-domain (avimer / maxibody) of protein A, serum transferrin (trans-body); designed ankyrin repeat proteins (DARPins), VNAR fragments, fibronectin (AdNectin), C-type lectin domains (tetranectins); variable domains of novel antigen receptor β-lactamase (VNAR fragments), human gamma-crystallin or ubiquitin (affilin molecules); Kunitz-type domains of human protease inhibitors, microbodies such as proteins from the knottin family, peptide aptamers and fibronectin (adnectin).

[0143] The term "antigen-binding domain" refers to a polypeptide or protein, or a portion of a polypeptide or protein, capable of specifically binding to an antigen. Exemplary antigen-binding domains include, but are not limited to, single-domain antibodies (e.g., VHH, (VHH)2), single-chain Fvs (e.g., scFv; (scFv)2), Fab fragments (e.g., Fab, single-chain Fab (scFab), F(ab')2), and disulfide-linked Fvs (sdFv). An antigen-binding domain may be part of a larger polypeptide or protein, e.g., a full-length antibody, an Fc fusion. In some embodiments, the antigen-binding domain is part of a full-length antibody. In some embodiments, the antigen-binding domain is operably linked to an Fc region. When an antigen-binding domain is referred to in the context of use with a target protein or polypeptide, the term "antigen" can be substituted with the name of the target protein or antigen. For example, an antigen-binding domain that specifically binds to hCAIX is also referred to herein as an "hCAIX-binding domain."

[0144] The terms "cancer" and "tumor" are used interchangeably herein to refer to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Malignant tumors form as a result of unregulated cell division and growth, and can invade adjacent tissues and metastasize to distant sites in the body, for example, via the lymphatic system or bloodstream.

[0145] As used herein, the term "CDR" or "complementarity-determining region" refers to the discontinuous antigen-binding sites found in the variable regions of both heavy and light chain polypeptides. These specific regions are described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991), the entire contents of each of which are incorporated herein by reference. Unless otherwise specified, the term "CDR" refers to the CDR defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest. (1991).

[0146] The terms "CH1" and "CH1 region" are used interchangeably herein to refer to the first constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hIgG1 CH1 region is set forth in SEQ ID NO: 119; the amino acid sequence of an exemplary reference hIgG4 CH1 region is set forth in SEQ ID NO: 132.

[0147] The terms "CH2" and "CH2 region" are used interchangeably herein to refer to the second constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hIgG1 CH2 region is set forth in SEQ ID NO: 121; the amino acid sequence of an exemplary reference hIgG4 CH2 region is set forth in SEQ ID NO: 134.

[0148] The terms "CH3" and "CH3 region" are used interchangeably herein to refer to the third constant region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hIgG1 CH3 region is set forth in SEQ ID NO: 122; the amino acid sequence of an exemplary reference hIgG4 CH3 region is set forth in SEQ ID NO: 135.

[0149] The terms "constant region" and "constant domain" are used interchangeably herein and refer to the carboxyl-terminal portions of the light and / or heavy chains of a full-length antibody that are not directly involved in binding the antibody to an antigen, but may exhibit various effector functions, such as interaction with Ig Fc receptors (e.g., Fcγ receptors). The constant regions of Ig molecules generally have more conserved amino acid sequences than Ig variable domains.

[0150] As used herein, the term "derived from" with respect to a polynucleotide refers to a polynucleotide having at least 70% (e.g., at least 85%) sequence identity to a reference polynucleotide (e.g., a naturally occurring polynucleotide) or a fragment thereof. The term "derived from" with respect to a polypeptide or protein refers to a polypeptide or protein comprising an amino acid sequence that has at least 70% (e.g., at least 85%) sequence identity to the amino acid sequence of a reference polypeptide or protein (e.g., a naturally occurring polypeptide or protein). The term "derived from," as used herein, does not refer to a particular process or method for obtaining the polynucleotide, polypeptide, or protein. For example, a polynucleotide, polypeptide, or protein can be recombinantly produced or chemically synthesized.

[0151] As used herein, the term "diagnosis" refers to the determination of the presence, absence, severity, or course of treatment of a disease (e.g., cancer, e.g., a cancer comprising cancer cells that express CAIX). The term "diagnosis" encompasses an initial determination as well as a secondary determination (e.g., follow-up) after the initial determination.

[0152] As used herein, the term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, regardless of the nature of the etiology.

[0153] The terms "hinge" or "hinge region" are used interchangeably herein to refer to the hinge region of an immunoglobulin heavy chain. The amino acid sequence of an exemplary reference hIgG1 hinge region is set forth in SEQ ID NO: 120; the amino acid sequence of an exemplary reference hIgG4 hinge region is set forth in SEQ ID NO: 133.

[0154] The terms "DNA" and "polydeoxyribonucleotide" are used interchangeably herein to refer to a polymer comprising multiple deoxyribonucleotides polymerized through phosphodiester bonds. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

[0155] The term "effector function," when used with respect to an antibody, refers to a biological activity attributable to the Fc region of the antibody and therefore varies depending on the antibody isotype. Antibody effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), Fc receptor binding (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa)), and Clq binding.

[0156] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as set out in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 5th ed., 1991 (the entire contents of each of which are incorporated herein by reference for any purpose).

[0157] As used herein, the term "Fab" refers to an antigen-binding domain comprising, from N- to C-terminus, a Fab heavy chain comprising a VH region and a CH1 region; and a light chain comprising, from N- to C-terminus, a VL region and a CL region; the Fab heavy and light chains associate to form the antigen-binding domain.

[0158] The term "Fab-Fc," as used herein, refers to an antibody comprising a Fab operably linked to an Fc region. For example, a full-length antibody comprises a first Fab operably linked to a first Fc region and a second Fab operably linked to a second Fc region.

[0159] As used herein, the term "Fc region" refers to the C-terminal region of an hIg heavy chain comprising, from N- to C-terminus, at least the CH2 region operably linked to the CH3 region. In some embodiments, the Fc region comprises an Ig hinge region or at least a portion of an Ig hinge region operably linked to the N-terminus of the CH2 region. In some embodiments, the Fc region has been engineered (e.g., contains one or more amino acid modifications) relative to a reference Fc region; see, e.g., Section 5.3.2.1. Further examples of proteins with engineered Fc regions can be found in Saunders 2019 (KO Saunders, "Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life," 2019, Frontiers in Immunology, Vol. 10, Art. 1296, pp. 1-20, the entire contents of which are incorporated herein by reference for any purpose).

[0160] As used herein, the term "Fc-modified fusion protein or polypeptide" refers to a fusion polypeptide or protein that includes an Fc region, wherein the Fc region has been modified (e.g., contains one or more amino acid modifications (e.g., one or more amino acid substitutions, deletions, or additions)).

[0161] As used herein, the terms "first" and "second," such as with respect to Fc regions, are used for convenience to distinguish between sites of each type when multiple sites of each type are present. The use of these terms is not intended to confer a particular order or orientation in the fusion protein unless explicitly stated.

[0162] As used herein, the term "framework region" or "FR region" refers to amino acid residues that are part of the variable region of an antibody but are not part of the CDRs (eg, using the Kabat definition of CDRs).

[0163] As used herein, the term "full-length antibody" refers to an antibody having a structure substantially similar to that of a naturally occurring antibody: (i) a first Ig light chain comprising, from N-terminus to C-terminus, a light chain variable (VL) region and a light chain constant (CL) region; (ii) a first Ig heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable (VH) region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (iii) a second Ig heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; and (iv) a second Ig light chain comprising, from N-terminus to C-terminus, a VL region and a VH region; the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and the first heavy chain and the second heavy chain associate to form a dimer. In some embodiments, the two heavy chains comprise substantially identical amino acid sequences; and the two light chains comprise substantially identical amino acid sequences. The amino acid sequences of the two heavy chains may differ, e.g., they may comprise one or more amino acid modifications that promote heterodimerization of the two heavy chains. In some embodiments, the two heavy chains comprise substantially identical amino acid sequences except for one or more amino acid modifications that promote heterodimerization of the appropriate heavy chains (e.g., as described herein); and the two light chains comprise substantially identical amino acid sequences. Antibody chains can be substantially identical, but not completely identical, if they differ due to post-translational modifications such as C-terminal truncation of lysine residues, alternative glycosylation patterns, etc. The amino acid sequence of any one of the chains of a full-length antibody can comprise one or more amino acid modifications relative to a reference (e.g., a wild-type antibody sequence).

[0164] The term "functional variant," as used herein with respect to a polypeptide or protein, refers to a polypeptide or protein that contains at least one, 15% or less, 12% or less, 10% or less, or 8% or less amino acid variation (e.g., substitution, deletion, addition) compared to the amino acid sequence of a reference polypeptide or protein, wherein the polypeptide or protein retains at least one specific function of the reference polypeptide or protein. Not all functions of the reference polypeptide or protein (e.g., wild-type) need be retained by a functional variant of the protein. In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein. For example, a functional variant of a hIL-12p40 polypeptide or protein can refer to a hIL-12p40 protein that contains amino acid substitutions compared to a reference hIL-12p40 protein (e.g., wild-type) that retain the ability to specifically bind to hIL-12R.

[0165] The term "functional fragment," as used herein with respect to a polypeptide or protein, refers to a fragment of a reference polypeptide or protein that retains at least one specific function. Not all functions of a reference polypeptide or protein need be retained by a functional fragment of a polypeptide or protein. In some cases, one or more functions are selectively reduced or eliminated. In some embodiments, the reference polypeptide or protein is a wild-type protein. For example, a functional fragment of hIL-12p40 can refer to a fragment of hIL-12p40 that retains the ability to specifically bind to IL-12R.

[0166] As used herein, "fusion" and its grammatical equivalents refer to the operative linkage of at least one polypeptide derived from a first polypeptide and another polypeptide derived from a second polypeptide, where the first and second polypeptides are different. The term fusion encompasses both direct linkage of at least two polypeptides via a peptide bond and indirect linkage via a linker (e.g., a peptide linker).

[0167] As used herein, "fusion polypeptide" or "fusion protein," and grammatical equivalents thereof, refer to a polypeptide or protein comprising at least one polypeptide derived from a first polypeptide operably linked to another polypeptide derived from a second polypeptide, wherein the first and second polypeptides are different. The at least two polypeptides of a fusion polypeptide or protein may be operably linked directly via a peptide bond or indirectly via a linker (e.g., a peptide linker). Thus, for example, the term fusion polypeptide encompasses embodiments in which polypeptide A is operably linked directly to polypeptide B via a peptide bond (polypeptide A-polypeptide B) and embodiments in which polypeptide A is operably linked to polypeptide B via a peptide linker (polypeptide A-peptide linker-polypeptide B).

[0168] As used herein, the term "heavy chain" refers to a portion of an immunoglobulin (e.g., human Ig) that generally comprises, from N- to C-terminus, the heavy chain variable region (VH), CH1 region, hinge region, CH2 region, and CH3 region. The constant region of the heavy chain (i.e., the CH1 region, hinge region, CH2 region, and CH3 region) can be of any of the different isotypes based on the amino acid sequence of the constant domain, such as human alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which result in human antibodies of the hIgA, hIgD, IgE, hIgG, and hIgM classes, respectively, including subclasses of hIgG, such as hIgG1, hIgG2, hIgG3, and hIgG4. As used herein, the term "heavy chain," when used in reference to a human antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to antibodies of the human IgA, IgD, IgE, IgG, and IgM classes, respectively, and include the subclasses of human IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0169] As used herein, the term "half-life extending moiety" refers to a moiety (e.g., a small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., PEG polymer), etc.) that, when conjugated or otherwise operably linked (e.g., fused) to a polypeptide or protein (a subject polypeptide or protein), extends the half-life of the subject polypeptide or protein in vitro when administered to a subject (e.g., a human subject). The pharmacokinetic properties of a polypeptide or protein can be assessed using in vitro models known in the art.

[0170] As used herein, the term "half-life extending polypeptide" or "half-life extending protein" refers to a polypeptide that, when operably linked to another polypeptide (a subject polypeptide or protein), extends the half-life of the subject polypeptide in vitro when administered to a subject (e.g., a human subject). The pharmacokinetic properties of a polypeptide or protein can be assessed using in vitro models known in the art.

[0171] As used herein, the term "heterologous," when used to describe a first element relative to a second element, means that the first element and second element are not present in the native configuration as described. For example, a polypeptide comprising a "heterologous moiety" refers to a polypeptide linked to a moiety (e.g., a small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., PEG polymer), etc.) that is not naturally linked to the polypeptide. For example, a non-limiting example of a heterologous moiety is a heterologous polypeptide (as defined herein).

[0172] Thus, as used herein, the term "heterologous signal peptide" refers to a signal peptide that is not naturally operably linked to a polypeptide or protein of interest. For example, with respect to a polypeptide comprising a signal peptide from human IL-2 (hIL-2) operably linked to hIL-12p40, the hIL-2 signal peptide constitutes a heterologous signal peptide.

[0173] As used herein, the term "cognate signal peptide" refers to the signal peptide that is naturally operably linked to a polypeptide or protein of interest. For example, with respect to a polypeptide comprising a signal peptide from human IL-2 operably linked to hIL-2, the hIL-2 signal peptide constitutes the cognate signal peptide.

[0174] The term "human carbonic anhydrase IX" or "CAIX" refers to the human carbonic anhydrase transmembrane dimeric metalloenzyme that promotes acid secretion in the gastrointestinal tract. CAIX is also referred to in the art as "carbonic anhydrase IX," "carbonic anhydrase 9," "CA9," and "CA-IX." As an exemplary reference, the amino acid sequence of the mature hCAIX protein can be found under Uniprot accession number Q16790 and is set forth herein as SEQ ID NO: 1.

[0175] As used herein, the term "human tumor-associated antigen" or "hTAA" refers to a protein expressed on the surface of human cancer cells that enables recruitment of a multispecific protein described herein to the human cancer cells. In some embodiments, the tumor-associated antigen is expressed by both normal cells and cancer cells. In some embodiments, the tumor-associated antigen is overexpressed in cancer cells compared to normal cells, e.g., greater than 1-fold, 2-fold, 3-fold, or more overexpression relative to normal cells. In some embodiments, the tumor-associated antigen is a protein that is inappropriately synthesized by cancer cells and contains, e.g., amino acid modifications (e.g., amino acid deletions, additions, and / or substitutions) compared to the protein expressed by normal cells. In some embodiments, the tumor-associated antigen is expressed only by cancer cells and not at detectable levels by normal cells. Methods for identifying and validating tumor-associated proteins are known to those skilled in the art and are described in the literature (see, for example, Bornstein, AAPS J. (2015), vol. 17(3), pp. 525-534; Hong et al., BMC Syst Biol. (2018), vol. 12 (Suppl 2), p. 17).

[0176] The term "human interleukin-12" or "hIL-12" refers to the human IL-12 protein or polypeptide.

[0177] The terms "interleukin-12" and "IL-12" are used interchangeably herein and are intended to refer to and encompass a functional IL-12 protein complex comprising the IL-12p35 subunit and the IL-12p40 subunit. In some embodiments, the IL-12p35 subunit and the IL-12p40 subunit are operably linked in a single polypeptide chain, e.g., single-chain IL-12 (scIL-12) (e.g., scIL-12 described herein). In some embodiments, the IL-12p35 subunit and the IL-12p40 subunit are not operably linked in a single polypeptide chain, but are encoded by two separate polypeptides.

[0178] The terms "single-chain IL-12" and "scIL-12" are used interchangeably herein and refer to a form of IL-12 that has been engineered to express an IL-12p40 polypeptide fused, either directly or indirectly via a peptide linker, to an IL-12p35 polypeptide, such that the IL-12p40 / IL-12p35 molecule is produced as a single polypeptide chain (i.e., a fusion polypeptide). scIL-12 can be configured in either order such that a single polypeptide is produced beginning with an IL-12p40 polypeptide as the amino-terminal ("N-terminal") portion fused, either directly or indirectly via a peptide linker, to an IL-12p35 polypeptide as the carboxyl-terminal ("C-terminal") portion of scIL-12. This configuration, when a peptide linker is utilized, can be represented by the abbreviation "IL-12p40-linker-IL-12p35." Conversely, in scIL-12 constructs, the IL-12p35 polypeptide can also be the N-terminal portion fused directly or via a peptide linker to IL-12p40 as the C-terminal portion of scIL-12, which configuration can be represented by the abbreviation "IL-12p35-linker-IL-12p40" when a peptide linker is utilized.

[0179] The term "hIL-12p35," as used herein, refers to the human α subunit of the heterodimeric IL-12 protein. The amino acid sequence of an exemplary reference IL-12p35 can be found as Uniprot Accession No. P29459 and is set forth herein as SEQ ID NO: 30. For purposes of this disclosure, the numbering of all amino acids (and, e.g., amino acid substitutions) of the hIL-12p35 polypeptides described herein is presented relative to the amino acid sequence of the immature form of hIL-12p35 (i.e., SEQ ID NO: 30), including the native signal peptide. As described herein, amino acids 1-22 of SEQ ID NO: 30 are the native signal peptide, which is cleaved in vivo to form the mature protein (SEQ ID NO: 31). The use of the immature form of hIL-12p35 to designate amino acid numbering is for consistency only and does not limit the scope of embodiments utilizing this numbering to polypeptides that include the hIL-12p35 signal peptide. For example, the hIL-12p35 polypeptide described herein as comprising the amino acid sequence of SEQ ID NO: 31 with the Y189A amino acid substitution (mature hIL-12p35) does not require the hIL-12p35 signal peptide, although the numbering of amino acid position Y189 is based on the immature form of the protein. It is common in the art to utilize mature proteins to produce variants and fusion proteins. One skilled in the art can readily determine the amino acid positions within the mature form of hIL-12p35 (SEQ ID NO: 31) based on the amino acid numbering for the immature form of hIL-12p35. As noted above, amino acids 1-22 of the immature form of hIL-12p35 protein constitute the signal sequence. Therefore, the amino acid position of a particular amino acid within the mature form of hIL-12p35 protein can be determined by subtracting 22 from the amino acid position of the particular amino acid designated for the immature form of hIL-12p35. For example, amino acid position Y189 (numbered relative to SEQ ID NO: 30) would correspond to amino acid position Y167 in the mature form of the protein (SEQ ID NO: 32).

[0180] The term "hIL-12p40," as used herein, refers to the human β subunit of the heterodimeric IL-12 protein. The amino acid sequence of an exemplary reference IL-12p40 can be found as Uniprot Accession No. P29460 and is set forth herein as SEQ ID NO: 32. For purposes of this disclosure, the numbering of all amino acids (and, e.g., amino acid substitutions) of the hIL-12p40 polypeptides described herein is presented relative to the amino acid sequence of the immature form of hIL-12p40 (i.e., SEQ ID NO: 32), including the native signal peptide. As described herein, amino acids 1-22 of SEQ ID NO: 32 are the native signal peptide, which is cleaved in vivo to form the mature protein (SEQ ID NO: 33). The use of the immature form of hIL-12p40 to designate amino acid numbering is for consistency only and does not limit the scope of embodiments utilizing this numbering to polypeptides that include the hIL-12p40 signal peptide. For example, the hIL-12p40 polypeptide described herein as comprising the amino acid sequence of SEQ ID NO: 33 with the W37A amino acid substitution (mature hIL-12p40) does not require the hIL-12 signal peptide, although the numbering of amino acid position W37 is based on the immature form of the protein. It is common in the art to utilize mature proteins to produce variants and fusion proteins. One of skill in the art can readily determine the amino acid position within the mature hIL-12p40 (SEQ ID NO: 33) based on the amino acid numbering for the immature form of hIL-12p40. As noted above, amino acids 1-22 of the immature hIL-12p40 protein constitute a signal sequence. Therefore, the amino acid position of a particular amino acid within the mature hIL-12p40 protein can be determined by subtracting 22 from the amino acid position of the particular amino acid designated for the immature form of hIL-12p40. For example, amino acid position W37 (numbered relative to SEQ ID NO: 32) corresponds to amino acid position W15 of the mature form of the protein (SEQ ID NO: 33).

[0181] As used herein, the term "isolated" with respect to a polypeptide, protein, or polynucleotide refers to a polypeptide, protein, or polynucleotide that is substantially free from other cellular components or other contaminants with which it is associated in nature.

[0182] As used herein, the term "Kabat numbering system" refers to the Kabat numbering convention for antibody variable regions. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Services, 5th ed., 1991, the entire contents of which are incorporated herein by reference for all purposes. Unless otherwise specified, the numbering of antibody variable regions is represented by the Kabat numbering system.

[0183] As used herein, the term "linker" refers to a linkage between two elements (e.g., polypeptide or protein domains). A linker can be a covalent bond or a peptide linker. The term "bond" refers to a chemical bond (e.g., an amide bond, a disulfide bond, or any type of bond resulting from a chemical reaction (e.g., chemical conjugation)). The term "peptide linker" refers to an amino acid or polypeptide that can be used to link two polypeptide or protein domains. In some embodiments, a peptide linker can be used to provide space and / or flexibility between two polypeptide or protein domains.

[0184] As used herein, the term "light chain" refers to a portion of an immunoglobulin (e.g., a human immunoglobulin) that comprises, from N- to C-terminus, a light chain variable region (VL), operably linked to a light chain constant region (CL). The CL can be of any of different types, such as, for example, kappa (κ) or gamma (λ), based on the amino acid sequence of the CL. In some embodiments, the multispecific proteins described herein comprise one or more light chains.

[0185] As used herein, the term "messenger RNA" or "mRNA" refers to any RNA that encodes at least one peptide or protein and can be translated in vitro, in vitro, in situ, or ex vivo to produce the encoded peptide or protein.

[0186] As used herein, the term "modified" with respect to a polynucleotide refers to a polynucleotide that contains at least one nucleotide substitution, alteration, inversion, addition, or deletion (e.g., one or more amino acid substitutions) relative to a reference polynucleotide. A modification may include the inclusion of a non-naturally occurring nucleotide residue. As used herein, the term "modified" with respect to an amino acid sequence refers to an amino acid sequence that contains at least one amino acid substitution, alteration, inversion, addition, or deletion relative to a reference amino acid sequence. A modification may include the inclusion of a non-naturally occurring amino acid residue. Naturally occurring amino acid derivatives are not considered modified amino acids for purposes of determining the percent identity of two amino acid sequences. For example, a naturally occurring modification of a glutamic acid amino acid residue to a pyroglutamic acid amino acid residue is not considered an amino acid modification for purposes of determining the percent identity of two amino acid sequences. Furthermore, for example, a naturally occurring modification of a glutamic acid amino acid residue to a pyroglutamic acid amino acid residue is not considered an amino acid "modification" as defined herein.

[0187] A "modification that promotes heterodimer formation between a first Fc region and a second Fc region" (or similar phrases) refers to a manipulation of the peptide backbone or post-translational modification of an Fc region that reduces or prevents the association of a polypeptide comprising the Fc region with an identical polypeptide to form a homodimer. As used herein, a modification that promotes association particularly includes separate modifications made to each of the two Fc regions (i.e., the first Fc region and the second Fc region) whose association is desired, where the modifications are complementary to each other so as to promote the association of the two Fc regions. For example, a modification that promotes association may alter the structure or charge of one or both of the Fc regions to sterically or electrostatically favor their association, respectively. Thus, heterodimerization occurs between a polypeptide comprising a first Fc region and a polypeptide comprising a second Fc region, which may be non-identical in the sense that the additional components (e.g., antigen-binding domains) fused to each of the Fc regions are not the same. In some embodiments, a modification that promotes association includes amino acid mutations, particularly amino acid substitutions, in the Fc region. In certain embodiments, the association-promoting modification comprises additional amino acid mutations, particularly one or more amino acid substitutions, in each of the first and second Fc regions. See, e.g., Section 5.3.2.2.

[0188] As used herein, the term "moiety" is used generally to refer to any macromolecule or micromolecule that can be operably linked to a polypeptide or protein described herein. Exemplary moieties include, but are not limited to, small molecules, polypeptides, polynucleotides (e.g., DNA, RNA), carbohydrates, lipids, synthetic polymers (e.g., PEG polymers).

[0189] As used herein, the term "operably linked" refers to the linkage of two moieties (e.g., two polypeptides or two polynucleotides) in a functional relationship. For example, a polypeptide is operably linked to another polypeptide when they are linked in frame (directly or indirectly via a peptide linker) such that both polypeptides are functional (e.g., a fusion protein or polypeptide described herein). Or, for example, a transcriptional regulatory polynucleotide, such as a promoter, enhancer, or other expression control element, is operably linked to a polynucleotide encoding a protein if it affects the transcription of the polynucleotide encoding the protein. The term "operably linked" can also refer to the conjugation of a moiety to, for example, a polynucleotide or polypeptide (e.g., the conjugation of a PEG polymer to a protein or polypeptide).

[0190] The determination of "percent identity" between two sequences (e.g., peptides or proteins (amino acid sequences) or polynucleotides (nucleic acid sequences)) can be achieved using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264-2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which are incorporated herein by reference in their entirety. BLAST nucleotide searches can be performed, for example, using the NBLAST nucleotide program parameters set to score=100 and word length=12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameters set, for example, to a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, the entire contents of which are incorporated herein by reference. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically, only exact matches are counted.

[0191] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to an animal, e.g., a human subject, comprising a therapeutic agent and a pharmaceutically acceptable carrier or diluent. A "pharmaceutically acceptable carrier or diluent" means a material for use in contact with the tissues of humans and / or non-human animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit / risk ratio.

[0192] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to refer to a polymer of DNA or RNA. Nucleic acid molecules can be single- or double-stranded; contain natural, non-natural, or modified nucleotides; and contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages instead of the phosphodiester linkages found between nucleotides in unmodified nucleic acid molecules. Nucleic acid molecules include all nucleic acid molecules obtained by any means available in the art, including, but not limited to, recombinant means, such as cloning nucleic acid molecules from recombinant libraries or cellular genomes using conventional cloning techniques and the polymerase chain reaction, as well as synthetic means. Those skilled in the art will understand that, unless otherwise specified, the nucleic acid sequences presented herein show thymidine (T) in representative DNA sequences, but when the sequences represent RNA (e.g., mRNA), thymidine (T) is substituted with uracil (U). Thus, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number may also include the corresponding RNA (e.g., mRNA) sequence encoded by that DNA in which each thymidine (T) in the DNA sequence is replaced with uracil (U).

[0193] As used herein, the term "polypeptide" refers to a polymer of at least two (e.g., at least five) amino acids linked by peptide bonds. The term "polypeptide" does not refer to a polymer chain of amino acids of a specific length. It is common in the art to refer to shorter polymers of amino acids (e.g., about 2 to 50 amino acids) as peptides and longer polymers of amino acids (e.g., more than about 50 amino acids) as polypeptides. However, the terms "peptide" and "polypeptide" are used interchangeably herein.

[0194] As used herein, the term "protein" refers to a polypeptide or a set (i.e., at least two) polypeptides. In embodiments where a protein comprises a set of polypeptides, the set of polypeptides associate to form a functional unit (i.e., a quaternary structure). In some embodiments, a polypeptide or set of polypeptides folds into a three-dimensional structure (i.e., a tertiary or quaternary structure). Where a polypeptide or set of polypeptides is contemplated herein, it should be understood that a protein comprising a polypeptide or set of polypeptides folded into a three-dimensional structure (i.e., a tertiary or quaternary structure) is also provided herein, and vice versa.

[0195] A "prophylactic" treatment is a treatment administered to a subject who shows no signs of disease, or who shows only early signs of disease, for the purpose of reducing the risk of developing the condition.

[0196] The terms "RNA" and "polyribonucleotide" are used interchangeably herein to refer to a polymer comprising multiple ribonucleotides polymerized through phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar is ribose. RNA may also contain modified nucleotides; including natural, non-natural, or altered internucleotide linkages, such as phosphoramidate or phosphorothioate linkages instead of the phosphodiester found between nucleotides in unmodified nucleic acid molecules.

[0197] As used herein, the term "sample" encompasses various biological specimens obtained from a subject. Exemplary sample types include, for example, blood and other liquid samples of biological origin (including, but not limited to, whole blood, peripheral blood mononuclear cells (PBMCs), serum, plasma, urine, saliva, amniotic fluid, feces, synovial fluid, etc.), nasopharyngeal swabs, solid tissue samples such as biopsies (or cells derived therefrom and their progeny), tissue cultures (or cells derived therefrom and their progeny), and cell cultures (or cells derived therefrom and their progeny). The term also includes samples that have been manipulated in any way after being obtained from a subject, such as by centrifugation, filtration, washing, precipitation, dialysis, chromatography, lysis, reagent treatment, enrichment for specific cell populations, refrigeration, freezing, staining, etc.

[0198] The term "scFv" or "single-chain variable fragment" refers to an antibody comprising a VH region operably linked to a VL region via a peptide linker, wherein the VH and VL regions associate to specifically bind to an antigen (e.g., form an antigen-binding domain). In some embodiments, an scFv comprises, from N- to C-terminus, a VH region, a peptide linker, and a VL region. In some embodiments, an scFv comprises, from N- to C-terminus, a VL region, a peptide linker, and a VH region.

[0199] The term "(scFv)2," as used herein, refers to an antibody comprising a first scFv operably linked to a second scFv (e.g., via a peptide linker). The first scFv and second scFv may specifically bind to the same or different antigens. In some embodiments, the first scFv and second scFv are operably linked via a peptide linker.

[0200] The term "scFv-Fc," as used herein, refers to an antibody comprising an scFv operably linked to an Fc region (e.g., via a peptide linker). In some embodiments, the scFv is operably linked only to the first Fc region of a protein comprising a first Fc region and a second Fc region. In some embodiments, the first scFv is operably linked to the first Fc region and the second scFv is operably linked to the second Fc region of a protein comprising a first Fc region and a second Fc region.

[0201] The term "(scFv)2-Fc," as used herein, refers to an (scFv)2 operably linked to an Fc region (e.g., via a peptide linker). In some embodiments, the (scFv)2 is operably linked only to the first Fc region of a protein comprising a first Fc region and a second Fc region. In some embodiments, the first (scFv)2 is operably linked to the first Fc region and the second (scFv)2 is operably linked to the second Fc region of a protein comprising a first Fc region and a second Fc region.

[0202] As used herein, the term "single domain antibody" or "sdAb" refers to an antibody having a single monomeric variable antibody domain. An sdAb can specifically bind to a particular antigen. A VHH (as defined herein) is an example of an sdAb.

[0203] As used herein, the term "signal peptide" or "signal sequence" refers to a sequence (e.g., an amino acid sequence) that can direct the transport or localization of a protein to a particular organelle, cellular compartment, or export out of a cell. The term encompasses both signal sequence peptides and nucleic acid sequences that encode signal peptides. Thus, when referring to a signal peptide in the context of a nucleic acid, it refers to the nucleic acid sequence that encodes the signal peptide.

[0204] As used herein, the term "specifically binds" refers to a preferential interaction, i.e., a significantly higher binding affinity, between a first protein (e.g., a ligand) and a second protein (e.g., the ligand's cognate receptor) compared to other amino acid sequences. Thus, when a first protein or polypeptide "specifically binds" to a second protein or polypeptide, it is understood that the first protein or polypeptide specifically binds to an epitope of the second protein or polypeptide. The term "epitope" refers to the portion of the second protein or polypeptide that the first protein or polypeptide specifically recognizes. The term "specifically binds" includes molecules that cross-react with the same epitope in different species. For example, an antibody that specifically binds to human CAIX may cross-react with CAIX from another species (e.g., cynomolgus monkey, mouse, etc.), but is still considered herein to specifically bind to human CAIX.

[0205] As used herein, the term "subject" includes any animal, such as a human or other animal. In some embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In some embodiments, the subject is a human. In some embodiments, the subject of the method is a non-human mammal. In some embodiments, the subject is a non-human mammal, such as a non-human primate (e.g., a monkey, an ape), an ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horse, donkey), a carnivore (e.g., a dog, a cat), a rodent (e.g., a rat, a mouse), or a lagomorph (e.g., a rabbit). In some embodiments, the subject is a bird, such as a member of the avian taxon Galliformes (e.g., chickens, turkeys, pheasants, quails), Anseriformes (e.g., ducks, geese), Paleognathiformes (e.g., ostriches, emus), Columbiformes (e.g., doves, pigeons), or Psittaciformes (e.g., parrots).

[0206] As used herein, the term "therapeutically effective amount of a therapeutic agent," when used alone or in combination with another therapeutic agent, refers to any amount of a therapeutic agent that protects a subject from developing a disease or promotes disease regression as evidenced by a reduction in the severity of symptoms of the disease or infection, an increase in the frequency and duration of symptom-free disease or infection, or prevention of disability or incapacity due to contraction of the disease or infection. The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to those skilled in the art, such as assaying the activity of an agent in human subjects in clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0207] As used herein, the term "treat / treating / treatment" refers to alleviating or ameliorating a disease and / or its associated symptoms, or achieving a desired pharmacological and / or physiological effect. It is understood that, although not exclusive, treating a disease does not require that the disease or its associated symptoms be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, eliminates, eliminates, attenuates, alleviates, reduces the intensity of, or cures the disease and / or adverse symptoms resulting from the disease. In some embodiments, the effect is prophylactic, i.e., the effect protects against or prevents the occurrence or recurrence of the disease. To this end, the methods of the present disclosure comprise administering a therapeutically effective amount of a composition as described herein.

[0208] As used herein, the term "variable region" refers to a portion of an antibody, typically a portion of either the light or heavy chain, typically the amino-terminal 110-120 or 110-125 amino acids in a mature heavy chain and 90-115 amino acids in a mature light chain. These regions vary extensively in sequence among antibodies and are responsible for the binding and specificity of a particular antibody to a particular antigen. Sequence variation is concentrated in regions called complementarity-determining regions (CDRs), while the more conserved regions of the variable domain are called framework regions (FRs). While not wishing to be bound by a particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antigen-antibody interaction and specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FR).

[0209] The terms "VL" and "VL region" are used interchangeably to refer to an immunoglobulin light chain variable region. A VL region can be incorporated into an antibody, such as an scFv, Fab, or full-length antibody. For example, an scFv comprises a VL region operably linked to a VH region via a peptide linker.

[0210] The terms "VH" and "VH region" are used interchangeably to refer to an immunoglobulin heavy chain variable region. A VH region can be incorporated into an antibody, such as an scFv, Fab, or full-length antibody. For example, an scFv contains a VH region operably linked to a VL region via a peptide linker.

[0211] The term "VHH" as used herein refers to a single domain antibody (sdAb) type having a single monomeric heavy chain variable antibody domain (VH). Such antibodies can be found in or produced from camelids (e.g., camels, llamas) that naturally lack light chains, or can be produced synthetically.

[0212] The term "(VHH)2," as used herein, refers to an antibody comprising a first VHH operably linked to a second VHH (e.g., via a peptide linker). The first VHH and second VHH may specifically bind to the same or different antigens. In some embodiments, the first VHH and second VHH are operably linked by a peptide linker.

[0213] The term "VHH-Fc," as used herein, refers to an antibody comprising a VHH operably linked to an Fc region (e.g., via a peptide linker). In some embodiments, the VHH is operably linked only to the first Fc region of a protein comprising a first Fc region and a second Fc region. In some embodiments, the first VHH is operably linked to the first Fc region and the second VHH is operably linked to the second Fc region of a protein comprising a first Fc region and a second Fc region.

[0214] The term "(VHH)2-Fc," as used herein, refers to a (VHH)2 operably linked to an Fc region (e.g., via a peptide linker). In some embodiments, the (VHH)2 is operably linked only to the first Fc region of a protein comprising a first Fc region and a second Fc region. In some embodiments, the first (VHH)2 is operably linked to the first region and the second (VHH)2 is operably linked to the second Fc domain of a protein comprising a first Fc region and a second Fc region.

[0215] 5.1.1 Humanized Anti-CAIX Antibodies Human CAIX (hCAIX) is a transmembrane dimeric metalloenzyme with an extracellular active site that promotes gastrointestinal acid secretion and is one of 14 carbonic anhydrase isoforms found in humans. The amino acid sequences of exemplary mature (SEQ ID NO: 1) and immature (SEQ ID NO: 2) reference hCAIX polypeptides are shown in Table 1. The N-terminal amino acids 1-37 (underlined) of SEQ ID NO: 2 represent a signal peptide.

[0216] [Table 1]

[0217] In one aspect, the present specification provides antibodies (and their functional fragments and variants (e.g., their antigen-binding domains)) that specifically bind to hCAIX, and such antibodies are also referred to herein as anti-CAIX antibodies.

[0218] The amino acid sequences of the VH and VL regions of exemplary anti-hCAIX antibodies are shown in Table 2.

[0219] [Table 2] TIFF2024534468000004.tif222169TIFF2024534468000005.tif16169

[0220] In some embodiments, the anti-hCAIX antibody (or functional fragment or variant thereof) comprises a VH and a VL.

[0221] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any VH polypeptide shown in Table 2; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any VL polypeptide shown in Table 2.

[0222] In some embodiments, the amino acid sequence of the VH comprises the amino acid sequence of any VH polypeptide shown in Table 2; and the amino acid sequence of the VL comprises the amino acid sequence of any VL polypeptide shown in Table 2.

[0223] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3-9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10-17.

[0224] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:3; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:3; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:3; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:12. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:3; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:13.In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:3; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:14. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:3; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:15. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:3; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:16. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:3; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:17.

[0225] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:4; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:10. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:4; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:11. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:4; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:12. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:4; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13.In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:4; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:14. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:4; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:15. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:4; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:16. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:4; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:17.

[0226] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:5; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:10. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:5; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:11. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:5; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:12. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:5; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:13.In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:5; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:14. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:5; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:15. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:5; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:16. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:5; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in SEQ ID NO:17.

[0227] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13.In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:6; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:17.

[0228] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13.In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:7; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:17.

[0229] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13.In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:8; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:17.

[0230] In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:12. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13.In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:17.

[0231] The nucleotide sequences of the VH and VL regions of exemplary anti-hCAIX antibodies are shown in Table 3.

[0232] [Table 3] TIFF2024534468000007.tif203170

[0233] In some embodiments, the VH nucleotide sequence comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any VH shown in Table 3; and the VL nucleotide sequence comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of any VL shown in Table 3.

[0234] In some embodiments, the nucleotide sequence of VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 18-23; and the nucleotide sequence of VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 24-29.

[0235] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:23; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:27.

[0236] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 18; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:24.

[0237] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 18; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:25.

[0238] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 18; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:26.

[0239] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 18; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:27.

[0240] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 18; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 28.

[0241] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 18; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:29.

[0242] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 19; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:24.

[0243] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 19; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:25.

[0244] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 19; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:26.

[0245] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 19; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:27.

[0246] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 19; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:28.

[0247] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 19; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 29.

[0248] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:20; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:24.

[0249] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:20; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:25.

[0250] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:20; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:26.

[0251] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:20; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:27.

[0252] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:20; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:28.

[0253] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:20; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:29.

[0254] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:21; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:24.

[0255] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:21; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:25.

[0256] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:21; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:26.

[0257] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:21; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:27.

[0258] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:21; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:28.

[0259] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:21; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:29.

[0260] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:22; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:24.

[0261] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:22; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:25.

[0262] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:22; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:26.

[0263] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:22; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:27.

[0264] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:22; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:28.

[0265] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:22; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:29.

[0266] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:23; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:24.

[0267] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:23; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:25.

[0268] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:23; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:26.

[0269] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:23; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:27.

[0270] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:23; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:28.

[0271] In some embodiments, the nucleotide sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:23; and the nucleotide sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence set forth in SEQ ID NO:29.

[0272] 5.2 hIL-12 Proteins and Polypeptides hIL-12 is a pleiotropic secreted cytokine composed of an α subunit, hIL-12p35, and a β subunit, hIL-12p40. The native hIL-12p35 and hIL-12p40 subunits are linked via disulfide bonds to form the physiologically active hIL12-p70 cytokine. See, for example, Sun, Lin et al. "Interleukin 12 (IL-12) family cytokines: Role in immune pathogenesis and treatment of CNS autoimmune disease," Cytokine, 75(2): 249-55 (2015) (hereinafter referred to as "Sun 2015"), the entire contents of which are incorporated herein by reference for any purpose. hIL-12 is, inter alia, proinflammatory and mediates its function via binding to the hIL-12 receptor (hIL-12R). The high-affinity hIL-12R is a heterodimer containing the hIL-12Rβ1 and hIL-12Rβ2 subunits. hIL-12R is constitutively or inducibly expressed in various immune cells, including NK cells, T cells, and B cells. When hIL-12 binds to hIL-12R expressed on activated T cells, NK cells, and DCs, for example, the TYK2, JAK2, and STAT pathways are activated. Among the STAT family of transcription factors, STAT4 is considered to be the most specific mediator of hIL-12-induced cellular responses. (See, e.g., Sun 2015).

[0273] The amino acid sequences of the reference immature and mature hIL-12p35 polypeptides are set forth in SEQ ID NOs: 30 and 31, respectively. The amino acid sequences of the reference immature and mature hIL-12p40 polypeptides are set forth in SEQ ID NOs: 32 and 33, respectively. The amino acid sequences of the reference immature and mature hIL-12Rβ1 (hIL-12Rβ1) polypeptides are set forth in SEQ ID NOs: 34 and 35, respectively. The amino acid sequences of the reference immature and mature hIL-12Rβ2 (hIL-12Rβ12) polypeptides are set forth in SEQ ID NOs: 36 and 37, respectively. See Table 4 herein.

[0274] [Table 4] TIFF2024534468000009.tif230168TIFF2024534468000010.tif31168

[0275] 5.2.1 hIL-12p40 Proteins and Polypeptides In one aspect, provided herein is a hIL-12p40 polypeptide that has reduced activity relative to a reference hIL-12p40 polypeptide (e.g., SEQ ID NO: 33). As described herein, any of the hIL-12p40 polypeptides described herein can be isolated and / or recombinant. In some embodiments, a hIL-12p40 polypeptide specifically binds to hIL-12R. In some embodiments, a hIL-12p40 polypeptide specifically binds to hIL-12Rβ1 (see also Section 5.2.3). In some embodiments, a hIL-12p40 polypeptide specifically binds to hIL-12Rβ2 (see also Section 5.2.2).

[0276] As noted above, for purposes of this disclosure, the numbering of all amino acids (and, e.g., amino acid substitutions) in the hIL-12p40 polypeptides described herein is shown relative to the amino acid sequence of the immature form of hIL-12p40, including the native hIL-12p40 signal peptide (i.e., SEQ ID NO: 32). The use of the immature form of hIL-12p40 to designate an amino acid number (e.g., W37) is for consistency only and does not limit the scope of embodiments utilizing this numbering system to polypeptides that include the hIL-12p40 signal peptide. For example, a hIL-12p40 polypeptide described herein that includes the amino acid sequence of SEQ ID NO: 33 with the W37A amino acid substitution does not require the hIL-12p40 signal peptide, although the numbering of amino acid position W37 is based on the immature form of the protein. It is common in the art to utilize mature forms of proteins to produce variants and fusion proteins.

[0277] Those skilled in the art can readily determine the amino acid positions within the mature hIL-12p40 (SEQ ID NO: 33) based on the amino acid numbering relative to the immature hIL-12p40. As noted above, amino acids 1-22 of the immature hIL-12p40 protein are a signal sequence (underlined). Therefore, the amino acid position of a particular amino acid within the mature hIL-12p40 protein can be determined by subtracting 22 from the amino acid position designated for the immature hIL-12p40. For example, amino acid position W37 (numbered relative to SEQ ID NO: 32) corresponds to amino acid position W15 of the mature protein (SEQ ID NO: 33).

[0278] In one aspect, provided herein is a fragment of a nucleotide sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) at amino acid positions (i) W37, F82, and K219; (ii) W37, F82, and K217; (iii) K106, K217, and K219; (iv) W37 and F82; (v) W37 and K217; (vi) W37 and K219; (vii) W37 and K106; (viii) F82 and K106; (xiv) F82 and K217; (xv) F82 and K219; (xvi) K217 and K219; (xvii) K106 and K217; or (xviii) K106 and K219 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0279] In some embodiments, the hIL-12p40 polypeptide is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) at amino acid positions (i) W37, F82, and K219; (ii) W37, F82, and K217; (iii) K106, K217, and K219; (iv) W37 and F82; (v) W37 and F82; and K217; (vi) W37 and K219; (vii) W37 and K106; (viii) F82 and K106; (xiv) F82 and K217; (xv) F82 and K219; (xvi) K217 and K219; (xvii) K106 and K217; or (xviii) K106 and K219 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32). In some embodiments, each of the amino acid substitutions is a substitution of the native amino acid residue with alanine.

[0280] In some embodiments, the hIL-12p40 polypeptide is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) has the following amino acid substitutions: (i) W37A, F82A, and K219A; (ii) W37A, F82A, and K217A; (iii) K106A, K217A, and K219A; (iv) W37A and F82A; (v) W37A and F82A; (xvi) K217A and K219A; (xvii) K106A and K217A; or (xviii) K106A and K219A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0281] In some embodiments, the hIL-12p40 polypeptide comprises or consists of an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) comprises or consists of an amino acid modification (e.g., substitution) at each of amino acid positions (i) W37, F82, and K219 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0282] In some embodiments, the hIL-12p40 polypeptide comprises or consists of an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) comprises or consists of each of the following amino acid substitutions:(i) W37A, F82A, and K219A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0283] The amino acid sequences of exemplary hIL-12p40 polypeptides described herein are shown in Table 5.

[0284] [Table 5] TIFF2024534468000012.tif230169TIFF2024534468000013.tif225169TIFF2024534468000014.tif229169TIFF2024534468000015.tif167169

[0285] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the amino acid sequence of a polypeptide set forth in Table 5. In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 38-65. In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38.

[0286] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions (relative to the amino acid sequence of SEQ ID NO:33) set forth in the amino acid sequence of any one of the polypeptides set forth in Table 5; and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 5.

[0287] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions (relative to the amino acid sequence of SEQ ID NO: 33) set forth in the amino acid sequence of any one of SEQ ID NOs: 38-65; and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-65.

[0288] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions (relative to the amino acid sequence of SEQ ID NO:33) set forth in the amino acid sequence of SEQ ID NO:38; and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO:38.

[0289] 5.2.2 Potency and Affinity of hIL-12p40 Proteins and Polypeptides In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates a lower increase in the level of STAT4 in cells expressing hIL-12R on their surface than the increase in STAT4 mediated by an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)). In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates a lower increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0290] In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates an increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)). In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates an increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is at least about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)). In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates an increase in levels of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is about 0.5-1000 fold, 0.5-100 fold, 0.5-10 fold, 0.5-5 fold, 0.5-2 fold, 1-1000 fold, 1-100 fold, 1-10 fold, 1-5 fold, 1-2 fold, 10-1000 fold, or 100-1000 fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0291] Suitable assays for measuring the EC50 of the hIL-12p40 polypeptides described herein are standard and well known to those of skill in the art. For example, the EC50 can be determined by generating a dose-response curve and examining the effect of various concentrations of hIL-12p40 protein or polypeptide in inducing activity in a particular functional assay (e.g., STAT4 signaling, STAT4 phosphorylation, STAT4-inducible SEAP expression (see, e.g., Example 4)). Section 6.4 describes an exemplary method for determining the EC50 of the hIL-12p40 polypeptides or proteins described herein (including the hIL-12 fusion proteins described herein) utilizing the hIL-12 HEKBlue reporter cell line (InvivoGen #hkb-IL12). The hIL-12 HEKBlue reporter cell line expresses the hIL-12Rβ1 and hIL-12Rβ2 subunits, human STAT4, and the STAT4-inducible SEAP (secreted embryonic alkaline phosphatase) reporter. Binding of the protein to hIL-12R thereby initiates JAK2 / STAT4 signaling and subsequent production of SEAP, which can be quantified using standard methods known in the art. Further, for example, the level of phosphorylated STAT4 can be assessed by contacting cells expressing hIL-12R with one or more concentrations of the hIL-12p40 protein or polypeptide described herein, lysing the cells, and assessing the level of phosphorylated STAT4, for example, by ELISA, Western blot, FRET-based assay, or chemiluminescence assay (e.g., ELISA-based assay). Cells in cell-based assays can be cells such as HEK293 cells recombinantly expressing hIL-12R and / or human STAT4; or cells naturally expressing hIL-12R and human STAT4.

[0292] In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates an increase in the level of interferon gamma (IFN-γ) produced by expressing hIL-12R on its surface that is less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0293] In some embodiments, the hIL-12p40 protein, when combined with the hIL-12p35 protein, mediates an increase in the level of IFN-γ produced by cells expressing hIL-12R on their surface that is 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12p40 protein (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0294] In some embodiments, the hIL-12p40 protein, when combined with the hIL-12p35 protein, mediates an increase in the level of IFN-γ produced by cells expressing hIL-12R on their surface that is at least about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in the level of IFN-γ produced in the presence of a suitable control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0295] In some embodiments, the hIL-12p40 protein, when combined with the hIL-12p35 protein, mediates an increase in the level of IFN-γ produced by cells expressing hIL-12R on their surface that is about 0.5-1000-fold, 0.5-100-fold, 0.5-10-fold, 0.5-5-fold, 0.5-2-fold, 1-1000-fold, 1-100-fold, 1-10-fold, 1-5-fold, 1-2-fold, 10-1000-fold, or 100-1000-fold less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

[0296] Suitable assays for measuring the level of a protein (e.g., IFN-γ) produced from cultured cells are standard and known to those of skill in the art. For example, the level of IFN-γ can be determined using an enzyme-linked immunosorbent assay (ELISA) (see, e.g., Example 5). Section 6.5 describes an exemplary method for determining the level of IFN-γ produced from cultured cells treated with a hIL-12p40 protein described herein or a reference hIL-12p40 protein.

[0297] In some embodiments, the hIL-12p40 protein binds to IL-12Rβ1 with lower affinity than a reference hIL-12p40 protein (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)). Binding affinity can be measured by standard assays known in the art. For example, binding affinity can be measured by surface plasmon resonance (SPR) (e.g., a BIAcore®-based assay), a common method known in the art (see, e.g., Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 55:2560, 1993; and U.S. Pat. Nos. 5,283,173 and 5,468,614, the entire contents of each of which are incorporated herein by reference for any purpose). SPR measures the change in molecular concentration at a sensor surface as molecules bind to and dissociate from the surface. The change in SPR signal is directly proportional to the change in mass concentration near the surface, allowing the binding kinetics between two molecules (e.g., proteins) to be measured. The dissociation constant of the complex can be determined by monitoring the change in refractive index versus time as buffer is passed over the chip.

[0298] Other suitable assays for measuring binding of one protein to another (e.g., binding of a protein described herein to hIL-12Rβ1) include, for example, immunoassays such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs), or determining binding by monitoring changes in the spectroscopic or optical properties of the proteins via fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blots, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, and other methods for detecting protein binding.

[0299] 5.2.3 hIL-12p35 Proteins and Polypeptides In one aspect, provided herein is a hIL-12p35 polypeptide that has reduced activity relative to a reference hIL-12p35 polypeptide (e.g., SEQ ID NO: 31). As described herein, any of the hIL-12p35 polypeptides described herein can be isolated and / or recombinant. In some embodiments, a hIL-12p35 polypeptide specifically binds to hIL-12R. In some embodiments, a hIL-12p35 polypeptide specifically binds to hIL-12Rβ1 (see also Section 5.2.3). In some embodiments, a hIL-12p35 polypeptide specifically binds to hIL-12Rβ2 (see also Section 5.2.3).

[0300] As noted above, for purposes of this disclosure, the numbering of all amino acids (and, e.g., amino acid substitutions) in the hIL-12p35 polypeptides described herein is shown relative to the amino acid sequence of the immature form of hIL-12p35 (i.e., SEQ ID NO: 30), including the native signal peptide. The use of the immature form of hIL-12p35 to designate an amino acid number (e.g., Y189) is for consistency only and does not limit the scope of embodiments utilizing this numbering system to polypeptides that include the hIL-12p35 signal peptide. For example, a hIL-12p35 polypeptide described herein that includes the amino acid sequence of SEQ ID NO: 31 with the Y189A amino acid substitution does not require the hIL-12p35 signal peptide, although the numbering of amino acid position Y189 is based on the immature form of the protein. It is common in the art to utilize mature forms of proteins to produce variants and fusion proteins.

[0301] Those skilled in the art can readily determine the amino acid positions within the mature form of hIL-12p35 (SEQ ID NO: 31) based on the amino acid numbering for the immature form of hIL-12p35. As noted above, amino acids 1-22 of the immature form of hIL-12p35 protein are a signal sequence. Therefore, the amino acid position of a particular amino acid within the mature form of hIL-12p35 protein can be determined by subtracting 22 from the amino acid position of that particular amino acid designated for the immature form of hIL-12p35. For example, amino acid position Y189 (numbered relative to SEQ ID NO: 30) corresponds to amino acid position Y167 of the mature form of the protein (SEQ ID NO: 31).

[0302] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31; and (b) comprises or consists of an amino acid modification (e.g., substitution, addition, deletion (e.g., substitution)) at one or more of the following amino acid positions: E60, F61, P63, K150, F188, Y189A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0303] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31; and (b) comprises or consists of an amino acid modification (e.g., substitution, addition, deletion (e.g., substitution)) at each of the following amino acid positions: (i) F188; (ii) Y189; (iii) F188 and Y189; or (iv) E60, F61, P63, K150, and F188 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0304] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31; and (b) comprises or consists of one or more of the following amino acid substitutions: E60K, F61H, P63S, K150H, F188P, F188A, and / or Y189A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0305] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31; and (b) comprises or consists of each of the following amino acid substitutions: (i) F188A; (ii) Y189A; (iii) F188A and Y189A; or (iv) E60K, F61H, P63S, K150H, and F188P (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0306] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31; and (b) amino acids A55 to K9 2, N50~K92, M51~K92, L52~K92, Q53~K92, K54~K92, N50~N93, M51~N93, L52~N93, Q53~N93, K54~N9 3, N50~E94, M51~E94, L52~E94, Q53~E94, K54~E94, N50~S95, M51~S95, L52~S95, Q53~S95, K54~S9 5, N50~C96, M51~C96, L52~C96, Q53~C96, K54~C96, N50~L97, M51~L97, L52~L97, Q53~L97, K54~L9 7, N50~P87, M51~P87, L52~P87, Q53~P87, K54~P87, N50~L88, M51~L88, L52~L88, Q53~L88, K54~L8 8, N50~E89, M51~E89, L52~E89, Q53~E89, K54~E89, N50~L90, M51~L90, L52~L90, Q53~L90, K54~L9 0, N50~T91, M51~T91, L52~T91, Q53~T91, K54~T91, R56~K92, Q57~K92, T58~K92, L59~K92, E60~K92A55~N93, R56~N93, Q57~N93, T58~N93, L59~N93, E60~N93, A55~E94, R56~E94, Q5 7~E94, T58~E94, L59~E94, E60~E94, A55~S95, R56~S95, Q57~S95, T58~S95, L59~ S95, E60~S95, A55~C96, R56~C96, Q57~C96, T58~C96, L59~C96, E60~C96, A55~L9 7, R56~L97, Q57~L97, T58~L97, L59~L97, E60~L97, A55~P87, R56~P87, Q57~P87, Comprises or consists of a deletion of T58 to P87, L59 to P87, E60 to P87, A55 to L88, R56 to L88, Q57 to L88, T58 to L88, L59 to L88, E60 to L88, A55 to E89, R56 to E89, Q57 to E89, T58 to E89, L59 to E89, E60 to E89, A55 to L90, R56 to L90, Q57 to L90, T58 to L90, L59 to L90, E60 to L90, A55 to T91, R56 to T91, Q57 to T91, T58 to T91, L59 to T91, or E60 to T91 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0307] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid sequence that is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31; and (b) comprises or consists of a deletion of amino acids A55 to K92 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

[0308] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is amino acids A55 - K92, N50 - K92, M51 - K92, L52 - K92, Q53 - K92, K54 - K92, N50 - N93, M51 - N93, L52 - N93, Q53 - N93, K54 - N93, N50 - E94, M51 - E94, L52 - E94, Q53 - E94, K54 - E94, N50 - S95, M51 - S95, L52 - S95, Q53 - S95, K54 - S95, N50 - C96, M51 - C96, L52 - C96, Q53 - C96, K54 - C96, N50 - L97, M51 - L97, L52 - L97, Q53 - L97, K54 - L97, N50 - P87, M51 - P87, L52 - P87, Q53 - P87, K54 - P87, N50 - L88, M51 - L88, L52 - L88, Q53 - L88, K54 - L88, N50 - E89, M51 - E89, L52 - E89, Q53 - E89, K54 - E89, N50 - L90, M51 - L90, L52 - L90, Q53 - L90, K54 - L90, N50 - T91, M51 - T91, L52 - T91, Q53 - T91, K54 - T91, R56 - K92, Q57 - K92, T58 - K92, L59 - K92, E60 - K92 A55 - N93, R56 - N93, Q57 - N93, T58 - N93, L59 - N93, E60 - N93, A55 - E94, R56 - E94, Q57 - E94, T58 - E94, L59 - E94, E60 - E94, A55 - S95, R56 - S95, Q57 - S95, T58 - S95, L59 - S95, E60 - S95, A55 - C96, R56 - C96, Q57 - C96, T58 - C96, L59 - C96, E60 - C96, A55 - L97, R56 - L97, Q57 - L97, T58 - L97, L59 - L97, E60 - L97, A55 - P87, R56 - P87, Q57 - P87, T58 - P87, L59 - P87, E60 - P87, A55 - L88, R56 - L88, Q57 - L88, T58 - L88, L59 - L88, E60 - L88, A55 - E89, R56 - E89, Q57 - E89, T58 - E89, L59 - E89, E60 - E89, A55 - L90, R56 - L90, Q57 - L90, T58 - L90, L59 - L90, E60 - L90, A55 - T91, R56 - T91, Q57 - T91, T58 - T91, L59 - T91,or comprising or consisting of a deletion of amino acids A55 to K92, N50 to K92, M51 to K92, L52 to K92, Q53 to K92, K54 to K92, N50 to N93, M51 to N93, L52 to N93, Q53 to N93, K54 to N93, N50 to E94, M51 to E94, L52 to E94, Q53 to E94, K54 to E94, N50 to S95, M51 to S95, L52 to S95, Q53 to S95, K54 to S95, N50 to C96, M51 to C96, L52 to C96, Q53 to C96, K54 to C96, N50 to L97, M51~L97, L52~L97, Q53~L97, K54~L97, N50~P87, M51~P87, L52~P87, Q53~P87, K54~P87, N50~L88, M51~L88, L52~L88, Q53~L88, K54~L88, N50~E89, M51~E89, L52~E 89, Q53~E89, K54~E89, N50~L90, M51~L90, L52~L90, Q53~L90, K54~L90, N50~T91, M5 1~T91, L52~T91, Q53~T91, K54~T91, R56~K92, Q57~K92, T58~K92, L59~K92, E60~K92 A55~N93, R56~N93, Q57~N93, T58~N93, L59~N93, E60~N93, A55~E94, R56~E94, Q57~E94, T58~E94, L59~E94, E60~E94, A55~S95, R56~S95, Q57~S95, T58~S95, L59~S95, E60~S95, A55~C96, R56~C96, Q57~C96, T58~C96, L59~C96, E60~C96, A55~L97, R56~L97, Q57~L97, T58~L97, L59~L97, E60~L97, A55~P87, R56~P87, Q57~P87, T58~P87, L59~P87, E60~P87, A55~L88, R56~L88, Q57~L88, T58~L88, L59~L88, E60~L88, A55~E89, R56~E89, Q57~E89, T58~E89, L59~E89, E60~E89, A55~L90, R56~L90, Q57~L90, T58~L90, L59~L90, E60~L90, A55~T91, R56~T91,Other than the deletion of Q57 to T91, T58 to T91, L59 to T91, or E60 to T91, the amino acids of the polypeptide are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31.

[0309] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of a deletion of amino acids A55 to K92 (amino acid numbering relative to the amino acid sequence of SEQ ID NO:30), and other than the deletion of amino acids A55 to K92, the amino acid sequence of the polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:31.

[0310] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at one or more of the following amino acid positions: E60, F61, P63, K150, F188, or Y189 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at one, two, three, four, five, six, or seven of the following amino acid positions: E60, F61, P63, K150, F188, or Y189 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30).

[0311] In some embodiments, the amino acid sequence of a hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at one or more of amino acid positions E60, F61, P63, K150, and F188 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of a hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at one or more of amino acid positions F188 and Y189 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of a hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at amino acid position F188 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at amino acid position Y189A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30).

[0312] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of one or more of the following amino acid substitutions: E60K, F61H, P63S, K150H, F188P, F188A, or Y189A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the following amino acid substitutions: E60K, F61H, P63S, K150H, and F188P (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the following amino acid substitutions: F188A and Y189A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the following amino acid substitution F188A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30): In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the following amino acid substitution Y189A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30).

[0313] In some embodiments, the amino acid sequence of the hIL12-p35 polypeptide comprises amino acid residues 50-95, 50-94, 50-93, 50-92, 50-91, 50-90, 50-89, 51-95, 51-94, 51-93, 51-92, 51-91, 51-90, 51-89, 52-95, 52-94, 52-93, 52-92, 52-91, 52-90, 52-89, In some embodiments, the amino acid sequence of the hIL12-p35 polypeptide comprises a deletion of amino acid residues A55 to K92 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30).

[0314] The amino acid sequences of exemplary hIL-12p35 polypeptides are shown in Table 6.

[0315] [Table 6]

[0316] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the polypeptides shown in Table 6.

[0317] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 31 or 110-114.

[0318] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the set of amino acid modifications (e.g., substitutions, deletions) (relative to the amino acid sequence of SEQ ID NO: 31) set forth in the amino acid sequence of any one of the polypeptides set forth in Table 6; and other than the set of amino acid modifications (e.g., substitutions, deletions), the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the polypeptide set forth in Table 6.

[0319] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of a set of amino acid modifications (e.g., substitutions, deletions) set forth in the amino acid sequence of any one of SEQ ID NOs: 110-114 (relative to the amino acid sequence of SEQ ID NO: 31); and other than the set of amino acid modifications (e.g., substitutions, deletions), the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 110-114.

[0320] 5.2.4 Potency and Affinity of hIL-12p35 Proteins and Polypeptides In some embodiments, when combined with hIL-12p40 protein, hIL-12p35 protein mediates a lower increase in the level of STAT4 in cells expressing hIL-12R on their surface than the increase in STAT4 mediated by an appropriate control (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 31)). In some embodiments, when combined with hIL-12p40 protein, hIL-12p35 protein mediates a lower increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 30)).

[0321] In some embodiments, when combined with hIL-12p40 protein, hIL-12p35 protein mediates an increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 30)). In some embodiments, when combined with hIL-12p40 protein, hIL-12p35 protein mediates an increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is at least about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 30)). In some embodiments, when combined with hIL-12p35 protein, hIL-12p40 protein mediates an increase in levels of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is about 0.5-1000 fold, 0.5-100 fold, 0.5-10 fold, 0.5-5 fold, 0.5-2 fold, 1-1000 fold, 1-100 fold, 1-10 fold, 1-5 fold, 1-2 fold, 10-1000 fold, or 100-1000 fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 30)).

[0322] Suitable assays for measuring the EC50 of the hIL-12p35 polypeptides described herein are standard and well known to those of skill in the art. For example, the EC50 can be determined by generating a dose-response curve and examining the effect of various concentrations of hIL-12p35 protein or polypeptide in inducing activity in a particular functional assay (e.g., STAT4 signaling, STAT4 phosphorylation, STAT4-inducible SEAP expression (see, e.g., Example 21)). Section 6.4 describes an exemplary method for determining the EC50 of the hIL-12p35 polypeptides or proteins described herein (including the hIL-12 fusion proteins described herein) utilizing the hIL-12 HEKBlue reporter cell line (InvivoGen #hkb-IL12). The hIL-12 HEKBlue reporter cell line expresses the hIL-12Rβ1 and hIL-12Rβ2 subunits, human STAT4, and the STAT4-inducible SEAP (secreted embryonic alkaline phosphatase) reporter. Binding of the protein to hIL-12R thereby initiates JAK2 / STAT4 signaling and subsequent production of SEAP, which can be quantified using standard methods known in the art. Further, for example, the level of phosphorylated STAT4 can be assessed by contacting cells expressing hIL-12R with one or more concentrations of the hIL-12p35 protein or polypeptide described herein, lysing the cells, and assessing the level of phosphorylated STAT4, for example, by ELISA, Western blot, FRET-based assay, or chemiluminescence assay (e.g., ELISA-based assay). Cells in cell-based assays can be cells such as HEK293 cells recombinantly expressing hIL-12R and / or human STAT4; or cells naturally expressing hIL-12R and human STAT4.

[0323] In some embodiments, when combined with hIL-12p40 protein, hIL-12p35 protein mediates a lesser increase in the level of interferon gamma (IFN-γ) produced by expressing hIL-12R on its surface than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 30)).

[0324] In some embodiments, when combined with hIL-12p40 protein, the hIL-12p35 protein mediates an increase in the level of IFN-γ produced by cells expressing hIL-12R on their surface that is 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12p35 protein (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 30)).

[0325] In some embodiments, when combined with hIL-12p40 protein, the hIL-12p35 protein mediates an increase in the level of IFN-γ produced by cells expressing hIL-12R on their surface that is at least about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in the level of IFN-γ produced in the presence of a suitable control (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 33)).

[0326] In some embodiments, the hIL-12p35 protein, when combined with the hIL-12p40 protein, mediates an increase in the level of IFN-γ produced by cells expressing hIL-12R on their surface that is about 0.5-1000-fold, 0.5-100-fold, 0.5-10-fold, 0.5-5-fold, 0.5-2-fold, 1-1000-fold, 1-100-fold, 1-10-fold, 1-5-fold, 1-2-fold, 10-1000-fold, or 100-1000-fold less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 30)).

[0327] Suitable assays for measuring the level of a protein (e.g., IFN-γ) produced from cultured cells are standard and known to those of skill in the art. For example, the level of IFN-γ can be determined using an enzyme-linked immunosorbent assay (ELISA) (see, e.g., Examples 22-23). ​​Section 6.5 describes an exemplary method for determining the level of IFN-γ produced from cultured cells treated with a hIL-12p40 protein described herein or a reference hIL-12p40 protein.

[0328] In some embodiments, the hIL-12p35 protein binds to IL-12Rβ1 with lower affinity than a reference hIL-12p35 protein (e.g., a reference hIL-12p35 protein (e.g., SEQ ID NO: 30)). Binding affinity can be measured by standard assays known in the art, see, e.g., Section 5.2.2.

[0329] 5.2.5 hIL-12 Single Chain Polypeptides and Proteins In one aspect, provided herein is a schIL-12 polypeptide comprising a hIL-12p40 polypeptide described in Section 5.2.1 herein and / or a hIL-12p35 polypeptide described in Section 5.2.3 herein. In some embodiments, where the schIL-12 polypeptide comprises a hIL-12p40 polypeptide described in Section 5.2.1 herein, the hIL-12p35 polypeptide is a hIL-12p35 polypeptide described in Section 5.3.1.2 herein. In some embodiments, where the schIL-12 polypeptide comprises a hIL-12p40 polypeptide described in Section 5.2.1 herein, the hIL-12p35 polypeptide is a hIL-12p35 polypeptide described in Section 5.2.3 herein.

[0330] In some embodiments, wherein the schIL-12 polypeptide comprises a hIL-12p35 polypeptide described in Section 5.2.3 herein, the hIL-12p40 polypeptide is a hIL-12p40 polypeptide described in Section 5.2.1 herein. In some embodiments, wherein the schIL-12 polypeptide comprises a hIL-12p35 polypeptide described in Section 5.2.3 herein, the hIL-12p40 polypeptide is a hIL-12p35 polypeptide described in Section 5.3.1.1 herein.

[0331] In some embodiments, schIL-12 comprises a hIL-12p35 polypeptide fused directly to a hIL-12p40 polypeptide. In some embodiments, the schIL-12 polypeptide comprises, from N- to C-terminus, a hIL-12p35 polypeptide, an optional peptide linker, and a hIL-12p40 polypeptide. In some embodiments, the schIL-12 polypeptide comprises, from N- to C-terminus, a hIL-12p40 polypeptide, an optional peptide linker, and a hIL-12p35 polypeptide.

[0332] In some embodiments, a schIL-12 polypeptide comprises a hIL-12p35 polypeptide operably linked directly via a peptide bond. In some embodiments, a schIL-12 polypeptide comprises a hIL-12p35 polypeptide indirectly fused to a hIL-12p40 polypeptide via a peptide linker. In some embodiments, a schIL-12 polypeptide comprises, from N- to C-terminus, a hIL-12p35 polypeptide, a peptide linker, and a hIL-12p40 polypeptide. In some embodiments, a schIL-12 polypeptide comprises, from N- to C-terminus, a hIL-12p40 polypeptide, a peptide linker, and a hIL-12p35 polypeptide.

[0333] In some embodiments, the hIL-12p40 polypeptide and the hIL-12p35 polypeptide are operably linked via a peptide linker. In some embodiments, the peptide linker is of sufficient length to allow association of the hIL-12p35 polypeptide and the hIL-12p40 polypeptide such that schIL-12 binds to the hIL-12 receptor. In some embodiments, the peptide linker comprises approximately 5-30, 5-25, 5-20, 5-15, 10-30, 10-25, 10-20, or 10-15 amino acids. In some embodiments, the peptide linker comprises or consists of glycine (G) and serine (S) amino acid residues.

[0334] The amino acid sequences of exemplary linkers for use in schIL-12 polypeptides (to operably link the hIL-12p35 and hIL-12p40 polypeptides of the schIL-12 polypeptides) are shown in Table 7.

[0335] [Table 7]

[0336] In some embodiments, the amino acid sequence of the peptide linker comprises the amino acid sequence of any one of the peptide linkers set forth in Table 7; or the amino acid sequence of any one of the peptide linkers set forth in Table 7 with one, two, or three amino acid modifications (e.g., substitutions, deletions, or additions). In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 66-81 or 369, or the amino acid sequence of any one of SEQ ID NOs: 66-81 or 369 with one, two, or three amino acid modifications (e.g., substitutions, deletions, or additions). In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 72, or the amino acid sequence of any one of SEQ ID NOs: 72 with one, two, or three amino acid modifications (e.g., substitutions, deletions, or additions). In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 369, or the amino acid sequence of any one of SEQ ID NOs: 369 with one, two, or three amino acid modifications (e.g., substitutions, deletions, or additions).

[0337] 5.2.6 Signal peptides In some embodiments, the hIL-12p40 and / or hIL-12p35 polypeptide comprises a homologous or heterologous signal peptide operably linked to the N-terminus of the hIL-12p40 and / or hIL-12p35 polypeptide.

[0338] In some embodiments, the hIL-12p40 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 38-65 and comprises a homologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, the hIL-12p40 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 38-65 and comprises a heterologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, the hIL-12p40 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 38 and comprises a homologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, the hIL-12p40 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 38 and comprises a heterologous signal peptide operably linked to the N-terminus of the polypeptide.

[0339] In some embodiments, the hIL-12p35 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 110-114 and comprises a homologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, the hIL-12p35 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 110-114 and comprises a heterologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, the hIL-12p35 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 110 and comprises a homologous signal peptide operably linked to the N-terminus of the polypeptide. In some embodiments, the hIL-12p35 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 110 and comprises a heterologous signal peptide operably linked to the N-terminus of the polypeptide.

[0340] Commonly used signal peptides are known in the art, such as the native signal peptides of human interleukin 2 (hIL-2), human oncostatin M (hOSM), human chymotrypsinogen (hCTRB1), human trypsinogen 2 (hTRY2), and human insulin (hINS). One of skill in the art can determine an appropriate signal peptide using standard methodologies known in the art. The amino acid sequences of exemplary signal peptides, along with the native signal sequence of hIL-12p40, are shown in Table 8.

[0341] [Table 8]

[0342] In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of the signal peptides shown in Table 8. In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of the signal peptides shown in Table 8, and further comprises one to less than 15% (less than 12%, less than 10%, less than 8%) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of the signal peptides shown in Table 8 with one, two, or three amino acid modifications (e.g., substitutions, deletions, additions).

[0343] In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 82-87 or 394. In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 82-87 or 394, and further comprises at least one but less than 15% (less than 12%, less than 10%, less than 8%) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of the signal peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 82-87 or 394 with one, two, or three amino acid modifications (e.g., substitutions, deletions, or additions).

[0344] 5.3 hIL-12 Fusion Proteins and Conjugates In one aspect, provided herein are fusion proteins comprising hIL-12 and a heterologous moiety (e.g., an antibody (e.g., a full-length antibody), an Fc region, etc.). In some embodiments, the fusion protein comprises an antibody and hIL-12 (see, e.g., Section 5.3.3). In some embodiments, the fusion protein comprises an Fc region and hIL-12 (see, e.g., Section 5.3.2).

[0345] In some embodiments, the fusion protein comprises a half-life extending moiety. Exemplary half-life extending moieties include, but are not limited to, human immunoglobulin (hIg), a fragment of hIg, a hIg constant region, a fragment of a hIg constant region, an Fc region, human transferrin, human serum albumin (HSA), an HSA-binding protein or peptide, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the fusion protein comprises a half-life extending polypeptide. Exemplary half-life extending polypeptides include, but are not limited to, hIg, a fragment of hIg, one or more hIg heavy chain constant regions, a fragment of a hIg constant region, an hIg Fc region, human transferrin, human serum albumin (HSA), and an HSA-binding protein or peptide. hIL-12 polypeptides or half-life extending moieties fused or conjugated to half-life extending moieties can be evaluated for their pharmacokinetic properties using standard in vitro methods known in the art.

[0346] 5.3.1 hIL-12 Proteins and Polypeptides As noted above, the fusion proteins described herein comprise a hIL-12 protein. In some embodiments, the amino acid sequence of at least one subunit of the hIL-12 protein (e.g., hIL-12p40 or hIL-12p35) comprises or consists of the amino acid sequence of a native subunit (e.g., hIL-12p40 or hIL-12p35). In some embodiments, the amino acid sequence of the hIL-12p35 subunit of the hIL-12 protein comprises or consists of the amino acid sequence of a native hIL-12p35 protein (e.g., SEQ ID NO: 31). In some embodiments, the amino acid sequence of the IL-12p40 subunit of the hIL-12 protein comprises or consists of the amino acid sequence of a native hIL-12p40 protein (e.g., SEQ ID NO: 33).

[0347] In some embodiments, the amino acid sequence of the hIL-12p40 of the hIL-12 protein comprises at least one amino acid modification relative to the amino acid sequence of a reference hIL-12p40 protein (e.g., a native hIL-12p40 protein, e.g., SEQ ID NO: 33). In some embodiments, the amino acid sequence of the hIL-12p35 subunit of the hIL-12 protein comprises at least one amino acid modification relative to the amino acid sequence of a reference hIL-12p35 protein (e.g., a native hIL-12p35 protein, e.g., SEQ ID NO: 31). In some embodiments, the amino acid sequence of the hIL-12p40 subunit of the hIL-12 protein comprises at least one amino acid modification relative to the amino acid sequence of a reference hIL-12p40 protein (e.g., a native hIL-12p40 protein, e.g., SEQ ID NO: 33); and the amino acid sequence of the hIL-12p35 subunit of the hIL-12 protein comprises at least one amino acid modification relative to the amino acid sequence of a reference hIL-12p35 protein (e.g., a native hIL-12p35 protein, e.g., SEQ ID NO: 31).

[0348] In some embodiments, the amino acid sequence of the hIL-12p35 subunit of the hIL-12 protein comprises or consists of the amino acid sequence of a native hIL-12p35 protein, and the amino acid sequence of the hIL-12p40 subunit of the hIL-12 protein comprises at least one amino acid modification relative to the amino acid sequence of a reference hIL-12p40 protein (e.g., a native hIL-12p40 protein, e.g., SEQ ID NO: 31). In some embodiments, the amino acid sequence of the hIL-12p40 subunit of the hIL-12 protein comprises or consists of the amino acid sequence of a native hIL-12p40 protein, and the amino acid sequence of the hIL-12p35 subunit of the hIL-12 protein comprises at least one amino acid modification relative to the amino acid sequence of a reference hIL-12p35 protein (e.g., a native hIL-12p35, e.g., SEQ ID NO: 31).

[0349] 5.3.1.1 hIL-12p40 subunit As noted above, the fusion proteins and polypeptides described herein include hIL-12 proteins that include the hIL-12p40 subunit.

[0350] In some embodiments, the amino acid sequence of IL-12p40 of the hIL-12 protein comprises or consists of the amino acid sequence of a native hIL-12p40 protein (e.g., SEQ ID NO: 33). In some embodiments, the amino acid sequence of IL-12p40 of the hIL-12 protein comprises or consists of the amino acid sequence of SEQ ID NO: 33. In some embodiments, the amino acid sequence of the hIL-12p40 subunit of the hIL-12 protein comprises at least one amino acid modification relative to the amino acid sequence of a reference hIL-12p40 protein (e.g., a native hIL-12p40 protein, e.g., SEQ ID NO: 33).

[0351] In some embodiments, the hIL-12p40 subunit is a hIL-12p40 polypeptide described herein, e.g., see Section 5.2 (e.g., Sections 5.2.1, 5.2.2, 5.2.5, and 5.2.6). The entire disclosure of Section 5.2 (e.g., Sections 5.2.1, 5.2.2, 5.2.5, and 5.2.6) is incorporated by reference as part of this Section 5.3.1.1. Any of the hIL-12p40 polypeptides and embodiments set forth in Section 5.2 can be incorporated into fusion proteins (e.g., antibody (e.g., anti-CAIX antibody) fusion proteins) described herein. In some embodiments, the hIL-12p40 subunit is a hIL-12p40 polypeptide described in Section 5.2 (e.g., Sections 5.2.1, 5.2.2, 5.2.5, and 5.2.6).

[0352] In some embodiments, the hIL-12p40 polypeptide is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) at least one of the amino acid positions (i) K280, K282, R283, K285, K286, and R288; (ii) E81, K121, and K286; (iii) S205, L206, P207, I208, E209, and V210; (iv) S205, L206, P207, I208, E209, and V210; (v) S205, L206, P207, I208, E209, and V210; (vi) K217, L218, K219, Y220, and E221; (vii) E81 and F82; (viii) E81, F82, and K106; (xiv) E81, F82, K106, and K217; (xv) P39, D40, E81, and F82; (xvi) W37, F82, and K217; (xvii) W37, F82, and K219; or (xviii) K106, K217, and K219; (xxix) W37, F82, K106, and K219; (xxx) H216, K217, and K219; (xxxi) P207; W37 and F82; (xxxii) W37 and K217; (xxxiii) W37 and K219; (xxxiv) W37 and K106; (xxxv) F82 and K106; (xxxvi) F (xli) W37; (xlii) F82; (xliii) K106; (xliv) K217; or (xlv) K219 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0353] In some embodiments, the hIL-12p40 polypeptide is (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) any of the following amino acid substitutions: (i) K280A, K282A, R283A, K285A, K286A, and R288A; (ii) E81K, K121E, and K286E; (iii) S205I, L184E, P207S, I208I , E209K, and V210S; (iv) S205I, L206V, P207S, I208I, E209K, V210I; (v) S205I, L206Q, P207S, I208I, E209K, V210G; (vi) K217S, L218I, K219T, Y220S, E221A; (vii) E81A and F82A; (viii) E81A, F82A, K106A; (xiv) E81A, F82A, K106A, and K217A; (xv) P39A, D40A, E81A, and F82A; (xvi ) W37A, F82A, and K217A; (xvii) W37A, F82A, and K219A; (xviii) K106A, K217A, and K219A; (xxix) W37A, F82A, K106A, and K219A; (xxx) H216A, K217A, and K219A; (xxxi) P207S; W37A and F82A; (xxxii) W37 and K217A; (xxxiii) W37A and K219A; (xxxiv) W37A and K106A; (xxxv) F82A and K1 (xli) W37A; (xlii) F82A; (xliii) K106A; (xliv) K217A; or (xlv) K219A; (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0354] In some embodiments, the hIL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 33 and further comprises a deletion of amino acid residues 23-127, (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0355] In some embodiments, the hIL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 33, and further comprises a deletion of amino acid residues 208-328 and amino acid substitutions at each of the following amino acid positions: S205, L206, and P207 (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32). In some embodiments, the hIL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 33, and further comprises a deletion of amino acid residues 208-328 and amino acid substitutions at each of the following positions: S205I, L206E, and P207S (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0356] In some embodiments, the hIL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 33 and further comprises substitutions of amino acids K217 and L218 with I (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

[0357] In some embodiments, the hIL-12p40 polypeptide comprises a modified heparin-binding domain. In some embodiments, the hIL-12p40 polypeptide comprises a modified heparin binding domain that disrupts, inhibits, or reduces the ability of the hIL-12p40 polypeptide to bind to heparin compounds compared to a reference hIL-12p40 polypeptide that does not comprise a modification in the heparin-binding domain. In some embodiments, the hIL-12p40 polypeptide comprises a modified heparin-binding domain and exhibits substantially the same, greater, or less immunostimulatory activity as that of a reference hIL-12p40 polypeptide that does not comprise a modification in the heparin-binding domain. In some embodiments, the hIL-12p40 polypeptide comprises a modified heparin-binding domain and exhibits substantially the same immunostimulatory activity as that of a reference hIL-12p40 polypeptide that does not comprise a modification in the heparin-binding domain.

[0358] In some embodiments, the unmodified heparin-binding domain of the hIL-12p40 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 88. In some embodiments, the modified heparin-binding domain comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 89.

[0359] The amino acid sequences of the hIL-12p40 reference heparin-binding domain and its variants are shown in Table 9.

[0360] [Table 9]

[0361] In some embodiments, in SEQ ID NO: 89, " * In some embodiments, one or more amino acid residues designated " in SEQ ID NO: 89 are alanine. * Each of the amino acid residues designated " is alanine. In some embodiments, amino acid residue X3 is alanine.

[0362] Exemplary hIL-12p40 polypeptides having modified heparin binding domains are described in US Pat. No. 8,617,557, the entire contents of which are incorporated herein by reference for all purposes.

[0363] The amino acid sequences of exemplary hIL-12p40 polypeptides that can be incorporated into the fusion proteins described herein are shown in Table 10.

[0364] [Table 10] TIFF2024534468000021.tif230168TIFF2024534468000022.tif232169TIFF2024534468000023.tif232169

[0365] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the polypeptides shown in Table 10.

[0366] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 38-51 or 90-109.

[0367] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the set of amino acid modifications (e.g., substitutions, deletions) (relative to the amino acid sequence of SEQ ID NO: 33) set forth in any one of the polypeptide amino acid sequences set forth in Table 10; and other than said set of amino acid modifications (e.g., substitutions, deletions), the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 10.

[0368] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid modifications (e.g., substitutions, deletions) (relative to the amino acid sequence of SEQ ID NO: 33) set forth in the amino acid sequence set forth in any one of SEQ ID NOs: 38-51 or 90-109; and other than the set of amino acid modifications (e.g., substitutions, deletions), the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-51 or 90-109.

[0369] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid modifications (e.g., substitutions, deletions) set forth in the amino acid sequence set forth in SEQ ID NO:38 (relative to the amino acid sequence of SEQ ID NO:33); and other than the set of amino acid modifications (e.g., substitutions, deletions), the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:38.

[0370] 5.3.1.2 hIL-12p35 subunit As noted above, the fusion proteins and polypeptides described herein include hIL-12 proteins that include the hIL-12p35 subunit.

[0371] In some embodiments, the amino acid sequence of the hIL-12p35 subunit of the hIL-12 protein comprises or consists of the amino acid sequence of a native hIL-12p35 protein (e.g., SEQ ID NO: 31). In some embodiments, the amino acid sequence of the hIL-12p35 subunit of the hIL-12 protein comprises at least one amino acid modification relative to the amino acid sequence of a reference hIL-12p35 protein (e.g., a native hIL-12p35 protein, e.g., SEQ ID NO: 31).

[0372] In some embodiments, the hIL-12p35 subunit is a hIL-12p35 polypeptide described herein, e.g., see Section 5.2 (e.g., Sections 5.2.3, 5.2.4, 5.2.5, and 5.2.6). The entire disclosure of Section 5.2 (e.g., Sections 5.2.3, 5.2.4, 5.2.5, and 5.2.6) is incorporated by reference as part of this Section 5.3.1.2. Any of the hIL-12p35 polypeptides and embodiments set forth in Section 5.2 (e.g., Sections 5.2.3, 5.2.4, 5.2.5, and 5.2.6) can be incorporated into fusion proteins (e.g., antibody (e.g., anti-CAIX antibody) fusion proteins) described herein. In some embodiments, the hIL-12p35 subunit is a hIL-12p35 polypeptide described in Section 5.2 (eg, Sections 5.2.3, 5.2.4, 5.2.5, and 5.2.6).

[0373] In some embodiments, at least one amino acid modification reduces the binding affinity of the hIL-12p35 subunit to hIL-12R. In some embodiments, at least one amino acid modification reduces the binding affinity of the hIL-12p35 subunit to hIL-12Rβ1. In some embodiments, at least one amino acid modification reduces the binding affinity of the hIL-12p35 subunit to hIL-12Rβ2. In some embodiments, at least one amino acid modification reduces the binding affinity of the hIL-12p35 subunit to hIL-12Rβ1 and hIL-12Rβ1.

[0374] As noted above, for purposes of this disclosure, the numbering of all amino acids (and, e.g., amino acid substitutions) in the hIL-12p35 polypeptides described herein is shown relative to the amino acid sequence of the immature form of hIL-12p35 (i.e., SEQ ID NO: 30), including the native signal peptide. The use of the immature form of hIL-12p35 to designate an amino acid number (e.g., Y189) is for consistency only and does not limit the scope of embodiments utilizing this numbering system to polypeptides that include the hIL-12p35 signal peptide. For example, a hIL-12p35 polypeptide described herein that includes the amino acid sequence of SEQ ID NO: 31 with the Y189A amino acid substitution does not require the hIL-12p35 signal peptide, although the numbering of amino acid position Y189 is based on the immature form of the protein. It is common in the art to utilize mature forms of proteins to produce variants and fusion proteins.

[0375] Those skilled in the art can readily determine the amino acid positions within the mature form of hIL-12p35 (SEQ ID NO: 31) based on the amino acid numbering for the immature form of hIL-12p35. As noted above, amino acids 1-22 of the immature form of hIL-12p35 protein are a signal sequence. Therefore, the amino acid position of a particular amino acid within the mature form of hIL-12p35 protein can be determined by subtracting 22 from the amino acid position of that particular amino acid designated for the immature form of hIL-12p35. For example, amino acid position Y189 (numbered relative to SEQ ID NO: 30) corresponds to amino acid position Y167 of the mature form of the protein (SEQ ID NO: 31).

[0376] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at one or more of the following amino acid positions: E60, F61, P63, K150, F188, or Y189 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at one or more of the following amino acid positions: E60, F61, P63, K150, F188, or Y189 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30).

[0377] In some embodiments, the amino acid sequence of a hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at one or more of amino acid positions E60, F61, P63, K150, and F188 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of a hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at one or more of amino acid positions F188 and Y189 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of a hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at amino acid position F188 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of an amino acid modification (e.g., substitution, addition, or deletion) at amino acid position Y189A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30).

[0378] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of one or more of the following amino acid substitutions: E60K, F61H, P63S, K150H, F188P, F188A, or Y189A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the following amino acid substitutions: E60K, F61H, P63S, K150H, and F188P (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the following amino acid substitutions: F188A and Y189A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30). In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the following amino acid substitution F188A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30): In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the following amino acid substitution Y189A (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30).

[0379] In some embodiments, the amino acid sequence of the hIL12-p35 polypeptide comprises amino acid residues 50-95, 50-94, 50-93, 50-92, 50-91, 50-90, 50-89, 51-95, 51-94, 51-93, 51-92, 51-91, 51-90, 51-89, 52-95, 52-94, 52-93, 52-92, 52-91, 52-90, 52-89, In some embodiments, the amino acid sequence of the hIL12-p35 polypeptide comprises a deletion of amino acid residues A55 to K92 (amino acid numbering relative to the amino acid sequence set forth in SEQ ID NO: 30).

[0380] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the polypeptides shown in Table 6.

[0381] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of SEQ ID NOs: 31 or 110-114.

[0382] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of the set of amino acid modifications (e.g., substitutions, deletions) (relative to the amino acid sequence of SEQ ID NO: 31) set forth in the amino acid sequence of any one of the polypeptides set forth in Table 6; and other than the set of amino acid modifications (e.g., substitutions, deletions), the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 6.

[0383] In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide comprises or consists of a set of amino acid modifications (e.g., substitutions, deletions) set forth in the amino acid sequence of any one of SEQ ID NOs: 110-114 (relative to the amino acid sequence of SEQ ID NO: 31); and other than the set of amino acid modifications (e.g., substitutions, deletions), the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 110-114.

[0384] 5.3.1.3 schIL-12 Polypeptides In some embodiments, the hIL-12 of the fusion proteins described herein is in the form of a single polypeptide chain (referred to herein as schIL-12). The schIL-12 polypeptide can include any of the hIL-12p40 polypeptides described herein (see, e.g., Section 5.2 (e.g., Section 5.2.1) and Section 5.3.1.1); and any of the hIL-12p35 polypeptides described herein (see, e.g., Section 5.2 (e.g., Section 5.2.3) and Section 5.3.1.2). The schIL-12 polypeptide can include a schIL-12 polypeptide described in Section 5.2.5 herein.

[0385] In some embodiments, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-51 or 90-109. In some embodiments, the amino acid sequence of the hIL-12p35 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 31 or 110-114.

[0386] In some embodiments, schIL-12 comprises a hIL-12p35 polypeptide fused directly to a hIL-12p40 polypeptide. In some embodiments, the schIL-12 polypeptide comprises, from N- to C-terminus, a hIL-12p35 polypeptide, an optional peptide linker, and a hIL-12p40 polypeptide. In some embodiments, the schIL-12 polypeptide comprises, from N- to C-terminus, a hIL-12p40 polypeptide, an optional peptide linker, and a hIL-12p35 polypeptide.

[0387] In some embodiments, a schIL-12 polypeptide comprises a hIL-12p35 polypeptide operably linked directly via a peptide bond. In some embodiments, a schIL-12 polypeptide comprises a hIL-12p35 polypeptide indirectly fused to a hIL-12p40 polypeptide via a peptide linker. In some embodiments, a schIL-12 polypeptide comprises, from N- to C-terminus, a hIL-12p35 polypeptide, a peptide linker, and a hIL-12p40 polypeptide. In some embodiments, a schIL-12 polypeptide comprises, from N- to C-terminus, a hIL-12p40 polypeptide, a peptide linker, and a hIL-12p35 polypeptide.

[0388] The amino acid sequences of exemplary linkers for use in schIL-12 polypeptides (to operably link hIL-12p35 and hIL-12p40 polypeptides) are shown in Table 11.

[0389] In some embodiments, the hIL-12p40 polypeptide and the hIL-12p35 polypeptide are operably linked via a peptide linker. In some embodiments, the peptide linker is of sufficient length to allow association of the hIL-12p35 polypeptide and the hIL-12p40 polypeptide such that schIL-12 binds to the hIL-12 receptor. In some embodiments, the peptide linker comprises approximately 5-30, 5-25, 5-20, 5-15, 10-30, 10-25, 10-20, or 10-15 amino acids. In some embodiments, the peptide linker comprises or consists of glycine (G) and serine (S) amino acid residues.

[0390] In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 66-81, or the amino acid sequence of any one of SEQ ID NOs: 66-81 with one, two, or three amino acid modifications (e.g., substitutions, deletions, or additions). In some embodiments, the peptide linker comprises the amino acid sequence of any one of SEQ ID NOs: 72, or the amino acid sequence of any one of SEQ ID NO: 72 with one, two, or three amino acid modifications (e.g., substitutions, deletions, or additions).

[0391] The amino acid sequences of exemplary schIL-12 polypeptides are shown in Table 11.

[0392] [Table 11] TIFF2024534468000025.tif167169

[0393] In some embodiments, the amino acid sequence of the schIL-12 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the polypeptides shown in Table 11.

[0394] In some embodiments, the amino acid sequence of the schIL-12 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs:115-118.

[0395] 5.3.1.4 Potency and Affinity of hIL-12 Fusion Proteins and Polypeptides In some embodiments, the hIL-12 fusion protein mediates a lesser increase in the level of STAT4 in cells expressing hIL-12R on their surface than the increase in STAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the hIL-12 fusion protein mediates a lesser increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the hIL-12 fusion protein mediates an increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the hIL-12 fusion protein mediates an increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is at least about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the hIL-12 fusion protein mediates an increase in the level of phosphorylated STAT4 (pSTAT4) in cells expressing hIL-12R on their surface that is about 0.5-1000-fold, 0.5-100-fold, 0.5-10-fold, 0.5-5-fold, 0.5-2-fold, 1-1000-fold, 1-100-fold, 1-10-fold, 1-5-fold, 1-2-fold, 10-1000-fold, or 100-1000-fold less than the increase in pSTAT4 mediated by an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). Suitable assays for determining the EC50 of the hIL-12 fusion proteins described herein are standard and well known to those of skill in the art, as described, inter alia, in Section 5.2.3.

[0396] In some embodiments, the hIL-12 fusion protein mediates an increase in the level of interferon gamma (IFN-γ) produced by expressing hIL-12R on its surface that is less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the hIL-12 fusion protein mediates an increase in the level of IFN-γ produced by expressing hIL-12R on its surface that is 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the hIL-12 fusion protein mediates an increase in the level of IFN-γ produced by expressing hIL-12R on its surface that is at least about 0.5-fold, 1-fold, 2-fold, 5-fold, 10-fold, 100-fold, or 1000-fold less than the increase in the level of IFN-γ produced in the presence of an appropriate control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). In some embodiments, the hIL-12 fusion protein mediates an increase in the level of IFN-γ produced by expressing hIL-12R on its surface that is about 0.5-1000-fold, 0.5-100-fold, 0.5-10-fold, 0.5-5-fold, 0.5-2-fold, 1-1000-fold, 1-100-fold, 1-10-fold, 1-5-fold, 1-2-fold, 10-1000-fold, or 100-1000-fold less than the increase in the level of IFN-γ produced in the presence of a suitable control (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). Suitable assays for measuring the level of a protein (e.g., IFN-γ) produced from cultured cells are standard and well known to those of skill in the art, as described, inter alia, in Section 5.2.3.

[0397] In some embodiments, the hIL-12 fusion protein binds to hIL-12Rβ1 with lower affinity than a reference hIL-12p40 protein (e.g., a reference hIL-12 fusion protein (e.g., SEQ ID NOs: 371, 372, 383)). Binding affinity can be measured by standard assays known in the art, as described, inter alia, in Section 5.2.25.2.3.

[0398] 5.3.2 Ig Fusion Proteins and Polypeptides In some embodiments, the fusion protein comprises one or more hIg heavy chain constant regions (e.g., CH1 region, hinge region, CH2 region, CH3 region, Fc region). In some embodiments, the one or more hIg heavy chain constant regions are part of an antibody (e.g., a full-length antibody) (see, e.g., Section 5.3.3). In some embodiments, the hIg is human IgG (hIgG). In some embodiments, the hIgG is hIgG1, IgG2, IgG3, or IgG4. In some embodiments, the hIgG is IgG1 or IgG4. In some embodiments, the hIgG is hIgG1. In some embodiments, the hIgG is hIgG4.

[0399] In some embodiments, the fusion protein comprises an Fc region. In some embodiments, the Fc region is a portion of an antibody. In some embodiments, the Fc region is a portion of a full-length antibody. In some embodiments, the Fc region comprises or consists of a CH2 region and a CH3 region. In some embodiments, the Fc region comprises or consists of at least a portion of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Fc region comprises or consists of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the Fc region comprises or consists of at least a portion of an hIgG CH2 region and an hIgG CH3 region. In some embodiments, the Fc region comprises or consists of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the Fc region comprises or consists of an hIgG hinge region, an hIgG CH2 region, and an hIgG CH3 region. In some embodiments, the Fc region comprises or consists of an hIgG1 CH2 region and an hIgG1 CH3 region. In some embodiments, the Fc region comprises or consists of at least a portion of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the Fc region comprises or consists of an hIgG1 hinge region, an hIgG1 CH2 region, and an hIgG1 CH3 region. In some embodiments, the Fc region comprises or consists of an hIgG4 CH2 region and an hIgG4 CH3 region. In some embodiments, the Fc region comprises or consists of at least a portion of an hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region. In some embodiments, the Fc region comprises or consists of an hIgG4 hinge region, an hIgG4 CH2 region, and an hIgG4 CH3 region.

[0400] Exemplary reference hIgG1 and hIgG4 heavy and light chain constant region amino acid sequences that can be incorporated into the embodiments described herein (e.g., one or more of the hIL-12 fusion proteins (or one or more polypeptides thereof) described herein (e.g., anti-CAIX antibody (e.g., full-length antibody) hIL-12 fusion proteins described herein)) are shown in Table 12.

[0401] [Table 12] TIFF2024534468000027.tif230168TIFF2024534468000028.tif230168TIFF2024534468000029.tif87168

[0402] In some embodiments, the fusion protein comprises one or more hIg constant regions, in some embodiments, the amino acid sequence of the one or more hIg constant regions comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 12.

[0403] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of a polypeptide set forth in Table 12, and further comprises one or more and less than 15% (less than 12%, less than 10%, less than 8%) amino acid variations (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of a polypeptide set forth in Table 12, which comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of a polypeptide set forth in Table 12, which comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence of a polypeptide shown in Table 12, containing or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions).

[0404] In some embodiments, the amino acid sequence of one or more hIg constant region amino acid sequences comprises or consists of a polypeptide set forth in Table 12, further comprising one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant region comprises or consists of an amino acid sequence of a polypeptide set forth in Table 12 comprising or consisting of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant region comprises or consists of an amino acid sequence of a polypeptide set forth in Table 12 comprising or consisting of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant region amino acid sequences comprises or consists of a polypeptide set forth in Table 12 comprising or consisting of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0405] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144).

[0406] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144), and further comprises at least one but less than 15% (less than 12%, less than 10%, less than 8%) amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144), which comprises or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144), which contains or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications (e.g., amino acid substitutions, deletions, or additions). In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144), which contains or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or fewer amino acid modifications (e.g., amino acid substitutions, deletions, or additions).

[0407] In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144), and further contains one or more but less than 15% (less than 12%, less than 10%, less than 8%) amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144), which contains or consists of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144), which comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid sequence of one or more hIg constant regions comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 119-146 (e.g., any one of SEQ ID NOs: 124-131 or 137-144), which comprises or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or less amino acid substitutions.

[0408] In some embodiments, the fusion protein comprises a light chain comprising an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acids set forth in any one of SEQ ID NOs: 145-146.

[0409] 5.3.2.1 Ig effector functions As described herein, in some embodiments, a fusion protein comprises an Fc region. In some embodiments, the Fc region of a fusion protein or polypeptide described herein exhibits a reduction in one or more Fc effector functions relative to a reference (e.g., wild-type) Fc region. Exemplary Fc effector functions include, but are not limited to, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and binding affinity to one or more human Fc receptors (e.g., Fcγ receptors (e.g., FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and / or FcγRIIIb (e.g., FcγRI, FcγIIa, and / or FcγIIIa))).

[0410] Standard in vitro and / or in vitro assays known in the art can be performed to assess Fc effector function, including any one or more of ADCC, CDC, ADCP, Fc receptor (e.g., Fcγ receptor) binding affinity, and C1q binding affinity.

[0411] For example, ADCC activity can be assessed using standard methods (both radioactive and non-radioactive) known in the art (see, e.g., WO2006 / 082515, WO2012 / 130831, the entire contents of each of which are incorporated herein by reference for all purposes). For example, ADCC activity can be assessed using chromium-5 ( 51 This can be assessed using a CD20 (Cr) assay. 51Cr is preloaded, NK cells are added to the culture, and radioactivity in the cell culture supernatant is assessed (indicating lysis of target cells by NK cells). Alternatively, a similar non-radioactive assay can be used in which target cells are preloaded with a fluorescent dye, such as calcein-AM, CFSE, BCECF, or a lanthanide fluorescent dye (europium). See, for example, Parekh, Bhavin S et al. "Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay." mAbs vol. 4,3 (2012): 310-8. Doi:10.4161 / mabs.19873, the entire contents of which are incorporated herein by reference. Exemplary commercially available non-radioactive assays include, for example, ACTI™ Non-Radioactive Cytotoxicity for Flow Cytometry (Cell Technology, Inc. Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Further non-limiting examples of in vitro assays that can be used to assess ADCC activity of the fusion proteins described herein include US5500362; US5821337; Hellstrom, I., et al., Proc. Nat'l Acad. Sci. USA 83 (1986) 7059-7063; Hellstrom, I., et al., Proc. Nat'l Acad. Sci. USA 82 (1985) 1499-1502; and Bruggemann, M., et al., J. Exp. Med. 166 (1987) 1351-1361, the entire contents of each of which are incorporated herein by reference.Alternatively, or in addition, the ADCC activity of the fusion proteins described herein can be assessed in vitro, for example, in an animal model such as that disclosed in Clynes, et al., Proc. Nat'l Acad. Sci. USA 95 (1998) 652-656, the entire contents of which are incorporated herein by reference for all purposes.

[0412] C1q binding assays can be used to assess the ability of the hIg fusion proteins or polypeptides described herein to bind to C1q (or bind with lower affinity than a reference fusion protein) and thus lack (or have reduced) CDC activity. The binding of the hIg fusion proteins or polypeptides described herein to C1q can be determined by a variety of in vitro assays (e.g., biochemical or immunological-based assays) known in the art for determining Fc-C1q interactions, including, for example, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., surface plasmon resonance (SPR) analysis), as well as other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labels on one or more of the components being examined and / or may employ a variety of detection methods, including, but not limited to, colorimetric, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinity and kinetics can be found, for example, in Fundamental Immunology, 4th ed., Paul, WE (ed.), Lippincott-Raven, Philadelphia (1999), the entire contents of which are incorporated herein by reference. See, for example, the C1q and C3c binding ELISAs described in WO2006 / 029879 and WO2005 / 100402, the entire contents of each of which are incorporated herein by reference for all purposes. Additional CDC activity assays include those described, for example, in Gazzano-Santoro, et al., J. Immunol. Methods 202 (1996) 163; Cragg, MS, et al., Blood 101 (2003) 1045-1052; and Cragg, MS, and Glennie, MJ, Blood 103 (2004) 2738-2743, the entire contents of each of which are incorporated herein by reference for all purposes.

[0413] ADCP activity can be measured by in vitro or in vitro methods known in the art, as well as by commercially available assays (see, e.g., van de Donk NW, Moreau P, Plesner T, et al. "Clinical efficacy and management of monoclonal antibodies targeting CD38 and SLAMF7 in multiple myeloma," Blood, 127(6):681-695 (2016), the entire contents of each of which are incorporated herein by reference). For example, ...

Claims

1. (a) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 33; and (b) at least one of the amino acid positions (i) W37, F82, and K219; (ii) W37, F82, and K217; (iii) K106, K217, and K219; (iv) W37 and F82; (v) W37 and K217; (vi) W37 and K219; (vii) W3 (xv) F82 and K219; (xvi) K217 and K219; (xvii) K106 and K217; or (xviii) K106 and K219, (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

2. The following amino acid substitutions were made: (i) W37A, F82A, and K219A; (ii) W37A, F82A, and K217A; (iii) K106A, K217A, and K219A; (iv) W37A and F82A; (v) W37A and K217A; (vi) W37A and K219A; (vii) W37A and K106A; (viii) F82A and K106A; (xiv) F82A and K217A; (xv) F82A and K219A; (xvi) K217A and K219A; (xvii) K106A and K217A; or (xviii) K106A and K219A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

3. 2. The hIL-12p40 polypeptide of claim 1, comprising or consisting of an amino acid substitution at each of amino acid positions W37, F82, and K219, (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

4. 2. The hIL-12p40 polypeptide of claim 1, comprising or consisting of each of the following amino acid substitutions: W37A, F82A, and K219A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 32).

5. 2. The hIL-12p40 polypeptide of claim 1, wherein the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of any one of the polypeptides set forth in Table 5 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a polypeptide set forth in Table 5.

6. 2. The hIL-12p40 polypeptide of claim 1, wherein the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of any one of SEQ ID NOs: 38-65 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-65.

7. 2. The hIL-12p40 polypeptide of claim 1, wherein the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of any one of SEQ ID NOs: 38-51 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 38-51.

8. 2. The hIL-12p40 polypeptide of claim 1, wherein the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of any one of SEQ ID NOs: 52-65 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 52-65.

9. 2. The hIL-12p40 polypeptide of claim 1, wherein the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of SEQ ID NO:38 or 52 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO:33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:38 or 52.

10. 2. The hIL-12p40 polypeptide of claim 1, wherein the amino acid sequence of the hIL-12p40 polypeptide comprises or consists of the set of amino acid substitutions set forth in the amino acid sequence of SEQ ID NO:38 (amino acid substitutions relative to the amino acid sequence of SEQ ID NO:33); and other than said set of amino acid substitutions, the amino acid sequence of the hIL-12p40 polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO:

38.

11. 2. The hIL-12p40 polypeptide of claim 1, wherein the amino acid sequence of the hIL-12p40 polypeptide is 100% identical to the amino acid sequence of SEQ ID NO:

38.

12. 2. The hIL-12p40 polypeptide of claim 1, wherein the hIL-12p40 polypeptide specifically binds to the hIL-12 receptor (hIL-12R).

13. 2. The hIL-12p40 polypeptide of claim 1, wherein when combined with a hIL-12p35 protein, the hIL-12p40 protein mediates an increase in phosphorylated STAT4 (pSTAT4) levels in cells expressing hIL-12R on their surface that is less than the increase in pSTAT4 mediated by a suitable control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

14. 2. The hIL-12p40 polypeptide of claim 1, wherein when combined with a hIL-12p35 protein, the hIL-12p40 protein mediates an increase in phosphorylated STAT4 (pSTAT4) levels in cells expressing hIL-12R on their surface that is about 0.5-1000 fold, 0.5-100 fold, 0.5-10 fold, 0.5-5 fold, 0.5-2 fold, 1-1000 fold, 1-100 fold, 1-10 fold, 1-5 fold, 1-2 fold, 10-1000 fold, or 100-1000 fold less than the increase in pSTAT4 mediated by a suitable control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

15. 2. The hIL-12p40 polypeptide of claim 1, wherein the hIL-12p40 protein, when combined with a hIL-12p35 protein, mediates an increase in the level of interferon gamma (IFN-γ) produced by expressing a hIL-12R on its surface that is less than the increase in the level of IFN-γ produced in the presence of a suitable control, such as a reference hIL-12p40 protein (e.g., SEQ ID NO: 33).

16. 2. The hIL-12p40 polypeptide of claim 1, wherein when combined with a hIL-12p35 protein, the hIL-12p40 protein mediates an increase in the level of IFN-γ produced by cells expressing a hIL-12R on their surface that is about 0.5-1000 fold, 0.5-100 fold, 0.5-10 fold, 0.5-5 fold, 0.5-2 fold, 1-1000 fold, 1-100 fold, 1-10 fold, 1-5 fold, 1-2 fold, 10-1000 fold, or 100-1000 fold less than the increase in the level of IFN-γ produced in the presence of a suitable control (e.g., a reference hIL-12p40 protein (e.g., SEQ ID NO: 33)).

17. 30. A human interleukin-12p35 (hIL-12p35) polypeptide comprising: (a) an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31; and (b) an amino acid sequence that comprises, or consists of, an amino acid modification (e.g., substitution, addition, deletion (e.g., substitution)) at one or more of the following amino acid positions: E60, F61, P63, K150, F188, Y189A (amino acid numbering relative to the amino acid sequence of SEQ ID NO: 30).

18. The amino acid sequence of the hIL-12p35 polypeptide is amino acids A55 - K92, N50 - K92, M51 - K92, L52 - K92, Q53 - K92, K54 - K92, N50 - N93, M51 - N93, L52 - N93, Q53 - N93, K54 - N93, N50 - E94, M51 - E94, L52 - E94, Q53 - E94, K54 - E94, N50 - S95, M51 - S95, L52 - S95, Q53 - S95, K54 - S95, N50 - C96, M51 - C96, L52 - C96, Q53 - C96, K54 - C96, N50 - L97, M51 - L97, L52 - L97, Q53 - L97, K54 - L97, N50 - P87, M51 - P87, L52 - P87, Q53 - P87, K54 - P87, N50 - L88, M51 - L88, L52 - L88, Q53 - L88, K54 - L88, N50 - E89, M51 - E89, L52 - E89, Q53 - E89, K54 - E89, N50 - L90, M51 - L90, L52 - L90, Q53 - L90, K54 - L90, N50 - T91, M51 - T91, L52 - T91, Q53 - T91, K54 - T91, R56 - K92, Q57 - K92, T58 - K92, L59 - K9or comprising or consisting of a deletion of amino acids A55 to K92, N50 to K92, M51 to K92, L52 to K92, Q53 to K92, K54 to K92, N50 to N93, M51 to N93, L52 to N93, Q53 to N93, K54 to N93, N50 to E94, M51 to E94, L52 to E94, Q53 to E94, K54 to E94, N50 to S95, M51 to S95, L52 to S95, Q53 to S95, K54 to S95, N50 to C96, M51 to C96, L52 to C96, Q53 to C96, K54 to C96, N50 to L97, M51~L97, L52~L97, Q53~L97, K54~L97, N50~P87, M51~P87, L52~P87, Q53~P87, K54~P87, N50~L88, M51~L88, L52~L88, Q53~L88, K54~L88, N50~E89, M51~E89, L52~E 89, Q53~E89, K54~E89, N50~L90, M51~L90, L52~L90, Q53~L90, K54~L90, N50~T91, M5 1~T91, L52~T91, Q53~T91, K54~T91, R56~K92, Q57~K92, T58~K92, L59~K92, E60~K92 A55-N93, R56-N93, Q57-N93, T58-N93, L59-N93, E60-N93, A55-E94, R56-E94, Q57-E94, T58-E94, L59-E94, E60-E94, A55-S95, R56-S95, Q57-S95, T58-S95, L59-S95, E60-S95, A55-C96, R56-C96, Q57-C96, T58-C96, L59-C96, E60-C96, A55-L97, R56-L97, Q57-L97, T58-L97, L59-L97, E60-L97, A55-P87, R56-P87, Q57-P87, T58-P87, L59-P87, E60-P87, A55-L88, R56-L88, Q57-L88, T58-L88, L59-L88, E60-L88, A55-E89, R56-E89, Q57-E89, T58-E89, L59-E89, E60-E89, A55-L90, R56-L90, Q57-L90, T58-L90, L59-L90, E60-L90, A55-T91, R56-T91,A hIL-12p35 polypeptide, wherein, other than the deletion of Q57 to T91, T58 to T91, L59 to T91, or E60 to T91, the amino acid sequence of the polypeptide is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:

31.

19. A single chain hIL-12 (schIL-12) polypeptide comprising the hIL-12p40 polypeptide of any one of claims 1 to 16 operably linked to a hIL-12p35 polypeptide.

20. 18. A schIL-12 polypeptide comprising the hIL-12p35 polypeptide of claim 17 operably linked to a hIL-12p40 polypeptide.

21. a. the hIL-12p40 polypeptide of any one of claims 1 to 16; b. hIL-12p35 polypeptide; and c. dissimilar parts A fusion protein comprising:

22. a. hIL-12p40 polypeptide; b. the hIL-12p35 polypeptide of claim 17; and c. dissimilar parts A fusion protein comprising:

23. a. A full-length antibody that specifically binds to hTAA, i. a first light chain comprising, from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL); ii. a first heavy chain comprising, from N-terminus to C-terminus, a heavy chain variable region (VH), a CH1 region, a hinge region, a CH2 region, and a CH3 region; iii. a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; iv. a second light chain comprising, from N-terminus to C-terminus, a VL region and a VH region; Including, the first light chain and the first heavy chain associate to form a first antigen-binding domain; the second light chain and the second heavy chain associate to form a second antigen-binding domain; and The first heavy chain and the second heavy chain associate to form a dimer. Full-length antibody; b. a hIL-12p40 polypeptide according to any one of claims 1 to 16; c. hIL-12p35 polypeptide; Includes: the CH3 region of the first heavy chain of the full-length antibody comprises one or more amino acid alterations (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 122) that does not contain the one or more amino acid alterations; the CH3 region of the second heavy chain of the full-length antibody comprises one or more amino acid alterations (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 122) that does not contain the one or more amino acid alterations; one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody and one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimer formation between the first heavy chain and the second heavy chain of the full-length antibody; the N-terminus of the hIL-12p40 polypeptide is operably linked to the C-terminus of the CH3 region of the first heavy chain via a first peptide linker; and the N-terminus of the hIL-12p35 polypeptide is operably linked to the C-terminus of the CH3 region of the second heavy chain via a second peptide linker; Fusion proteins.

24. a. a first polypeptide comprising, from N-terminus to C-terminus, a first light chain comprising a VL region and a CL region; b. From N-terminus to C-terminus: (i) a first heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) a first peptide linker; and (iii) a second polypeptide comprising the hIL-12p40 polypeptide of any one of claims 1-16; c. From N-terminus to C-terminus: (i) a second heavy chain comprising, from N-terminus to C-terminus, a VH region, a CH1 region, a hinge region, a CH2 region, and a CH3 region; (ii) a second peptide linker; and (iii) a third polypeptide comprising a hIL-12p35 polypeptide; and d. a fourth polypeptide comprising, from N-terminus to C-terminus, a second light chain comprising a VL region and a CL region. Including, the VL of the first light chain and the VH of the first heavy chain associate to form a first antigen-binding domain that specifically binds to the first hTAA; the CH3 region of the first heavy chain comprises one or more amino acid alterations (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 122) that does not contain the one or more amino acid alterations; the CH3 region of the second heavy chain comprises one or more amino acid alterations (e.g., substitutions) relative to the amino acid sequence of a reference CH3 region (e.g., a wild-type CH3 region, e.g., SEQ ID NO: 122) that does not contain the one or more amino acid alterations; one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody are different from one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody; one or more amino acid modifications in the CH3 region of the first heavy chain of the full-length antibody and one or more amino acid modifications in the CH3 region of the second heavy chain of the full-length antibody promote heterodimer formation between the first heavy chain and the second heavy chain of the full-length antibody; Fusion proteins.

25. An antibody (or antigen-binding domain thereof) that specifically binds to hCAIX and comprises a VH and a VL, wherein the amino acid sequence of the VH is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 3 to 9; and the amino acid sequence of the VL is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10 to 17.

26. A polynucleotide encoding the hIL-12p40 polypeptide of any one of claims 1 to 16.

27. 27. An expression vector comprising the polynucleotide of claim 26.

28. A host cell comprising the hIL-12p40 polypeptide of any one of claims 1 to 16.

29. A carrier comprising the hIL-12p40 polypeptide of any one of claims 1 to 16.

30. 30. The carrier of claim 29, wherein the carrier is a lipid nanoparticle, a liposome, a lipoplex, or a nanoliposome.

31. A pharmaceutical composition comprising the hIL-12p40 polypeptide of any one of claims 1 to 16, and a pharmaceutically acceptable excipient.

32. A kit comprising the hIL-12p40 polypeptide of any one of claims 1 to 16.

33. 2. A method for producing the hIL-12p40 polypeptide of claim 1, comprising: a. introducing the polynucleotide of claim 26 into a population of cells in vitro or ex vivo; b. culturing the cell population under conditions sufficient for the cell population to express the multispecific protein; and c. Optionally, isolating and / or purifying the hIL-12p40 polypeptide. A method comprising:

34. A pharmaceutical composition comprising a hIL-12p40 polypeptide according to any one of claims 1 to 16 for stimulating effector functions of T cells or NK cells in a subject.

35. A pharmaceutical composition comprising a hIL-12p40 polypeptide according to any one of claims 1 to 16 for preventing or treating cancer in a subject.

36. 36. The pharmaceutical composition of claim 35, wherein the cancer is a solid tumor.

37. 36. The pharmaceutical composition of claim 35, wherein the cancer is lung cancer, central nervous system cancer (e.g., brain cancer or spinal cancer, e.g., astrocytoma, glioblastoma), breast cancer, colorectal cancer, colon cancer, rectal cancer, esophageal cancer, kidney cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, skin cancer, bladder cancer, uterine cancer, brain cancer, endometrial cancer, lip cancer, oral cancer, mesothelioma, sarcoma, thyroid cancer, thymus cancer, renal cancer, anal cancer, head cancer, neck cancer, or head and neck cancer.

38. 36. The pharmaceutical composition of claim 35, wherein the cancer is renal cancer (e.g., renal cell carcinoma), bladder cancer, colon cancer, small intestine cancer, esophageal / gastric junction (GEJ) cancer, central nervous system cancer (e.g., brain or spinal cord cancer, e.g., glioblastoma), cervical cancer, gastric cancer, lung cancer (e.g., small cell lung cancer), or gastrointestinal cancer.

39. 36. The pharmaceutical composition of claim 35, wherein the subject is a human.

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