UPAR antibodies and their fusion proteins

Abstract

The present disclosure provides urokinase plasminogen activator receptor (uPAR) binding polypeptides (e.g., uPAR antigen binding proteins) and fusion proteins comprising the uPAR binding polypeptides and a cytokine or variant thereof. The present disclosure further provides pharmaceutical compositions comprising the uPAR binding polypeptides and fusion proteins of the present disclosure, and methods of treating cancer therewith. The present disclosure further provides engineered IL-12 p35 and p40 polypeptides.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 165,313, filed March 24, 2021, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Immunotherapy is a rapidly emerging treatment for cancer. Immunotherapies fall into the general categories of (i) immune checkpoint molecule modulators, which can agonize or antagonize regulatory molecules as appropriate to enhance a desired immune response (e.g., agonize stimulatory immune receptors) or inhibit an undesired immune response (e.g., antagonize inhibitory immune checkpoint molecules), (ii) cytokines, which are protein molecules that help regulate and guide the immune system, and (iii) cancer vaccines, which are molecules (or combinations) that provide an immunological memory response against a specific antigen / pathogen. Although there are currently promising immunotherapies in clinical use and a thriving pipeline of new drugs / agents in development, new immunotherapies are desirable for use alone or in combination with other categories of immunotherapies, targeted therapies, and traditional cytotoxic therapies. Summary of the Invention

[0003] In one aspect, the present disclosure provides an isolated antigen binding protein that specifically binds to urokinase plasminogen activator receptor (uPAR), comprising: (a) i) the HCDR1 amino acid sequence of GFNIKDEY (SEQ ID NO: 3), GFSLTNYG (SEQ ID NO: 11), GNTFTDYG (SEQ ID NO: 19), GTTFTDYG (SEQ ID NO: 41), or GYTFTSYG (SEQ ID NO: 27); ii) the HCDR2 amino acid sequence of IDPENGDT (SEQ ID NO: 4), IWSDGGT (SEQ ID NO: 12), INTNTGEP (SEQ ID NO: 20), or IYPRSGNT (SEQ ID NO: 28); and iii) an antibody heavy chain variable (VH) domain comprising an HCDR3 amino acid sequence of TGGNYVGWFPY (SEQ ID NO: 5), ARGGRSDLFAY (SEQ ID NO: 13), AHYSFDY (SEQ ID NO: 21), or AGKDYGSTYADY (SEQ ID NO: 29); (b) iv) the LCDR1 amino acid sequences of SSVSY (SEQ ID NO: 6), QSIVHSNGNTY (SEQ ID NO: 14), ENIYSN (SEQ ID NO: 22), SSVSSRY (SEQ ID NO: 30); v) the LCDR2 amino acid sequence of DTS (SEQ ID NO: 7), KVS (SEQ ID NO: 15), AAT (SEQ ID NO: 23), or GTS (SEQ ID NO: 31), and vi) an antibody light chain variable (VL) domain comprising the LCDR3 amino acid sequence of QQWSSNPPY (SEQ ID NO: 8), FQGSHVPYT (SEQ ID NO: 16), QHFWGTPWT (SEQ ID NO: 24), or QQYHSDPLT (SEQ ID NO: 32); The present invention provides an antigen-binding protein comprising:

[0004] In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:42, or SEQ ID NO:43, and the VL domain comprises the amino acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:44.

[0005] In certain embodiments of the antigen binding protein, a) the VH domain comprises an HCDR1 amino acid sequence of GFNIKDEY (SEQ ID NO: 3), an HCDR2 amino acid sequence of IDPENGDT (SEQ ID NO: 4), and an HCDR3 amino acid sequence of TGGNYVGWFPY (SEQ ID NO: 5); b) The VL domain comprises an LCDR1 amino acid sequence of SSVSY (SEQ ID NO: 6), an LCDR2 amino acid sequence of DTS (SEQ ID NO: 7), and an LCDR3 amino acid sequence of QQWSSNPPY (SEQ ID NO: 8).

[0006] In certain embodiments of the antigen binding protein, a) the VH domain comprises an HCDR1 amino acid sequence of GFSLTNYG (SEQ ID NO: 11), an HCDR2 amino acid sequence of IWSDGGT (SEQ ID NO: 12), and an HCDR3 amino acid sequence of ARGGRSDLFAY (SEQ ID NO: 13); b) The VL domain comprises an LCDR1 amino acid sequence of QSIVHSNGNTY (SEQ ID NO: 14), an LCDR2 amino acid sequence of KVS (SEQ ID NO: 15), and an LCDR3 amino acid sequence of FQGSHVPYT (SEQ ID NO: 16).

[0007] In certain embodiments of the antigen binding protein, a) the VH domain comprises an HCDR1 amino acid sequence of GNTFTDYG (SEQ ID NO: 19) or GTTFTDYG (SEQ ID NO: 41), an HCDR2 amino acid sequence of INTNTGEP (SEQ ID NO: 20), and an HCDR3 amino acid sequence of AHYSFDY (SEQ ID NO: 21); b) The VL domain comprises the LCDR1 amino acid sequence of ENIYSN (SEQ ID NO: 22), the LCDR2 amino acid sequence of AAT (SEQ ID NO: 23), and the LCDR3 amino acid sequence of QHFWGTPWT (SEQ ID NO: 24).

[0008] In certain embodiments of the antigen binding protein, a) the VH domain comprises an HCDR1 amino acid sequence of GYTFTSYG (SEQ ID NO: 27), an HCDR2 amino acid sequence of IYPRSGNT (SEQ ID NO: 28), and an HCDR3 amino acid sequence of AGKDYGSTYADY (SEQ ID NO: 29); b) The VL domain comprises an LCDR1 amino acid sequence of SSVSSRY (SEQ ID NO: 30), an LCDR2 amino acid sequence of GTS (SEQ ID NO: 31), and an LCDR3 amino acid sequence of QQYHSDPLT (SEQ ID NO: 32).

[0009] In certain embodiments of the antigen binding protein, a) the VH domain comprises the amino acid sequence of SEQ ID NO: 33 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; b) the VH domain comprises the amino acid sequence of SEQ ID NO: 33 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; c) the VH domain comprises the amino acid sequence of SEQ ID NO: 34 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; d) the VH domain comprises the amino acid sequence of SEQ ID NO: 34 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; e) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 38; f) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 39; g) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 40; h) the VH domain comprises the amino acid sequence of SEQ ID NO: 42 and the VL domain comprises the amino acid sequence of SEQ ID NO: 44; or i) the VH domain comprises the amino acid sequence of SEQ ID NO: 43 and the VL domain comprises the amino acid sequence of SEQ ID NO: 44.

[0010] In certain embodiments, the VH domain and the VL domain are linked by an amino acid linker.

[0011] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 148), where n is an integer from 1 to 5.

[0012] In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 149), GGSGGGGSGGGSGGGGSGGGGSGGGSGG (SEQ ID NO: 150), or GGGSGGGGSG (SEQ ID NO: 246).

[0013] In certain embodiments, the antigen binding protein comprises a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single chain Fv (scFv), a dAb fragment, a single domain antibody, or a nanobody.

[0014] In certain embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 56-71.

[0015] In certain embodiments, the antigen binding protein further comprises the amino acid sequence of any one of SEQ ID NOs: 72-78.

[0016] In certain embodiments, the antigen binding protein comprises the antibody heavy chain (HC) amino acid sequence of SEQ ID NO: 45, 46, 49, 53, or 54, and the antibody light chain (LC) amino acid sequence of SEQ ID NO: 47, 48, 50, 51, 52, and 55.

[0017] In certain embodiments of the antigen binding protein, a) the antibody HC comprises the amino acid sequence of SEQ ID NO: 45 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 47; b) the antibody HC comprises the amino acid sequence of SEQ ID NO: 45 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 48; c) the antibody HC comprises the amino acid sequence of SEQ ID NO: 46 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 47; d) the antibody HC comprises the amino acid sequence of SEQ ID NO: 46 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 48; e) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 50; f) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 51; g) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 52; h) the antibody HC comprises the amino acid sequence of SEQ ID NO: 53 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 55; or i) the antibody HC comprises the amino acid sequence of SEQ ID NO:54 and the antibody LC comprises the amino acid sequence of SEQ ID NO:55.

[0018] In a specific embodiment, the antigen binding protein comprises the amino acid sequence of SEQ ID NO:189.

[0019] In a specific embodiment, the antigen binding protein comprises the amino acid sequence of SEQ ID NO:213.

[0020] In a specific embodiment, the antigen binding protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:178 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:159.

[0021] In a specific embodiment, the antigen binding protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:193 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:161.

[0022] In a specific embodiment, the antigen binding protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:179 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:159.

[0023] In a specific embodiment, the antigen binding protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:194 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:162.

[0024] In a specific embodiment, the antigen binding protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:238, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:159, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:239.

[0025] In certain embodiments, the antigen binding protein is about 5×10 -8 Less than M, approx. 1×10 -8 Less than M, about 5 x 10 -9 Less than M, approx. 1×10 -9 Less than M, about 5 x 10 -10 Less than M or about 1×10 -10 Binds to human uPAR with a KD of less than M.

[0026] In certain embodiments, the antigen binding protein binds to human uPAR in the presence of a uPAR ligand.

[0027] In a specific embodiment, the antigen binding protein binds to human uPAR bound to a uPAR ligand.

[0028] In certain embodiments, the antigen binding protein binds to human uPAR on human uPAR-expressing cells, thereby generating a fluorescent signal that is detected by a fluorescently labeled antibody that binds to the antigen binding protein, and has an EC50 value of about 0.1 nM to about 3.0 nM.

[0029] In certain embodiments, the antigen binding protein binds to human uPAR on human uPAR-expressing cells in the presence of a uPAR ligand.

[0030] In a specific embodiment, the antigen binding protein binds to the human uPAR DII-DIII domain or the human uPAR DIII domain.

[0031] In one aspect, the disclosure provides an isolated antigen binding protein that competes for binding with the antigen binding protein described above.

[0032] In another aspect, the disclosure provides an isolated antigen binding protein thereof that binds to the same epitope as the antigen binding protein described above.

[0033] In one aspect, the disclosure provides a pharmaceutical composition comprising an antigen binding protein as described above and a pharma- ceutically acceptable carrier or diluent.

[0034] In one aspect, the disclosure provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding an antigen binding protein as described above.

[0035] In one aspect, the disclosure provides an expression vector comprising the above-described polynucleotide sequence.

[0036] In one aspect, the disclosure provides a host cell comprising the above-described expression vector.

[0037] In another aspect, the disclosure provides a method of producing an antigen binding protein as described above, comprising culturing a host cell as described above under conditions to express the antigen binding protein.

[0038] In certain embodiments, the method further comprises isolating the antigen binding protein from the host cell.

[0039] In one aspect, the disclosure provides for the use of an antigen binding protein that specifically binds to uPAR for treating or preventing a disease or disorder associated with uPAR activity or expression, comprising administering a therapeutically effective amount of the pharmaceutical composition described above to a subject in need of such treatment.

[0040] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the pharmaceutical composition described above.

[0041] In one aspect, the disclosure provides a fusion protein comprising: (i) an antigen binding protein as described above; and (ii) a cytokine or a variant thereof.

[0042] In certain embodiments of the fusion protein, the antigen binding protein and the cytokine or variant thereof are linked by an amino acid linker.

[0043] In certain embodiments of the fusion protein, the cytokine or variant thereof is selected from the group consisting of IL-2, circularly permuted IL-2 (cpIL-2), IL-15, circularly permuted IL15 (cpIL-15), IL-6, circularly permuted IL-6 (cpIL-6), IL-10, or IL-12.

[0044] In certain embodiments of the fusion protein, the cytokine or variant thereof comprises at least one interleukin-12 (IL-12) subunit.

[0045] In certain embodiments of the fusion protein, the IL-12 subunits include one or both of p35 and p40.

[0046] In certain embodiments of the fusion protein, p35 comprises one or more amino acid substitutions at positions M12, S27, N28, Q35, F39, S44, C74, M111, V114, M119, M145, F150, L161, and D188 of SEQ ID NO:81.

[0047] In certain embodiments of the fusion protein, the amino acid substitutions include M12S, S27D, N28R, Q35E, F39D or F39H, S44K, C74K, C74D, C74W, C74Y or C74Q, M111F or M111W, V114I, M119L, M145L, F150Y, L161D or L161Q, D188N, or a combination thereof.

[0048] In a particular embodiment of the fusion protein, the amino acid substitutions include N28R and Q35E.

[0049] In a particular embodiment of the fusion protein, the amino acid substitutions include S27D and D188N.

[0050] In a particular embodiment of the fusion protein, the amino acid substitution comprises C74K.

[0051] In a particular embodiment of the fusion protein, the amino acid substitutions include S27D, C74K, and D188N.

[0052] In certain embodiments of the fusion protein, p35 comprises the amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.

[0053] In a particular embodiment of the fusion protein, p40 comprises one or more amino acid substitutions at positions C177 and C252 of SEQ ID NO:84.

[0054] In certain embodiments of the fusion protein, the amino acid substitution at C177 is selected from the group consisting of C177S, C177F, C177M, C177H, C177I, or C177Q.

[0055] In a particular embodiment of the fusion protein, the amino acid substitution at C252 is C252S.

[0056] In certain embodiments of the fusion protein, p40 comprises the amino acid sequence of SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87.

[0057] In certain embodiments of the fusion protein, the cytokine or variant thereof comprises a single chain IL-12 (scIL-12) comprising the p35 and p40 subunits joined by an amino acid linker.

[0058] In a particular embodiment of the fusion protein, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 156).

[0059] In certain embodiments of the fusion protein, the scIL-12 comprises the amino acid sequence of SEQ ID NO:88 or SEQ ID NO:89.

[0060] In one aspect, the present disclosure provides a pharmaceutical composition comprising the fusion protein described above and a pharma- ceutically acceptable carrier or diluent.

[0061] In one aspect, the disclosure provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding the above-described fusion protein.

[0062] In one aspect, the disclosure provides an expression vector comprising the above-described polynucleotide sequence.

[0063] In one aspect, the disclosure provides a host cell comprising the above-described expression vector.

[0064] In another aspect, the disclosure provides a method of producing the above-mentioned fusion protein, comprising culturing the above-mentioned host cell under conditions to express the fusion protein.

[0065] In certain embodiments, the method further comprises isolating the fusion protein from the host cell.

[0066] In one aspect, the present disclosure provides the use of a fusion protein that specifically binds to uPAR for treating or preventing a disease or disorder associated with uPAR activity or expression, comprising administering a therapeutically effective amount of the above-mentioned pharmaceutical composition or the above-mentioned fusion protein to a subject in need of such treatment.

[0067] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the above-described pharmaceutical composition or the above-described fusion protein.

[0068] In certain embodiments, the pharmaceutical composition or fusion protein is administered simultaneously with a cytokine or a variant thereof or a second fusion protein, where the second fusion protein comprises a different cytokine or variant thereof than the first fusion protein.

[0069] In certain embodiments, the pharmaceutical composition or fusion protein is administered sequentially with a cytokine or a variant thereof or a second fusion protein, where the second fusion protein comprises a different cytokine or variant thereof than the first fusion protein.

[0070] In certain embodiments, the cytokine or variant thereof comprises the IL-12 p35 subunit or the IL-12 p40 subunit.

[0071] In certain embodiments, the clearance rate of the fusion protein in the patient's tumor is slower than the clearance rate of the fusion protein in the patient's serum.

[0072] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a fusion protein and an unfused IL-12 subunit; (a) The fusion protein comprises: (i) an antigen-binding protein that specifically binds to the urokinase plasminogen activator receptor (uPAR); (ii) an IL-12 p35 subunit or a variant thereof, or an IL-12 p40 subunit or a variant thereof; (b) the non-fused IL-12 subunit comprises an IL-12 p35 subunit or a variant thereof, or an IL-12 p40 subunit or a variant thereof; Herein, methods are provided wherein when the fusion protein comprises the IL-12 p35 subunit or a variant thereof, the unfused IL-12 subunit comprises the IL-12 p40 subunit or a variant thereof, and when the fusion protein comprises the IL-12 p40 subunit or a variant thereof, the unfused IL-12 subunit comprises the IL-12 p35 subunit or a variant thereof.

[0073] In certain embodiments, the fusion protein and the unfused IL-12 subunit are administered sequentially.

[0074] In a specific embodiment, one or both of the fusion protein and the unfused IL-12 subunit are administered intravenously.

[0075] In certain embodiments, the fusion protein is administered first, followed by administration of the unfused IL-12 subunit after a sufficient amount of time has passed to allow the fusion protein to accumulate in the patient's tumor.

[0076] In certain embodiments, the fusion protein is administered first, followed by administration of the unfused IL-12 subunit after a sufficient amount of time has passed such that the concentration of the fusion protein has substantially decreased in the patient's serum.

[0077] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a first fusion protein and a second fusion protein; (a) a first fusion protein comprising: (i) an antigen binding protein that specifically binds to a first epitope on a urokinase plasminogen activator receptor (uPAR); (ii) an IL-12 p35 subunit or a variant thereof; (b) the second fusion protein is (i) an antigen binding protein that specifically binds to a second epitope on uPAR; (ii) an IL-12 p40 subunit or a variant thereof.

[0078] In certain embodiments, the first epitope and the second epitope are the same.

[0079] In certain embodiments, the first epitope and the second epitope are different.

[0080] In certain embodiments, the first fusion protein and the second fusion protein are administered sequentially.

[0081] In certain embodiments, one or both of the first and second fusion proteins are administered intravenously.

[0082] In certain embodiments, a first fusion protein is administered first, followed by administration of a second fusion protein after a sufficient amount of time has passed to allow the first fusion protein to accumulate in the patient's tumor.

[0083] In certain embodiments, a first fusion protein is administered first, followed by administration of a second fusion protein after a sufficient amount of time has passed for the concentration of the first fusion protein to be substantially reduced in the patient's serum.

[0084] In certain embodiments, the second fusion protein is administered first, followed by administration of the first fusion protein after a sufficient amount of time has passed to allow the second fusion protein to accumulate in the patient's tumor.

[0085] In certain embodiments, the second fusion protein is administered first, followed by the first fusion protein after a sufficient amount of time has passed for the concentration of the second fusion protein to be substantially reduced in the patient's serum.

[0086] In certain embodiments, one or both of the first and second fusion proteins comprises: a) a VH domain comprising an HCDR1 amino acid sequence of GFNIKDEY (SEQ ID NO: 3), an HCDR2 amino acid sequence of IDPENGDT (SEQ ID NO: 4), and an HCDR3 amino acid sequence of TGGNYVGWFPY (SEQ ID NO: 5); b) an antigen-binding protein comprising a VL domain comprising an LCDR1 amino acid sequence of SSVSY (SEQ ID NO: 6), an LCDR2 amino acid sequence of DTS (SEQ ID NO: 7), and an LCDR3 amino acid sequence of QQWSSNPPY (SEQ ID NO: 8).

[0087] In certain embodiments, one or both of the first and second fusion proteins comprises: a) a VH domain comprising an HCDR1 amino acid sequence of GNTFTDYG (SEQ ID NO: 19) or GTTFTDYG (SEQ ID NO: 41), an HCDR2 amino acid sequence of INTNTGEP (SEQ ID NO: 20), and an HCDR3 amino acid sequence of AHYSFDY (SEQ ID NO: 21); b) an antigen-binding protein comprising a VL domain comprising an LCDR1 amino acid sequence of ENIYSN (SEQ ID NO: 22), an LCDR2 amino acid sequence of AAT (SEQ ID NO: 23), and an LCDR3 amino acid sequence of QHFWGTPWT (SEQ ID NO: 24).

[0088] In certain embodiments, one or both of the first and second fusion proteins comprises: a) a VH domain comprising an HCDR1 amino acid sequence of GYTFTSYG (SEQ ID NO: 27), an HCDR2 amino acid sequence of IYPRSGNT (SEQ ID NO: 28), and an HCDR3 amino acid sequence of AGKDYGSTYADY (SEQ ID NO: 29); b) an antigen-binding protein comprising a VL domain comprising an LCDR1 amino acid sequence of SSVSSRY (SEQ ID NO: 30), an LCDR2 amino acid sequence of GTS (SEQ ID NO: 31), and an LCDR3 amino acid sequence of QQYHSDPLT (SEQ ID NO: 32).

[0089] In one aspect, the disclosure provides a combination comprising a first fusion protein and a second fusion protein, (a) a first fusion protein comprising: (i) an antigen binding protein that specifically binds to a first epitope on a urokinase plasminogen activator receptor (uPAR); (ii) an IL-12 p35 subunit or a variant thereof; (b) the second fusion protein is (i) an antigen binding protein that specifically binds to a second epitope on uPAR; (ii) IL-12 p40 subunit or a variant thereof.

[0090] In certain embodiments, the first epitope and the second epitope are the same.

[0091] In certain embodiments, the first epitope and the second epitope are different.

[0092] In one aspect, the disclosure provides a fusion protein comprising: (i) an antigen binding protein that specifically binds to urokinase plasminogen activator receptor (uPAR); and (ii) a cytokine or a variant thereof.

[0093] In certain embodiments, the antigen binding protein and the cytokine or variant thereof are linked by an amino acid linker.

[0094] In certain embodiments, the cytokine or variant thereof is selected from the group consisting of IL-2, circularly permuted IL-2 (cpIL-2), IL-15, circularly permuted IL15 (cpIL-15), IL-6, circularly permuted IL-6 (cpIL-6), IL-10, or IL-12.

[0095] In certain embodiments, the cytokine or variant thereof comprises at least one interleukin-12 (IL-12) subunit.

[0096] In certain embodiments, the IL-12 subunits include one or both of p35 and p40.

[0097] In certain embodiments, p35 comprises one or more amino acid substitutions at positions M12, S27, N28, Q35, F39, S44, C74, M111, V114, M119, M145, F150, L161, and D188 of SEQ ID NO:81.

[0098] In certain embodiments, the amino acid substitutions include M12S, S27D, N28R, Q35E, F39D or F39H, S44K, C74K, C74D, C74W, C74Y or C74Q, M111F or M111W, V114I, M119L, M145L, F150Y, L161D or L161Q, D188N, or a combination thereof.

[0099] In certain embodiments, the amino acid substitutions include N28R and Q35E.

[0100] In a particular embodiment, the amino acid substitutions include S27D and D188N.

[0101] In certain embodiments, the amino acid substitution comprises C74K.

[0102] In certain embodiments, the amino acid substitutions include S27D, C74K, and D188N.

[0103] In certain embodiments, p35 comprises the amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.

[0104] In certain embodiments, p40 comprises one or more amino acid substitutions at positions C177 and C252 of SEQ ID NO:84.

[0105] In certain embodiments, the amino acid substitution at C177 is selected from the group consisting of C177S, C177F, C177M, C177H, C177I, or C177Q.

[0106] In a specific embodiment, the C252 amino acid substitution is C252S.

[0107] In certain embodiments, p40 comprises the amino acid sequence of SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87.

[0108] In certain embodiments, the cytokine or variant thereof comprises a single chain IL-12 (scIL-12) comprising the p35 and p40 subunits linked by an amino acid linker.

[0109] In certain embodiments, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 156).

[0110] In certain embodiments, the scIL-12 comprises the amino acid sequence of SEQ ID NO:88 or SEQ ID NO:89.

[0111] In certain embodiments, the antigen binding protein is (a) i) the HCDR1 amino acid sequence of GFNIKDEY (SEQ ID NO: 3), GFSLTNYG (SEQ ID NO: 11), GNTFTDYG (SEQ ID NO: 19), GTTFTDYG (SEQ ID NO: 41), or GYTFTSYG (SEQ ID NO: 27); ii) the HCDR2 amino acid sequence of IDPENGDT (SEQ ID NO: 4), IWSDGGT (SEQ ID NO: 12), INTNTGEP (SEQ ID NO: 20), or IYPRSGNT (SEQ ID NO: 28); and iii) an antibody heavy chain variable (VH) domain comprising an HCDR3 amino acid sequence of TGGNYVGWFPY (SEQ ID NO: 5), ARGGRSDLFAY (SEQ ID NO: 13), AHYSFDY (SEQ ID NO: 21), or AGKDYGSTYADY (SEQ ID NO: 29); (b) iv) LCDR1 amino acid sequences of SSVSY (SEQ ID NO: 6), QSIVHSNGNTY (SEQ ID NO: 14), ENIYSN (SEQ ID NO: 22), SSVSSRY (SEQ ID NO: 30); v) the LCDR2 amino acid sequence of DTS (SEQ ID NO: 7), KVS (SEQ ID NO: 15), AAT (SEQ ID NO: 23), or GTS (SEQ ID NO: 31), and vi) an antibody light chain variable (VL) domain comprising an LCDR3 amino acid sequence of QQWSSNPPY (SEQ ID NO: 8), FQGSHVPYT (SEQ ID NO: 16), QHFWGTPWT (SEQ ID NO: 24), or QQYHSDPLT (SEQ ID NO: 32).

[0112] In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:42, or SEQ ID NO:43, and the VL domain comprises the amino acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:44.

[0113] In certain embodiments of the fusion protein, a) the VH domain comprises an HCDR1 amino acid sequence of GFNIKDEY (SEQ ID NO: 3), an HCDR2 amino acid sequence of IDPENGDT (SEQ ID NO: 4), and an HCDR3 amino acid sequence of TGGNYVGWFPY (SEQ ID NO: 5); b) The VL domain comprises an LCDR1 amino acid sequence of SSVSY (SEQ ID NO: 6), an LCDR2 amino acid sequence of DTS (SEQ ID NO: 7), and an LCDR3 amino acid sequence of QQWSSNPPY (SEQ ID NO: 8).

[0114] In certain embodiments of the fusion protein, a) the VH domain comprises an HCDR1 amino acid sequence of GFSLTNYG (SEQ ID NO: 11), an HCDR2 amino acid sequence of IWSDGGT (SEQ ID NO: 12), and an HCDR3 amino acid sequence of ARGGRSDLFAY (SEQ ID NO: 13); b) The VL domain comprises an LCDR1 amino acid sequence of QSIVHSNGNTY (SEQ ID NO: 14), an LCDR2 amino acid sequence of KVS (SEQ ID NO: 15), and an LCDR3 amino acid sequence of FQGSHVPYT (SEQ ID NO: 16).

[0115] In certain embodiments of the fusion protein, a) the VH domain comprises an HCDR1 amino acid sequence of GNTFTDYG (SEQ ID NO: 19) or GTTFTDYG (SEQ ID NO: 41), an HCDR2 amino acid sequence of INTNTGEP (SEQ ID NO: 20), and an HCDR3 amino acid sequence of AHYSFDY (SEQ ID NO: 21); b) The VL domain comprises the LCDR1 amino acid sequence of ENIYSN (SEQ ID NO: 22), the LCDR2 amino acid sequence of AAT (SEQ ID NO: 23), and the LCDR3 amino acid sequence of QHFWGTPWT (SEQ ID NO: 24).

[0116] In certain embodiments of the fusion protein, a) the VH domain comprises an HCDR1 amino acid sequence of GYTFTSYG (SEQ ID NO: 27), an HCDR2 amino acid sequence of IYPRSGNT (SEQ ID NO: 28), and an HCDR3 amino acid sequence of AGKDYGSTYADY (SEQ ID NO: 29); b) The VL domain comprises an LCDR1 amino acid sequence of SSVSSRY (SEQ ID NO: 30), an LCDR2 amino acid sequence of GTS (SEQ ID NO: 31), and an LCDR3 amino acid sequence of QQYHSDPLT (SEQ ID NO: 32).

[0117] In certain embodiments of the fusion protein, a) the VH domain comprises the amino acid sequence of SEQ ID NO: 33 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; b) the VH domain comprises the amino acid sequence of SEQ ID NO: 33 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; c) the VH domain comprises the amino acid sequence of SEQ ID NO: 34 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; d) the VH domain comprises the amino acid sequence of SEQ ID NO: 34 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; e) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 38; f) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 39; g) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 40; h) the VH domain comprises the amino acid sequence of SEQ ID NO: 42 and the VL domain comprises the amino acid sequence of SEQ ID NO: 44; or i) the VH domain comprises the amino acid sequence of SEQ ID NO: 43 and the VL domain comprises the amino acid sequence of SEQ ID NO: 44.

[0118] In certain embodiments, the VH domain and the VL domain are linked by an amino acid linker.

[0119] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 148), where n is an integer from 1 to 5.

[0120] In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 149), GGSGGGGSGGGSGGGGSGGGGSGGGSGG (SEQ ID NO: 150), or GGGSGGGGSG (SEQ ID NO: 246).

[0121] In certain embodiments, the antigen binding protein comprises a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single chain Fv (scFv), a dAb fragment, a single domain antibody, or a nanobody.

[0122] In certain embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 56-71.

[0123] In certain embodiments, the antigen binding protein further comprises the amino acid sequence of any one of SEQ ID NOs: 72-78.

[0124] In certain embodiments, the fusion protein comprises an antibody heavy chain (HC) amino acid sequence of SEQ ID NO: 45, 46, 49, 53, or 54, and an antibody light chain (LC) amino acid sequence of SEQ ID NO: 47, 48, 50, 51, 52, and 55.

[0125] In certain embodiments of the fusion protein, a) the antibody HC comprises the amino acid sequence of SEQ ID NO: 45 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 47; b) the antibody HC comprises the amino acid sequence of SEQ ID NO: 45 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 48; c) the antibody HC comprises the amino acid sequence of SEQ ID NO: 46 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 47; d) the antibody HC comprises the amino acid sequence of SEQ ID NO: 46 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 48; e) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 50; f) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 51; g) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 52; h) the antibody HC comprises the amino acid sequence of SEQ ID NO: 53 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 55; or i) the antibody HC comprises the amino acid sequence of SEQ ID NO:54 and the antibody LC comprises the amino acid sequence of SEQ ID NO:55.

[0126] In a specific embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:189.

[0127] In a specific embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:213.

[0128] In a specific embodiment, the fusion protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:178 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:159.

[0129] In a specific embodiment, the fusion protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:193 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:161.

[0130] In a specific embodiment, the fusion protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:179 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:159.

[0131] In a specific embodiment, the fusion protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:194 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:162.

[0132] In a specific embodiment, the fusion protein comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:238, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:159, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:239.

[0133] In certain embodiments, the antigen binding protein is about 5×10 -8 Less than M, approx. 1×10 -8 Less than M, about 5 x 10 -9 Less than M, approx. 1×10 -9 Less than M, about 5 x 10 -10 Less than M or about 1×10 -10 Binds to human uPAR with a KD of less than M.

[0134] In certain embodiments, the antigen binding protein binds to human uPAR in the presence of a uPAR ligand.

[0135] In certain embodiments, the antigen binding protein binds to human uPAR bound to a uPAR ligand.

[0136] In certain embodiments, the antigen binding protein binds to human uPAR on human uPAR-expressing cells, thereby generating a fluorescent signal that is detected by a fluorescently labeled antibody that binds to the antibody or a fragment thereof, and has an EC50 value of about 0.1 nM to about 3.0 nM.

[0137] In certain embodiments, the antigen binding protein binds to human uPAR on human uPAR-expressing cells in the presence of a uPAR ligand.

[0138] In a specific embodiment, the antigen binding protein binds to the human uPAR DII-DIII domain or the human uPAR DIII domain.

[0139] In one aspect, the disclosure provides an engineered IL-12 p35 polypeptide comprising one or more amino acid substitutions at positions S27, N28, Q35, F39, S44, C74, M111, V114, M119, M145, F150, and L161 of SEQ ID NO:81.

[0140] In certain embodiments, the engineered IL-12 p35 polypeptide further comprises an amino acid substitution at one or both of S27 and D188.

[0141] In certain embodiments, the amino acid substitutions include M12S, S27D, N28R, Q35E, F39D or F39H, S44K, C74K, C74D, C74W, C74Y or C74Q, M111F or M111W, V114I, M119L, M145L, F150Y, L161D or L161Q, D188N, or a combination thereof.

[0142] In certain embodiments, the amino acid substitutions include N28R and Q35E.

[0143] In certain embodiments, the amino acid substitution comprises C74K.

[0144] In certain embodiments, the amino acid substitutions include S27D, C74K, and D188N.

[0145] In certain embodiments, the engineered IL-12 p35 polypeptide comprises the amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.

[0146] In another aspect, the disclosure provides a fusion protein comprising (i) an engineered IL-12 p35 polypeptide as described above, and (ii) one or more fusion partners.

[0147] In certain embodiments, one or more of the fusion partners includes a moiety that reduces aggregation of the engineered IL-12 p35 polypeptide.

[0148] In certain embodiments, one or more of the fusion partners includes a moiety that increases expression of the engineered IL-12 p35 polypeptide.

[0149] In certain embodiments, one or more of the fusion partners comprises a moiety that increases the serum half-life of the engineered IL-12 p35 polypeptide.

[0150] In certain embodiments, one or more of the fusion partners comprises a targeting moiety.

[0151] In certain embodiments, the one or more fusion partners comprises serum albumin, polyethylene glycol (PEG), or an antigen binding protein.

[0152] In certain embodiments, the antigen binding protein specifically binds to urokinase plasminogen activator receptor (uPAR).

[0153] In one aspect, the disclosure provides an engineered IL-12 p40 polypeptide comprising one or more amino acid substitutions at positions C177 and C252 of SEQ ID NO:84.

[0154] In certain embodiments, the amino acid substitution at C177 is selected from the group consisting of C177S, C177F, C177M, C177H, C177I, or C177Q.

[0155] In a specific embodiment, the C252 amino acid substitution is C252S.

[0156] In certain embodiments, p40 comprises the amino acid sequence of SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87.

[0157] In another aspect, the disclosure provides a fusion protein comprising (i) an engineered IL-12 p40 polypeptide as described above, and (ii) one or more fusion partners.

[0158] In certain embodiments, one or more of the fusion partners includes a moiety that reduces aggregation of the engineered IL-12 p40 polypeptide.

[0159] In certain embodiments, one or more of the fusion partners includes a moiety that increases expression of the engineered IL-12 p40 polypeptide.

[0160] In certain embodiments, one or more of the fusion partners comprises a moiety that increases the serum half-life of the engineered IL-12 p40 polypeptide.

[0161] In certain embodiments, one or more of the fusion partners comprises a targeting moiety.

[0162] In certain embodiments, the one or more fusion partners comprises serum albumin, polyethylene glycol (PEG), or an antigen binding protein.

[0163] In certain embodiments, the antigen binding protein specifically binds to urokinase plasminogen activator receptor (uPAR).

[0164] In one aspect, the disclosure provides a fusion protein comprising: (i) a urokinase plasminogen activator receptor (uPAR) binding polypeptide; and (ii) a cytokine or a variant thereof.

[0165] In certain embodiments, the uPAR binding polypeptide is linked to the cytokine by an amino acid linker.

[0166] In certain embodiments, the cytokine comprises a functional fragment thereof, a functional variant thereof, or a circularly permuted variant thereof.

[0167] In certain embodiments, the cytokine is linked, via one or more optional amino acid linkers, to all or a portion of the receptor for which the cytokine has binding specificity.

[0168] In certain embodiments, the uPAR binding polypeptide comprises a urokinase plasminogen activator (uPA) polypeptide or a variant thereof capable of specifically binding to uPAR.

[0169] In certain embodiments, the uPA variant is an amino terminal fragment (ATF) of uPA.

[0170] In certain embodiments, the ATF has an amino acid sequence set forth in SEQ ID NO:91, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:91.

[0171] In certain embodiments, the ATF of uPA contains one or more amino acid substitutions or deletions.

[0172] In certain embodiments, uPA has an amino acid sequence set forth in SEQ ID NO:90, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:90.

[0173] In a specific embodiment, the cytokine is circularly permuted IL-2 and the receptor is IL-2Rα.

[0174] In a specific embodiment, the cytokine is circularly permuted IL-15 and the receptor is IL-15Rα.

[0175] In a particular embodiment, the cytokine is circularly permuted IL-1 and the receptor is IL-1RI, IL-1RII.

[0176] In certain embodiments, the uPAR binding polypeptide is an anti-uPAR antigen binding protein.

[0177] In certain embodiments, the uPAR binding polypeptide is an scFv fragment of an anti-uPAR antibody.

[0178] In certain embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 98, 99, 100, 106, 107, 111, and 113.

[0179] In certain embodiments, the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 98, 99, 111, and 112.

[0180] In certain embodiments, the uPAR binding agents, cytokines and receptors are of human origin.

[0181] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the fusion protein described above.

[0182] In certain embodiments, the cancer is selected from the group consisting of melanoma, carcinoma, blastoma, and lymphoma.

[0183] In one aspect, the disclosure provides a method of treating an inflammatory disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the fusion protein described above.

[0184] In certain embodiments, the inflammatory disease is selected from the group consisting of IBD and RA.

[0185] In one aspect, the present disclosure provides a pharmaceutical composition comprising at least one fusion protein as described above, and optionally a pharma- ceutically acceptable excipient.

[0186] In one aspect, the present disclosure provides: via one or more optional amino acid linkers, (i) a cytokine or a variant thereof, or (ii) providing a fusion protein comprising a urokinase plasminogen activator receptor (uPAR) binding polypeptide linked to a checkpoint molecule modulator;

[0187] In certain embodiments, the cytokine comprises a functional fragment thereof, a functional variant thereof, or a circularly permuted variant thereof.

[0188] In certain embodiments, the cytokine is linked, via one or more optional amino acid linkers, to all or a portion of the receptor for which the cytokine has binding specificity.

[0189] In certain embodiments, the uPAR binding polypeptide comprises a urokinase plasminogen activator (uPA) polypeptide or a variant thereof capable of specifically binding to uPAR.

[0190] In certain embodiments, the uPA variant is an amino terminal fragment (ATF) of uPA.

[0191] In certain embodiments, the ATF has an amino acid sequence set forth in SEQ ID NO:91, or an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:91.

[0192] In certain embodiments, the ATF of uPA contains one or more amino acid substitutions or deletions.

[0193] In certain embodiments, the cytokine or checkpoint molecule modulator is selected from the group consisting of IL-2, circularly permuted IL-2 (cpIL-2), IL-15, circularly permuted IL15 (cpIL-15), IL-6, circularly permuted IL-6 (cpIL-6), IL-10, IL-12, IL-15, GM-CSF, IFNγ, IFNα, 4-1BBB (CD131) or its ligand, OX40 or its ligand, PD-1, PD-L1, an anti-PD-1 antibody, CTLA-4, an anti-CTLA4 antibody, GITR or its ligand, ICOS or its ligand, CD27, CD70, CD40 or its ligand.

[0194] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the fusion protein described above.

[0195] In certain embodiments, the cancer is selected from the group consisting of melanoma and lymphoma.

[0196] In one aspect, the disclosure provides a method of treating an inflammatory disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the fusion protein described above.

[0197] In certain embodiments, the inflammatory disease is selected from the group consisting of IBD and RA.

[0198] In one aspect, the present disclosure provides a pharmaceutical composition comprising at least one fusion protein as described above, and optionally a pharma- ceutically acceptable excipient.

[0199] In one aspect, the disclosure provides a method of immunotherapy for treating cancer in a patient, comprising administering to a patient in need thereof the fusion protein described above.

[0200] In certain embodiments, the method further comprises administering a targeted cancer therapy to the patient.

[0201] In certain embodiments, the method further comprises administering to the patient a conventional cytotoxic therapy selected from chemotherapy, radiation therapy, or both.

[0202] In certain embodiments, the method further comprises administering an immunotherapy selected from a cancer vaccine, a checkpoint inhibitor, or a monoclonal antibody.

[0203] In certain embodiments, the uPAR binding polypeptide comprises M25 or an amino acid sequence homologous to M25 with at least 80% identity at the amino acid level.

[0204] In certain embodiments, the uPAR binding polypeptide comprises P25 or an amino acid sequence homologous to P25 with at least 80% identity at the amino acid level. [Brief description of the drawings]

[0205] [Figure 1] Binding data of anti-uPAR antibodies to the DII-DIII domains of human uPAR (A) or the DIII domain alone (B) are shown. [Diagram 2] Binding data of anti-uPAR antibodies to human (A) and mouse (B) uPAR expressed on the surface of HEK293 cells engineered to express human or mouse uPAR are shown. Titrations of anti-uPAR antibodies were incubated with the cells and mean fluorescence intensity (MFI) values ​​were determined across the antibody concentrations tested. [Diagram 3] 1 shows Octet binding data between p35 C74 and p40 C177 variants. Along the X-axis, the first amino acid listed at each data point corresponds to the p35 C74 substitution, and the second amino acid listed at each data point corresponds to the p40 C177 substitution (i.e., the "KS" data point is the binding affinity between the p35 C74K and p40 C177S variants). [Figure 4] Schematic diagrams of several exemplary IL-12 subunit fusion proteins are shown. A shows a single chain human IL-12 p35 / p40 fusion protein. B shows a p35 / serum albumin (e.g., human serum albumin or mouse serum albumin) fusion protein. C shows a p35 / Fc CH2-CH3 domain fusion protein paired with a Fab / Fc CH2-CH3 domain fusion protein. Each Fc domain contains a knob-into-hole (KiH) mutation to facilitate heterodimerization. D shows a p40 / scFv fusion protein. E shows a p35 / tandem scFv (scFv2) fusion protein. F shows a p35 / Fc CH2-CH3 domain fusion protein paired with a scFv / Fc CH2-CH3 domain fusion protein. Each Fc domain contains a KiH mutation to facilitate heterodimerization. G shows a p40 / Fab domain fusion protein. H indicates a p40 / IgG fusion protein, in which the p40 protein is linked to the C-terminus of each CH3 domain of IgG. [Diagram 5] Tumor and serum pharmacokinetics of radiolabeled tumor-targeted IL-12 subunits p40 (A) and p35 (B) by PET / CT imaging. Tumor-targeted subunits were measured over time in tumors and blood of mice to determine % of maximum exposure. Tumor-targeted p40 was a fusion of anti-uPAR antibody M37 with p40 in Fab format. Tumor-targeted p35 was a fusion of anti-uPAR antibody M37 with p35 in KiH format. [Figure 6] Cell-based in vitro IL-12 activity data as measured by % Stat4 phosphorylation in CD8+ (A) and CD4+ (B) T cells are shown. IL-12 subunits were added to T cells at a 1:1 ratio across the indicated concentrations. [Figure 7] Cell-based in vitro IL-12 activity data, as measured by interferon (IFN) gamma secretion by T cells, are shown. IL-12 subunits were added to T cells at a 1:1 ratio across the indicated concentrations. [Figure 8] Shown is the accumulation of IL-12 subunits in tumors (A) and liver (B) of mouse tumor models. The IL-12 subunit molecules were non-targeted scIL12, targeted Fab-p40, non-targeted p35-MSA, targeted IgG-p40, and targeted p35-KiH. "%ID / g" corresponds to the percent of injected dose per gram of tissue. [Figure 9] Serum interferon (IFN) gamma concentrations in mice receiving scIL-12 or two doses of the combination of p35-MSA and p40-Fab are shown. [Figure 10] Cell-based in vitro IL-12 activity data as measured by % Stat4 phosphorylation in CD8+ T cells in uPAR-expressing HEK / T cell co-cultures are shown. IL-12 subunits were added at a 1:1 ratio across the indicated concentrations to uPAR-expressing HEK cells and then co-incubated with CD8+ T cells. [Figure 11]1 shows the accumulation of specific fusion proteins in tumor tissues in an RKO xenograft tumor mouse model. [Figure 12] 1 shows accumulation of specific fusion proteins in serum in an RKO xenograft tumor mouse model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0206] Provided herein are urokinase plasminogen activator receptor (uPAR) binding polypeptides (e.g., uPAR antigen binding proteins) and fusion proteins comprising the uPAR binding polypeptides and a cytokine or variant thereof. The disclosure further provides pharmaceutical compositions comprising the uPAR binding polypeptides and fusion proteins of the disclosure, and methods of treating cancer therewith. The disclosure further provides engineered IL-12 p35 and p40 polypeptides.

[0207] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification, unless otherwise specified. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0208] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or extrinsic definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" is not limiting, unless otherwise indicated, the use of the term "including" to mean "and / or", as well as other forms such as "include" and "included".

[0209] In order that the present invention may be more readily understood, certain terms are first defined.

[0210] Antigen-binding proteins As used herein, the term "antibody" or "antigen binding protein" refers to an immunoglobulin molecule or immunoglobulin-derived molecule that specifically binds or is immunologically reactive with an antigen or epitope, including both polyclonal and monoclonal antibodies, and also includes functional antibody fragments, including, but not limited to, fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain variable fragments (scFv), and single domain antibody (e.g., sdAb, sdFv, nanobody, VHH) fragments. Thus, an antibody is a single domain antibody or comprises at least one variable light chain and at least one variable heavy chain. In one embodiment, at least one variable light chain and at least one variable heavy chain are linked as a single polypeptide chain. The term "antibody" or "antigen binding protein" includes germline derived antibodies. The term "antibody" or "antigen binding protein" includes engineered or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, tandem tri-scFv), etc. Unless otherwise indicated, the term "antibody" or "antigen binding protein" should be understood to encompass functional antibody fragments thereof.

[0211] In certain embodiments, the antigen binding protein is multispecific (i.e., binds to two or more different target molecules or to two or more epitopes on the same target molecule). In certain embodiments, the antigen binding protein is bispecific, e.g., binds to two different target molecules or to two epitopes on the same target molecule. In certain embodiments, the antibody is trispecific, e.g., binds to at least three different target molecules.

[0212] Antigen-binding proteins can be monovalent or multivalent, i.e., have one or more antigen-binding sites. Non-limiting examples of monovalent antigen-binding proteins include scFv, Fab, scFab, dAb, VHH, V(NAR), DARPin, affilin, and nanobody. Multivalent antigen-binding proteins can have two, three, four or more antigen-binding sites. Non-limiting examples of multivalent antigen-binding proteins include full-length immunoglobulins, F(ab')2 fragments, bis-scFv (or tandem scFv or BiTE), DART, diabody, scDb, DVD-Ig, IgG-scFab, scFab-Fc-scFab, IgG-scFv, scFv-Fc, scFv-fc-scFv, Fv2-Fc, FynomAB, quadroma, CrossMab, DuoBody, triabody, and tetrabody. In some embodiments, the multivalent antigen-binding protein is bivalent, i.e., there are two binding sites. In some embodiments, the multivalent antigen-binding protein is bispecific, i.e., the antigen-binding protein is directed to two different targets or two different target sites on one target molecule. In some embodiments, the multivalent antigen-binding protein comprises more than two, e.g., three or four different binding sites for three or four different antigens, respectively. Such antigen-binding proteins are multivalent and multispecific, in particular trispecific or tetraspecific, respectively.

[0213] As used herein, a "single-chain variable fragment" (scFv) is an antigen-binding protein comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL). The VH and VL domains of the scFv are linked via any suitable linker recognized in the art. Such linkers include, but are not limited to, repeating GGGGS amino acid sequences or variants thereof. Although scFvs generally do not comprise an antibody constant domain region, the scFvs of the present disclosure may be linked or conjugated to an antibody constant domain region (e.g., an antibody Fc domain) to modify various properties of the scFv, for example, but not limited to, to increase serum or tissue half-life. An scFv generally has a molecular weight of about 25 kDa and a hydrodynamic radius of about 2.5 nm. In certain embodiments, the antigen-binding protein comprises a tandem scFv, i.e., a first scFv is linked to a second scFv. The first and second scFv may be of the same amino acid sequence and / or may bind to the same epitope. Alternatively, the first and second scFv may be different amino acid sequences and / or may bind to different epitopes.

[0214] As used herein, a "Fab fragment" or "Fab" is an antibody fragment containing the variable light chain (VL) domain and the light chain constant domain (CL), and the variable heavy chain (VH) domain and the first constant domain of the heavy chain (CH1).

[0215] As used herein, a "VHH", "nanobody" or "heavy chain only antibody" is an antigen binding protein that contains a single heavy chain variable domain derived from a species of the Camelidae family, including camels, llamas and alpacas. VHHs generally have a molecular weight of about 15 kDa.

[0216] In one embodiment, the antigen binding protein comprises an Fc domain. The presence of an Fc domain may be advantageous for inducing a cytotoxic immune response and / or activating complement (e.g., ADCC / ADCP or CDC effector function). Exemplary antibody formats that include an Fc domain include, but are not limited to, full-length immunoglobulins, DVD-Ig, scFv-Fc and scFv-Fc.scFv fusions, IgG-scFab, scFab-dsscFv, Fv2-Fc, IgG-scFv fusions (e.g., bsAb, Bs1Ab, Bs2Ab, Bs3Ab, Ts1Ab, Ts2Ab, knob-into-hole (KiH), etc.), DuoBody, and / or CrossMab. An active Fc domain may increase the likelihood of pro-inflammatory cytokine release by T cells and other effector cells in the tumor microenvironment, which is believed to be part of the therapeutic mechanism of action. The Fc domain may be fully activated or partially silenced to avoid overstimulation of the immune system. In some embodiments, the Fc domain is inactive and does not stimulate pro-inflammatory cytokine release, but maintains improved half-life and / or stability of the antigen binding protein. In some embodiments, the antigen binding protein comprises a constant region selected from the group consisting of human IgG1, IgG2, IgG3 or IgG4 isotypes. In other embodiments, the antigen binding protein comprises a constant region selected from the group consisting of mouse IgG1, IgG2A, IgG2B or IgG3 isotypes.

[0217] The antigen binding proteins of the disclosure may comprise one or more linkers to link domains of the antigen binding protein (e.g., linking a VH and a VL to form an scFv, or linking multiple binding domains to form a multispecific antigen binding protein).

[0218] Illustrative examples of linkers include glycine polymers (Gly). n glycine-serine polymer (Gly n Ser) nwhere n is an integer of at least 1, 2, 3, 4, 5, 6, 7, or 8; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art.

[0219] Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to function as neutral tethers between domains of fusion proteins such as the antigen binding proteins described herein. Glycine has access to much more Φ-Ψ space than other small side chain amino acids and is much less restrictive than residues with longer side chains (Scheraga, Rev. Computational Chem. 1:1173-142 (1992)). Those skilled in the art will recognize that the design of antigen binding proteins in certain embodiments may include fully or partially flexible linkers, such that the linker may include not only stretches of flexible linkers but also one or more stretches that confer low flexibility to provide the desired structure.

[0220] However, the linker sequence can be selected to resemble a natural linker sequence, for example using an amino acid stretch corresponding to the first part of the human CH1 and Cκ sequences or the lower part of the hinge region of human IgG.

[0221] In the scFv portion, the peptide linker design connecting the VL and VH domains is generally a flexible linker consisting of small non-polar or polar residues such as, for example, Gly, Ser and Thr. A particular exemplary linker connecting the variable domains of the scFv portion is the (Gly4Ser)4 linker, where 4 is an exemplary number of repeats of the motif. In a particular embodiment, the linker comprises the amino acid sequence GGSGGGGSGGGGSGGGGSGGGSGGGSGG, GGGGSGGGGSGGGGGS, or GGGSGGGGSG (SEQ ID NO: 246).

[0222] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 148), where n is an integer from 1 to 5. In certain embodiments, the amino acid linker comprises (GGGS)n (SEQ ID NO: 247), where n is an integer from 1 to 5.

[0223] Other exemplary linkers include the following amino acid sequences: GGG; DGGGS; TGEKP (Liu et al., Proc. Natl. Acad. Sci. 94:5525-5530 (1997)); GGRR; (GGGGS) n (SEQ ID NO: 148) (n=1, 2, 3, 4 or 5) (Kim et al, Proc. Natl. Acad. Sci. 93:1156-1160(1996)); EGKSSGSGSESKVD (Chaudhary et al., Proc. Natl. Acad. Sci. 87:1066-1070(1990)); KESGSVGSSEQLAQFRSLD (Bird et al., Science 242:423-426(1988)), GGRRGGGS; LQRDGERP; LRQKDGGGSERP; and GSTSGSGKPGSGEGSTKG (Cooper et al., Blood, 101(4):1637-1644(2003)). Alternatively, flexible linkers can be rationally designed using computer programs capable of modeling the 3D structures of proteins and peptides, or by phage display methods.

[0224] An antibody may comprise a variable light (VL) domain and a variable heavy (VH) domain, each of which further comprises a set of three CDRs.

[0225] As used herein, the term "complementarity determining region" or "CDR" refers to a non-contiguous sequence of amino acids in an antibody variable region that confers antigen specificity and binding affinity. Generally, there are three CDRs in each heavy chain variable domain (HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable domain (LCDR1, LCDR2, LCDR3). "Framework region" or "FR" is known in the art to refer to the portion of the heavy and light chain variable domains other than the CDRs. Generally, there are four FRs in each heavy chain variable domain (HFR1, HFR2, HFR3, and HFR4) and four FRs in each light chain variable domain (LFR1, LFR2, LFR3, and LFR4). Thus, an antibody variable region amino acid sequence can be represented by the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Each segment of the formula, i.e., FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, represents a separate amino acid sequence (or a polynucleotide sequence encoding same), and may be mutated, including one or more amino acid substitutions, deletions, and insertions. In certain embodiments, the antibody variable light chain amino acid sequence may be represented by the formula LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. In certain embodiments, the antibody variable heavy chain amino acid sequence may be represented by the formula HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0226] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of several well-known schemes, including those described in Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (the “Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (the “Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745. (the “Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol,2003 January;27(1):55-77 (the "IMGT" numbering scheme), and Honegger A and Pluckthun A,“Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool,” J Mol Biol,2001 June.8;309(3):657-70, (the "AHo" numbering scheme).

[0227] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence length, and some antibodies have insertions and deletions provided by the insertion letter, e.g., "30a". The two schemes place certain insertions and deletions ("indels") at different positions, which results in numbering differences. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0228] Thus, unless otherwise specified, the "CDR" or "complementarity determining region" of an antibody or fragment thereof, such as the variable domain thereof, or each particular CDR (e.g., HCDR1, HCDR2) is understood to encompass the complementarity determining region (or particular complementarity determining region) defined by any of the known schemes. Similarly, unless otherwise specified, the "FR" or "framework region" of an antibody or fragment thereof, such as the variable domain thereof, or each particular FR (e.g., "HFR1", "HFR2") is understood to encompass the framework region (or particular framework region) defined by any of the known schemes. In some cases, the scheme for identifying a particular CDR or FR is designated as the CDR defined by Kabat, Chothia, or Contact methods. In other cases, the particular amino acid sequence of the CDR or FR is given.

[0229] In certain embodiments, the antigen-binding proteins disclosed herein are humanized. As used herein, the term "humanized" or "humanization" refers to antigen-binding proteins that have been modified to be more similar to human antibodies. Non-human antigen-binding proteins, such as mouse antigen-binding proteins disclosed herein, would provoke a negative immune response when administered to humans for treatment. Therefore, humanizing non-human (e.g., mouse) antigen-binding proteins is advantageous for subsequent therapeutic use.

[0230] In certain embodiments, antigen binding proteins are humanized by resurfacing (i.e., remodeling solvent-exposed residues of a non-human framework to make them more human-like). Resurfacing strategies are detailed in WO2004 / 016740, WO2008 / 144757, and WO2005 / 016950, each of which is incorporated herein by reference.

[0231] In certain embodiments, antigen binding proteins are humanized by CDR grafting (i.e., inserting mouse antigen binding protein CDRs into a human antibody acceptor framework).

[0232] As used herein, the term "affinity" refers to the strength of interaction between the antigen-binding site of an antibody and the epitope to which it binds. As will be readily understood by those skilled in the art, the affinity of an antibody or antigen-binding protein can be reported as a dissociation constant (KD) in molar units (M). The antibodies of the present disclosure have an affinity of 10 -8 ~10 -14 A high affinity antibody may have a KD value in the range of 10 -9 M (1 nanomolar, nM) or less. For example, a high affinity antibody may have a KD value in the range of about 1 nM to about 0.01 nM. A high affinity antibody may have a KD value of about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, or about 0.1 nM. An antibody with very high affinity may have a KD value of 10 -12M (1 picomolar, pM) or less. Weak or low affinity antibodies have a KD value of 10 -1 ~10 -4 Low affinity antibodies can have K values ​​in the range of 10 -4 M or more, e.g., 10 -4 M, 10 -3 M, 10 -2 M, or 10 -1 It may have a KD value of M.

[0233] The ability of an antibody to bind to a particular antigenic determinant (e.g., uPAR) can be measured by either enzyme-linked immunosorbent assay (ELISA) or other techniques known to those skilled in the art, such as surface plasmon resonance (SPR) technology (analysis on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and conventional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0234] The amino acid sequences of various antibody constant domains used in this disclosure are shown in Table 1 below. [Table 1-1] [Table 1-2]

[0235] Antigen-binding protein heterodimerization In one embodiment of the present disclosure, antigen-binding proteins comprising two Fc domains are heterodimerized by knob-into-hole pairing (KiH). This dimerization technique utilizes engineered protrusions ("knobs") and cavities ("holes") at the interface of the CH3 domains. If a knob or hole of suitable position and dimensions exists at the interface of either the first or second CH3 domain, one only needs to engineer a corresponding hole or knob, respectively, at the adjacent interface, thereby facilitating and enhancing pairing of the Fc domains at the CH3 / CH3 domain interface. A "knob" refers to at least one amino acid side chain, typically a large side chain, that protrudes from the interface of the CH3 portion of the first Fc domain. This protrusion is complementary to a "hole" in the CH3 portion of the second Fc domain, forming a "knob" that is received by the "hole". A "hole" is at least one amino acid side chain, typically a small side chain, that is recessed from the interface of the CH3 portion of the second Fc domain. This technique is described, for example, in U.S. Patent No. 5,821,333; Ridgway et al., Protein Engineering 9:617-621 (1996); and Carter P., J. Immunol. Methods 248:7-15 (2001).

[0236] Exemplary amino acid residues that may function as knobs include arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Amino acid residues present in the CH3 domain may be replaced or substituted with the amino acid residues of the knob. Preferred amino acids for substitution may include any amino acid that contains a small side chain, such as alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), or valine (V).

[0237] Exemplary amino acid residues that may function as holes include alanine (A), serine (S), threonine (T), and valine (V). Amino acid residues present in the CH3 domain may be replaced or substituted with hole amino acid residues. Preferred amino acids for substitution may include any amino acid that contains a large or bulky side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0238] The CH3 domain may be derived from a human IgG1 antibody. Exemplary amino acid substitutions for the CH3 domain include T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, and combinations thereof. A particular exemplary combination is a knob mutation T366Y or T366W for the first CH3 domain and a hole mutation Y407T or Y407V for the second CH3 domain.

[0239] In certain embodiments, the KiH Fc domain comprises a knob Fc portion comprising the amino acid substitution T366W and a hole Fc portion comprising the amino acid substitutions T366S, L368A, Y407V according to Eu numbering. In certain embodiments, the hole Fc portion further comprises the amino acid substitutions H435R and Y436F according to Eu numbering.

[0240] In certain embodiments of the present disclosure, antigen binding proteins comprising two Fc domains are heterodimerized by Fab arm exchange (FAE). Human IgG1 carrying a P228S hinge mutation may contain a F405L or K409R CH3 domain mutation. Mixing the two antibodies with a reducing agent results in FAE. This technique is described in U.S. Pat. No. 9,212,230 and Labrijn AF, Proc Natl Acad Sci USA 110(13):5145-5150 (2013).

[0241] In certain embodiments of the present disclosure, antigen-binding proteins comprising two Fc domains are heterodimerized by electrostatic steering effects. This dimerization technique utilizes electrostatic steering to promote and strengthen pairing of Fc domains at the CH3 / CH3 domain interface. The charge complementarity between the two CH3 domains is altered to favor heterodimerization (opposite charge pairing) over homodimerization (same charge pairing). In this method, electrostatic repulsion prevents homodimerization. Exemplary amino acid residue substitutions can include K409D, K392D, and / or K370D in the first CH3 domain, and D399K, E356K, and / or E357K in the second CH3 domain. This technique is described in U.S. Patent Application Publication No. 2014 / 0154254A1 and Gunasekaran K., J Biol Chem 285(25):19637-19646 (2010).

[0242] In certain embodiments of the present disclosure, antigen binding proteins comprising two Fc domains are heterodimerized by hydrophobic interactions. This dimerization technique utilizes hydrophobic interactions instead of electrostatic interactions to promote and strengthen the pairing of the Fc domains at the CH3 / CH3 domain interface. Exemplary amino acid residue substitutions can include K409W, K360E, Q347E, Y349S, and / or S354C in the first CH3 domain, and D399V, F405T, Q347R, E357W, and / or Y349C in the second CH3 domain. Preferred pairs of amino acid residue substitutions between the first and second CH3 domains include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technique is described in US Patent Application Publication No. 2015 / 0307628A1.

[0243] In a specific embodiment of the present disclosure, antigen binding proteins comprising two Fc domains are heterodimerized by using leucine zipper fusion. A leucine zipper domain fused to the C-terminus of each CH3 domain of the antibody chain forces heterodimerization. This technique is described in Wranik B., J Biol Chem 287(52):43331-43339(2012).

[0244] In a specific embodiment of the present disclosure, antigen binding proteins containing two Fc domains are heterodimerized by using a strand-exchange engineered domain (SEED) construct. Heterodimerization is forced by CH3 domains derived from IgG and IgA formats. This technique is described in Muda M., Protein Eng. Des. Sel. 24(5):447-454 (2011).

[0245] Unless otherwise indicated, antibody constant region numbering employed herein corresponds to the EU numbering scheme described in Edelman et al., Proc. Natl. Acad. Sci. USA 63(1):78-85 (1969).

[0246] Further methods for heterodimerization of heavy and / or light chains and for the production and purification of asymmetric antibodies are known in the art, see, e.g., Klein C., mABs 4(6):653-663 (2012), and U.S. Patent No. 9,499,634, each of which is incorporated herein by reference.

[0247] Additional Fc domain mutations are contemplated to alter Fc function, including but not limited to, alterations in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0248] In a particular embodiment, the antigen binding protein comprises an IgG1 constant domain comprising the L234A and L235A amino acid substitutions according to Eu numbering.

[0249] In a specific embodiment, the antigen binding protein comprises an IgG1 constant domain comprising a P329G amino acid substitution, according to Eu numbering.

[0250] Urokinase plasminogen activator receptor (uPAR) binding polypeptide As used herein, the terms "uPAR" or "urokinase plasminogen activator receptor" or "urokinase receptor" or "uPA receptor" or "CD87" or "cluster of differentiation 87" refer to targets of antigen-binding proteins described herein. uPAR is composed of three distinct domains of the Ly-6 / uPAR / alpha-neurotoxin family. All three domains are involved in the high affinity binding of its primary ligand, urokinase. In addition to its primary ligand, urokinase, uPAR interacts with several other proteins, including vitronectin, uPAR-associated protein (uPARAP), and the integrin family of membrane proteins.

[0251] As used herein, the term "binding polypeptide" or "binding protein" corresponds to a polypeptide that specifically binds to a target protein (e.g., uPAR). Binding polypeptides include, but are not limited to, the above antigen-binding proteins as well as ligands of target proteins. In certain embodiments, the binding polypeptide is a urokinase plasminogen activator (uPA) polypeptide or a variant thereof that can specifically bind to uPAR. In certain embodiments, the uPA variant is an amino acid terminal fragment (ATF) of uPA. In certain embodiments, the ATF has an amino acid sequence set forth in SEQ ID NO: 91 or an amino acid sequence having at least 80% identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the amino acid sequence set forth in SEQ ID NO: 91. In certain embodiments, the ATF of uPA includes one or more amino acid substitutions or deletions. In certain embodiments, uPA has an amino acid sequence set forth in SEQ ID NO:90, or an amino acid sequence having at least 80% identity (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the amino acid sequence set forth in SEQ ID NO:90.

[0252] In one aspect, the present disclosure provides an isolated antigen binding protein that specifically binds to urokinase plasminogen activator receptor (uPAR), comprising: (a) i) the HCDR1 amino acid sequence of GFNIKDEY (SEQ ID NO: 3), GFSLTNYG (SEQ ID NO: 11), GNTFTDYG (SEQ ID NO: 19), GTTFTDYG (SEQ ID NO: 41), or GYTFTSYG (SEQ ID NO: 27); ii) the HCDR2 amino acid sequence of IDPENGDT (SEQ ID NO: 4), IWSDGGT (SEQ ID NO: 12), INTNTGEP (SEQ ID NO: 20), or IYPRSGNT (SEQ ID NO: 28); and iii) an antibody heavy chain variable (VH) domain comprising an HCDR3 amino acid sequence of TGGNYVGWFPY (SEQ ID NO: 5), ARGGRSDLFAY (SEQ ID NO: 13), AHYSFDY (SEQ ID NO: 21), or AGKDYGSTYADY (SEQ ID NO: 29); (b) iv) the LCDR1 amino acid sequences of SSVSY (SEQ ID NO: 6), QSIVHSNGNTY (SEQ ID NO: 14), ENIYSN (SEQ ID NO: 22), SSVSSRY (SEQ ID NO: 30); v) the LCDR2 amino acid sequence of DTS (SEQ ID NO: 7), KVS (SEQ ID NO: 15), AAT (SEQ ID NO: 23), or GTS (SEQ ID NO: 31), and vi) an antibody light chain variable (VL) domain comprising an LCDR3 amino acid sequence of QQWSSNPPY (SEQ ID NO: 8), FQGSHVPYT (SEQ ID NO: 16), QHFWGTPWT (SEQ ID NO: 24), or QQYHSDPLT (SEQ ID NO: 32).

[0253] In certain embodiments, the antigen binding proteins of the disclosure comprise at least about 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence similarity or identity to any of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 amino acid sequences set forth above and in Tables 3 and 8.

[0254] In certain embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:42, or SEQ ID NO:43, and the VL domain comprises the amino acid sequence of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:44.

[0255] In certain embodiments, the antigen binding proteins of the disclosure comprise at least about 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence similarity or identity to any of the VH or VL amino acid sequences set forth above and in Tables 3 and 8.

[0256] In certain embodiments, a) the VH domain comprises an HCDR1 amino acid sequence of GFNIKDEY (SEQ ID NO: 3), an HCDR2 amino acid sequence of IDPENGDT (SEQ ID NO: 4), and an HCDR3 amino acid sequence of TGGNYVGWFPY (SEQ ID NO: 5); b) The VL domain comprises an LCDR1 amino acid sequence of SSVSY (SEQ ID NO: 6), an LCDR2 amino acid sequence of DTS (SEQ ID NO: 7), and an LCDR3 amino acid sequence of QQWSSNPPY (SEQ ID NO: 8).

[0257] In certain embodiments, a) the VH domain comprises an HCDR1 amino acid sequence of GFSLTNYG (SEQ ID NO: 11), an HCDR2 amino acid sequence of IWSDGGT (SEQ ID NO: 12), and an HCDR3 amino acid sequence of ARGGRSDLFAY (SEQ ID NO: 13); b) The VL domain comprises an LCDR1 amino acid sequence of QSIVHSNGNTY (SEQ ID NO: 14), an LCDR2 amino acid sequence of KVS (SEQ ID NO: 15), and an LCDR3 amino acid sequence of FQGSHVPYT (SEQ ID NO: 16).

[0258] In certain embodiments, a) the VH domain comprises an HCDR1 amino acid sequence of GNTFTDYG (SEQ ID NO: 19) or GTTFTDYG (SEQ ID NO: 41), an HCDR2 amino acid sequence of INTNTGEP (SEQ ID NO: 20), and an HCDR3 amino acid sequence of AHYSFDY (SEQ ID NO: 21); b) The VL domain comprises the LCDR1 amino acid sequence of ENIYSN (SEQ ID NO: 22), the LCDR2 amino acid sequence of AAT (SEQ ID NO: 23), and the LCDR3 amino acid sequence of QHFWGTPWT (SEQ ID NO: 24).

[0259] In certain embodiments, a) the VH domain comprises an HCDR1 amino acid sequence of GYTFTSYG (SEQ ID NO: 27), an HCDR2 amino acid sequence of IYPRSGNT (SEQ ID NO: 28), and an HCDR3 amino acid sequence of AGKDYGSTYADY (SEQ ID NO: 29); b) The VL domain comprises an LCDR1 amino acid sequence of SSVSSRY (SEQ ID NO: 30), an LCDR2 amino acid sequence of GTS (SEQ ID NO: 31), and an LCDR3 amino acid sequence of QQYHSDPLT (SEQ ID NO: 32).

[0260] In certain embodiments, a) the VH domain comprises the amino acid sequence of SEQ ID NO: 33 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; b) the VH domain comprises the amino acid sequence of SEQ ID NO: 33 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; c) the VH domain comprises the amino acid sequence of SEQ ID NO: 34 and the VL domain comprises the amino acid sequence of SEQ ID NO: 35; d) the VH domain comprises the amino acid sequence of SEQ ID NO: 34 and the VL domain comprises the amino acid sequence of SEQ ID NO: 36; e) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 38; f) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 39; g) the VH domain comprises the amino acid sequence of SEQ ID NO: 37 and the VL domain comprises the amino acid sequence of SEQ ID NO: 40; h) the VH domain comprises the amino acid sequence of SEQ ID NO: 42 and the VL domain comprises the amino acid sequence of SEQ ID NO: 44; or i) the VH domain comprises the amino acid sequence of SEQ ID NO: 43 and the VL domain comprises the amino acid sequence of SEQ ID NO: 44.

[0261] In certain embodiments, the VH domain and the VL domain are linked by an amino acid linker.

[0262] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 148), where n is an integer from 1 to 5.

[0263] In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 149) or GGSGGGGSGGGSGGGGSGGGGSGGGSGG (SEQ ID NO: 150).

[0264] In certain embodiments, the antigen binding protein comprises a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single chain Fv (scFv), a dAb fragment, a single domain antibody, or a nanobody.

[0265] In certain embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 56-71.

[0266] In certain embodiments, the antigen binding protein further comprises the amino acid sequence of any one of SEQ ID NOs: 72-78.

[0267] In certain embodiments, the antigen binding protein comprises the antibody heavy chain (HC) amino acid sequence of SEQ ID NO: 45, 46, 49, 53, or 54, and the antibody light chain (LC) amino acid sequence of SEQ ID NO: 47, 48, 50, 51, 52, and 55.

[0268] In certain embodiments, a) the antibody HC comprises the amino acid sequence of SEQ ID NO: 45 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 47; b) the antibody HC comprises the amino acid sequence of SEQ ID NO: 45 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 48; c) the antibody HC comprises the amino acid sequence of SEQ ID NO: 46 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 47; d) the antibody HC comprises the amino acid sequence of SEQ ID NO: 46 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 48; e) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 50; f) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 51; g) the antibody HC comprises the amino acid sequence of SEQ ID NO: 49 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 52; h) the antibody HC comprises the amino acid sequence of SEQ ID NO: 53 and the antibody LC comprises the amino acid sequence of SEQ ID NO: 55; or i) the antibody HC comprises the amino acid sequence of SEQ ID NO:54 and the antibody LC comprises the amino acid sequence of SEQ ID NO:55.

[0269] In certain embodiments, the antigen binding protein is about 5×10 -8 Less than M, approx. 1×10 -8 Less than M, about 5 x 10 -9 Less than M, approx. 1×10 -9 Less than M, about 5 x 10 -10 Less than M or about 1×10 -10 Binds to human uPAR with a KD of less than M.

[0270] In certain embodiments, the antigen binding protein binds to human uPAR in the presence of a uPAR ligand.

[0271] In a specific embodiment, the antigen binding protein binds to human uPAR bound to a uPAR ligand.

[0272] In certain embodiments, the antigen binding protein binds to human uPAR on human uPAR-expressing cells, thereby generating a fluorescent signal that is detected by a fluorescently labeled antibody that binds to the antigen binding protein, and has an EC50 value of about 0.1 nM to about 3.0 nM.

[0273] In certain embodiments, the antigen binding protein binds to human uPAR on human uPAR-expressing cells in the presence of a uPAR ligand.

[0274] In a specific embodiment, the antigen binding protein binds to the human uPAR DII-DIII domain or the human uPAR DIII domain.

[0275] In one aspect, the disclosure provides an isolated antigen binding protein that competes for binding with the antigen binding protein described above.

[0276] In one aspect, the disclosure provides an isolated antigen binding protein that binds to the same epitope as the antigen binding proteins described above.

[0277] In one aspect, the disclosure provides a pharmaceutical composition comprising an antigen binding protein as described above and a pharma- ceutically acceptable carrier or diluent.

[0278] In one aspect, the disclosure provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding an antigen binding protein as described above.

[0279] In one aspect, the disclosure provides an expression vector comprising the above-described polynucleotide sequence.

[0280] In one aspect, the disclosure provides a host cell comprising the above-described expression vector.

[0281] In another aspect, the present disclosure provides a method of producing an antigen binding protein as described above, comprising: (i) culturing said host cell under conditions allowing expression of the antigen binding protein; (ii) recovering the antigen binding protein, and optionally (iii) further purifying and / or modifying and / or formulating the antigen binding protein.

[0282] Cytokines and their variants As used herein, "cytokine" is a general term that includes "lymphokines" (cytokines made by lymphocytes), "monokines" (cytokines made by monocytes), "chemokines" (cytokines with chemotactic activity), and "interleukins" (cytokines made by one white blood cell that act on other white blood cells). Cytokines may act on the cell that secretes them (autocrine action), on neighboring cells (paracrine action), or possibly on distant cells (endocrine action). Proinflammatory cytokines are produced primarily by activated macrophages and dendritic cells and are involved in upregulating inflammatory responses, and include, but are not limited to, IL-1β, IL-6, and TNF-α. Chemokines are usually assigned to four groups depending on the spacing of the first two cysteine ​​residues: CC chemokines (e.g., RANTES, monocyte chemoattractant protein or MCP-1, monocyte inflammatory protein or MIP-1α, and MIP-1β), CXC chemokines (e.g., IL-8, also called growth-related oncogene or GRO / KC), C chemokines (e.g., lymphotactin), and CXXXC chemokines (e.g., fractalkine). Anti-inflammatory cytokines are a set of immunoregulatory molecules that suppress or control inflammatory responses. Cytokines cooperate with certain cytokine inhibitors and soluble cytokine receptors to regulate immune responses. Major anti-inflammatory cytokines include, but are not limited to, interleukin (IL)-1 receptor antagonist, IL-4, IL-10, IL-11, and IL-13. Leukemia inhibitory factor, interferon-alpha, IL-6, IL-10, and transforming growth factor (TGF)-β are classified as either anti- or pro-inflammatory cytokines under various circumstances. Specific cytokine receptors for IL-1, TNF-α, and IL-18 also function as inhibitors of pro-inflammatory cytokines.

[0283] The cytokine polypeptide of the fusion proteins disclosed herein can be any cytokine, including variants thereof, such as functional fragments thereof, or circularly permuted variants thereof. Useful cytokines include IL-1α, IL-1β, IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12p35, IL-12p40, IL-13, IL-15, IL-17 family members, IL-18, IL-21, IL-22, IL-23, IL-23p19, IL-30 (IL-27p28), IL-33, IL-34, IL-35, IL-35p35, IL-36Ra, IL-36a, IL-36b, IL-36g, IL-37, IL-38, LIF, CNTF, Oncostatin, The cytokine or variant thereof includes, but is not limited to, M, CLCF-1, GM-CSF, Oxferritin, apolipoprotein e, tumor necrosis factor alpha (TNFα), interferon-alpha (IFNα), interferon-beta (IFNβ), or interferon-gamma (IFNγ). In certain embodiments, the cytokine or variant thereof is selected from the group consisting of IL-2, circularly permuted IL-2 (cpIL-2), IL-15, circularly permuted IL15 (cpIL-15), IL-6, circularly permuted IL-6 (cpIL-6), IL-10, or IL-12. In certain embodiments, the cytokine or variant thereof comprises at least one interleukin-12 (IL-12) subunit (e.g., p35 and p40). In certain embodiments, the IL-12 subunit comprises one or both of p35 and p40. In certain embodiments, the cytokine or variant thereof comprises a single chain IL-12 (scIL-12) comprising a p35 polypeptide and a p40 polypeptide joined by an amino acid linker.

[0284] As used herein, the term "variant" refers to a polypeptide that differs from a reference polypeptide but retains essential properties. A typical variant of a polypeptide is one whose primary amino acid sequence differs from another reference polypeptide. Generally, the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions, with limited differences. A variant and a reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. A variant of a polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally. In addition, the term "variant" as used herein includes circular permutations of proteins and peptides.

[0285] As used herein, the terms "circularly permuted" and "circular permutation" (CP or cp) refer to the conceptual process of taking a linear protein or its cognate nucleic acid sequence, fusing the native N- and C-termini to form a circular molecule (either directly or via a linker using protein or recombinant DNA methods), and then cleaving (opening) the circular molecule at a different location to form a new linear protein or cognate nucleic acid molecule with termini different from those of the original molecule. Thus, circular permutation preserves the sequence, structure, and function of the protein (other than the optional linker), while generating new C- and N-termini at different locations, resulting in an improved orientation for fusion with a desired polypeptide fusion partner compared to the original ligand, according to one aspect of the present disclosure. Circular permutation also includes any process that results in a circularly permuted linear molecule, as defined herein. Generally, circularly permuted molecules are expressed de novo as linear molecules and do not formally undergo the steps of cyclization and opening. As used herein, a particular circular permutation of a molecule is indicated by brackets containing amino acid residues with peptide bonds removed between them, in the case of a circularly permuted protein. For example, the designation IL6(Q182 / Q180) refers to a circularly permuted IL6 growth factor in which a ring opening site (where a peptide bond is removed) has been created between residues Q182 and Q180 of unsubstituted or unmodified native IL6, such that the newly generated N-terminus is a glutamine that was previously residue 182, and the newly generated C-terminus is a glutamine that was previously residue 180. Circularly permuted proteins, and methods for making them, are described in detail in US 9,359,415, which is incorporated herein by reference.

[0286] The terms "unsubstituted," "native," "wild-type," or "unmodified" ligand, polypeptide, protein, cytokine are used herein to provide a reference point for a cytokine prior to reconstitution into a circularly permuted molecule, e.g., as described above. Typically, an unmodified cytokine has an amino- and carboxy-terminus and an amino acid sequence that generally corresponds substantially to the amino- and carboxy-terminus and amino acid sequence of an independent domain of the cytokine or protein as it occurs in vivo. An unmodified cytokine may be a complete mature form or a precursor to the mature form (such as a proprotein).

[0287] As used herein, a "fragment" refers to a segment of a polypeptide that is shorter than the reference protein. A protein fragment may have terminal (carboxy or amino terminal) and / or internal deletions. Generally, a protein fragment is at least 4 (e.g., at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 100 or more) amino acids in length. Biologically active fragments or biologically active variants of any of the proteins, fusion proteins or fragments described herein have at least 25% (e.g., at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% or more) of the activity of the wild-type full-length reference protein. In the case of uPAR-binding polypeptides, the relevant activity is the ability of the uPAR-binding polypeptide to bind to the target uPAR receptor. In the case of cytokine polypeptides, the relevant activity is the ability to bind to the receptor and agonize or antagonize it accordingly. Depending on its intended use, the polypeptide, biologically active fragment, or biologically active variant thereof can be of any species, but is preferably a mammal (e.g., mouse, rat, rabbit, hamster, gerbil, dog, cat, goat, pig, cow, horse, whale, monkey, or human), and most preferably a human.

[0288] Therefore, it is understood that the present disclosure provides (i) biologically active variants and (ii) biologically active fragments or biologically active variants of full-length polypeptides (e.g., various polypeptides of the fusion proteins of the present invention, including uPAR-binding polypeptides, cytokines, and corresponding receptors or receptor fragments described herein). Biologically active variants of full-length, preferably mature wild-type proteins or fragments of proteins can contain additions, deletions, or substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, or 50 conservative amino acid substitutions. A conservative substitution is the replacement of one amino acid with another amino acid with similar properties. Conservative substitutions include those within the following groups: valine, alanine and glycine; leucine, valine and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine ​​and threonine; lysine and arginine; and phenylalanine and tyrosine. Non-polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitution of one member of the above polar, basic or acidic groups with another member of the same group can be considered a conservative substitution. In contrast, a non-conservative substitution is a substitution of one amino acid with another amino acid with different characteristics. A deletion variant may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments or non-contiguous single amino acids (of two or more amino acid segments). An addition (addition variant) includes a fusion protein containing (a) a full-length wild-type polypeptide or a fragment thereof containing at least 5 amino acids, and (b) an unrelated or heterologous amino acid sequence at the internal or terminal (C or N).In the context of such fusion proteins, the term "heterologous amino acid sequence" refers to an amino acid sequence other than (a). Thus, fusion proteins containing the peptides described herein and heterologous amino acid sequences do not correspond in sequence to all or a portion of a naturally occurring protein. The heterologous sequence can be, for example, a sequence used in the purification of recombinant proteins (e.g., FLAG or polyhistidine (hexahistidine or 6xHis)).

[0289] Engineered Interleukin-12 Subunits IL-12 is a heterodimer composed of a p35 subunit (i.e., IL-12 p35) and a p40 subunit (i.e., IL-12 p40). Optimal activity of IL-12 is achieved when the heterodimer is formed and binds to the IL-12 receptor (e.g., IL-12Rβ1 / IL-12Rβ2 heterodimer). The two subunits (p35 and p40) can be linked together to form single-chain IL-12 (scIL-12), thereby eliminating the need for heterodimer assembly and enhancing the activity of IL-12. However, scIL-12 exhibits high toxicity when administered systemically. Therefore, it is advantageous to supply each IL-12 subunit separately.

[0290] Unfortunately, IL-12 subunits are difficult to express and purify for therapeutic purposes. p35 is poorly expressed. Expression can be improved by linking p35 to a fusion partner, e.g., an antibody or fragment thereof, or human serum albumin (HSA). p35 is also prone to aggregation. p40 presents similar challenges. p40 can be expressed without a fusion partner, but the purified composition is often contaminated with p40 homodimers (about 15-20%). p40 is also prone to dimerization and aggregation over time at 37°C. The present disclosure provides engineered IL-12 subunits (i.e., p35 and p40 variants) that can overcome these challenges while maintaining binding affinity for each other.

[0291] In one aspect, the disclosure provides an engineered IL-12 p35 polypeptide comprising one or more amino acid substitutions at positions S27, N28, Q35, F39, S44, C74, M111, V114, M119, M145, F150, and L161 of SEQ ID NO:81.

[0292] In certain embodiments, the engineered IL-12 p35 polypeptide further comprises an amino acid substitution at one or both of S27 and D188.

[0293] In certain embodiments, the amino acid substitutions include M12S, S27D, N28R, Q35E, F39D or F39H, S44K, C74K, C74D, C74W, C74Y or C74Q, M111F or M111W, V114I, M119L, M145L, F150Y, L161D or L161Q, D188N, or a combination thereof.

[0294] In certain embodiments, the amino acid substitutions include N28R and Q35E.

[0295] In certain embodiments, the amino acid substitution comprises C74K.

[0296] In certain embodiments, the amino acid substitutions include S27D, C74K, and D188N.

[0297] In certain embodiments, the engineered IL-12 p35 polypeptide comprises the amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83, or an amino acid sequence having at least about 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence similarity or identity to the amino acid sequence set forth in SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.

[0298] In a further aspect, the disclosure provides a fusion protein comprising (i) an engineered IL-12 p35 polypeptide as described above, and (ii) one or more fusion partners (e.g., functional moieties or anti-uPAR antigen binding proteins).

[0299] In certain embodiments, one or more of the fusion partners includes a moiety that reduces aggregation of the engineered IL-12 p35 polypeptide.

[0300] In certain embodiments, one or more of the fusion partners includes a moiety that increases expression of the engineered IL-12 p35 polypeptide.

[0301] In certain embodiments, one or more of the fusion partners comprises a moiety that increases the serum half-life of the engineered IL-12 p35 polypeptide.

[0302] In certain embodiments, one or more of the fusion partners comprises a targeting moiety.

[0303] In certain embodiments, the one or more fusion partners comprises serum albumin, polyethylene glycol (PEG), or an antigen binding protein.

[0304] In certain embodiments, the antigen binding protein specifically binds to urokinase plasminogen activator receptor (uPAR).

[0305] In one aspect, the disclosure provides an engineered IL-12 p40 polypeptide comprising one or more amino acid substitutions at positions C177 and C252 of SEQ ID NO:84.

[0306] In certain embodiments, the amino acid substitution at C177 is selected from the group consisting of C177S, C177F, C177M, C177H, C177I, or C177Q.

[0307] In a specific embodiment, the C252 amino acid substitution is C252S.

[0308] In certain embodiments, p40 comprises the amino acid sequence of SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87, or an amino acid sequence comprising at least about 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence similarity or identity to the amino acid sequence set forth in SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87.

[0309] In a further aspect, the disclosure provides a fusion protein comprising (i) an engineered IL-12 p40 polypeptide as described above, and (ii) one or more fusion partners (e.g., functional moieties or anti-uPAR antigen binding proteins).

[0310] In certain embodiments, one or more of the fusion partners includes a moiety that reduces aggregation of the engineered IL-12 p40 polypeptide.

[0311] In certain embodiments, one or more of the fusion partners includes a moiety that increases expression of the engineered IL-12 p40 polypeptide.

[0312] In certain embodiments, one or more of the fusion partners comprises a moiety that increases the serum half-life of the engineered IL-12 p40 polypeptide.

[0313] In certain embodiments, one or more of the fusion partners comprises a targeting moiety.

[0314] In certain embodiments, the one or more fusion partners comprises serum albumin, polyethylene glycol (PEG), or an antigen binding protein.

[0315] In certain embodiments, the antigen binding protein specifically binds to urokinase plasminogen activator receptor (uPAR).

[0316] Exemplary IL-12 amino acid sequences are shown in Table 2 below. [Table 2-1] [Table 2-2]

[0317] Fusion proteins The urokinase plasminogen activator receptor (uPAR) binding polypeptides described herein, including the uPAR antigen binding proteins described above, can be linked (i.e., bound or fused) to one or both of 1) a cytokine or variant thereof and 2) a checkpoint molecule modulator. The uPAR antigen binding proteins are useful for targeting the linked protein (i.e., the cytokine or checkpoint molecule modulator) to uPAR-expressing tumors.

[0318] The phrase "checkpoint molecule modulator" refers to a protein or peptide capable of agonizing or antagonizing a checkpoint molecule, as appropriate, to enhance a desired immune response (e.g., agonizing an immunostimulatory checkpoint molecule or antagonizing an immunosuppressive checkpoint molecule) or inhibit an undesirable immune response (e.g., antagonizing an immunostimulatory checkpoint molecule against an inappropriate response). Stimulatory or costimulatory checkpoint molecules include, but are not limited to, CD27, CD70, CD28, CD40, CD40 ligand, CD122, CD137, OX40, GITR and its ligand, ICOS, CD131 (4-1BBB and its ligand). Inhibitory checkpoint molecules include, but are not limited to, adenosine A2A receptor (A2AR), B7-H3, B7-H4, BTLA (B and T lymphocyte attenuator), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), IDO (indoleamine 2,3, dioxygenase), KIR (killer cell immunoglobulin-like receptor), LAG3 (lymphocyte activation gene 3), PD-1 (programmed cell death 1 receptor) and its ligands PD-L1 (programmed death ligand 1) and PD-L2 (programmed death ligand 2), TIM-3 (T cell immunoglobulin and mucin domain-containing protein 3), VISTA (v domain Ig inhibitor of T cell activation). Checkpoint molecule modulators for use in the fusion proteins of the present disclosure include inhibitory or stimulatory checkpoint molecules, functional fragments thereof, functional derivatives thereof or circularly permuted variants thereof.

[0319] In addition to antigen-binding proteins, uPAR-binding polypeptides can include peptides derived from integrins that block uPAR integration interactions. One such peptide is M25, which has the amino acid sequence PRYQHIGLVAMFRQNTG (SEQ ID NO: 157), a peptide derived from the β2 subunit of Mac-1 that inhibits lymphocyte adhesion to fibrinogen, vitronectin, and cytokine-stimulated endothelial cells. M25 also blocks the association of uPAR with β1, and inhibits the β1-integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen (Simon DI, et al. (2000) J Biol Chem. 275: 10228-34). A second peptide that inhibits uPAR-integrin complex formation, P25, having the amino acid sequence AESTYHHLSLGYMYTLN (sequence number 158), was able to reduce tumor cell adhesion to vitronectin and increase tumor cell adhesion to fibronectin (van der Pluijm G, et al. (2001) Am J Pathol. 159:971-82).

[0320] In one aspect, the disclosure provides a fusion protein comprising: (i) an antigen binding protein that specifically binds to urokinase plasminogen activator receptor (uPAR); and (ii) a cytokine or a variant thereof.

[0321] In certain embodiments, the antigen binding protein and the cytokine or variant thereof are linked by an amino acid linker.

[0322] In certain embodiments, the cytokine or variant thereof is selected from the group consisting of IL-2, circularly permuted IL-2 (cpIL-2), IL-15, circularly permuted IL15 (cpIL-15), IL-6, circularly permuted IL-6 (cpIL-6), IL-10, or IL-12.

[0323] In certain embodiments, the cytokine or variant thereof comprises at least one interleukin-12 (IL-12) subunit (e.g., p35 and p40). In certain embodiments, the IL-12 subunit comprises one or both of p35 and p40. For example, and without limitation, the fusion protein comprises (i) an antigen binding protein that specifically binds to uPAR and (ii) a p35 polypeptide. Alternatively, the fusion protein comprises (i) an antigen binding protein that specifically binds to uPAR and (ii) a p40 polypeptide.

[0324] In certain embodiments, the p35 polypeptide comprises one or more amino acid substitutions at positions M12, S27, N28, Q35, F39, S44, C74, M111, V114, M119, M145, F150, L161, and D188 of SEQ ID NO: 81. In certain embodiments, the amino acid substitutions comprise M12S, S27D, N28R, Q35E, F39D or F39H, S44K, C74K, C74D, C74W, C74Y or C74Q, M111F or M111W, V114I, M119L, M145L, F150Y, L161D or L161Q, D188N, or combinations thereof. In certain embodiments, the amino acid substitutions comprise N28R and Q35E. In certain embodiments, the amino acid substitutions include S27D and D188N. In certain embodiments, the amino acid substitutions include C74K. In certain embodiments, the amino acid substitutions include S27D, C74K, and D188N. In certain embodiments, p35 comprises an amino acid sequence of SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83, or an amino acid sequence that comprises at least about 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence similarity or identity to the amino acid sequence set forth in SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.

[0325] In certain embodiments, the p40 polypeptide comprises one or more amino acid substitutions at positions C177 and C252 of SEQ ID NO: 84. In certain embodiments, the C177 amino acid substitution is selected from the group consisting of C177S, C177F, C177M, C177H, C177I, or C177Q. In certain embodiments, the C252 amino acid substitution is C252S. In certain embodiments, the p40 polypeptide comprises an amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87, or an amino acid sequence comprising at least about 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence similarity or identity to the amino acid sequence set forth in SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87.

[0326] In certain embodiments, the cytokine or variant thereof comprises a single chain IL-12 (scIL-12) comprising a p35 polypeptide and a p40 polypeptide linked by an amino acid linker. In certain embodiments, the amino acid linker comprises the sequence GGSGGGGSGG (SEQ ID NO: 156). In certain embodiments, the scIL-12 comprises an amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 89, or an amino acid sequence comprising at least about 80% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence similarity or identity to the amino acid sequence set forth in SEQ ID NO: 88 or SEQ ID NO: 89.

[0327] In certain embodiments, the antigen binding protein of the fusion protein is a) a VH domain comprising an HCDR1 amino acid sequence of GFNIKDEY (SEQ ID NO: 3), an HCDR2 amino acid sequence of IDPENGDT (SEQ ID NO: 4), and an HCDR3 amino acid sequence of TGGNYVGWFPY (SEQ ID NO: 5); b) an antigen-binding protein comprising a VL domain comprising an LCDR1 amino acid sequence of SSVSY (SEQ ID NO: 6), an LCDR2 amino acid sequence of DTS (SEQ ID NO: 7), and an LCDR3 amino acid sequence of QQWSSNPPY (SEQ ID NO: 8).

[0328] In certain embodiments, the antigen binding protein of the fusion protein is a) a VH domain comprising an HCDR1 amino acid sequence of GFSLTNYG (SEQ ID NO: 11), an HCDR2 amino acid sequence of IWSDGGT (SEQ ID NO: 12), and an HCDR3 amino acid sequence of ARGGRSDLFAY (SEQ ID NO: 13); b) a VL domain comprising an LCDR1 amino acid sequence of QSIVHSNGNTY (sequence number 14), an LCDR2 amino acid sequence of KVS (sequence number 15), and an LCDR3 amino acid sequence of FQGSHVPYT (sequence number 16).

[0329] In certain embodiments, the antigen binding protein of the fusion protein is a) a VH domain comprising an HCDR1 amino acid sequence of GNTFTDYG (SEQ ID NO: 19) or GTTFTDYG (SEQ ID NO: 41), an HCDR2 amino acid sequence of INTNTGEP (SEQ ID NO: 20), and an HCDR3 amino acid sequence of AHYSFDY (SEQ ID NO: 21); b) an antigen-binding protein comprising a VL domain comprising an LCDR1 amino acid sequence of ENIYSN (SEQ ID NO: 22), an LCDR2 amino acid sequence of AAT (SEQ ID NO: 23), and an LCDR3 amino acid sequence of QHFWGTPWT (SEQ ID NO: 24).

[0330] In certain embodiments, the antigen binding protein of the fusion protein is a) a VH domain comprising an HCDR1 amino acid sequence of GYTFTSYG (SEQ ID NO: 27), an HCDR2 amino acid sequence of IYPRSGNT (SEQ ID NO: 28), and an HCDR3 amino acid sequence of AGKDYGSTYADY (SEQ ID NO: 29); b) an antigen-binding protein comprising a VL domain comprising an LCDR1 amino acid sequence of SSVSSRY (SEQ ID NO: 30), an LCDR2 amino acid sequence of GTS (SEQ ID NO: 31), and an LCDR3 amino acid sequence of QQYHSDPLT (SEQ ID NO: 32).

[0331] In certain embodiments, the antigen binding protein of the fusion protein is a) a VH domain comprising the amino acid sequence of SEQ ID NO: 33, and a VL domain comprising the amino acid sequence of SEQ ID NO: 35; b) a VH domain comprising the amino acid sequence of SEQ ID NO: 33, and a VL domain comprising the amino acid sequence of SEQ ID NO: 36; c) a VH domain comprising the amino acid sequence of SEQ ID NO: 34, and a VL domain comprising the amino acid sequence of SEQ ID NO: 35; d) a VH domain comprising the amino acid sequence of SEQ ID NO: 34, and a VL domain comprising the amino acid sequence of SEQ ID NO: 36; e) a VH domain comprising the amino acid sequence of SEQ ID NO: 37, and a VL domain comprising the amino acid sequence of SEQ ID NO: 38; f) a VH domain comprising the amino acid sequence of SEQ ID NO: 37, and a VL domain comprising the amino acid sequence of SEQ ID NO: 39; g) a VH domain comprising the amino acid sequence of SEQ ID NO: 37, and a VL domain comprising the amino acid sequence of SEQ ID NO: 40; h) a VH domain comprising the amino acid sequence of SEQ ID NO: 42 and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or i) comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 43, and a VL domain comprising the amino acid sequence of SEQ ID NO: 44.

[0332] In certain embodiments, the VH domain and the VL domain are joined by an amino acid linker. In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 148), where n is an integer from 1 to 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 149) or GGSGGGGSGGGSGGGGSGGGGSGGGSGG (SEQ ID NO: 150).

[0333] In certain embodiments, the antigen binding protein comprises a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a single chain Fv (scFv), a dAb fragment, a single domain antibody, or a nanobody.

[0334] In certain embodiments, the antigen binding protein comprises the amino acid sequence of any one of SEQ ID NOs: 56-71.

[0335] In certain embodiments, the antigen binding protein further comprises the amino acid sequence of any one of SEQ ID NOs: 72-78.

[0336] In certain embodiments, the antigen binding protein comprises the antibody heavy chain (HC) amino acid sequence of SEQ ID NO: 45, 46, 49, 53, or 54, and the antibody light chain (LC) amino acid sequence of SEQ ID NO: 47, 48, 50, 51, 52, and 55.

[0337] In certain embodiments, the antigen binding protein is a) antibody HC comprising the amino acid sequence of SEQ ID NO: 45, and antibody LC comprising the amino acid sequence of SEQ ID NO: 47; b) an antibody HC comprising the amino acid sequence of SEQ ID NO: 45, and an antibody LC comprising the amino acid sequence of SEQ ID NO: 48; c) antibody HC comprising the amino acid sequence of SEQ ID NO: 46, and antibody LC comprising the amino acid sequence of SEQ ID NO: 47; d) an antibody HC comprising the amino acid sequence of SEQ ID NO: 46, and an antibody LC comprising the amino acid sequence of SEQ ID NO: 48; e) antibody HC comprising the amino acid sequence of SEQ ID NO: 49, and antibody LC comprising the amino acid sequence of SEQ ID NO: 50; f) antibody HC comprising the amino acid sequence of SEQ ID NO: 49, and antibody LC comprising the amino acid sequence of SEQ ID NO: 51; g) antibody HC comprising the amino acid sequence of SEQ ID NO: 49, and antibody LC comprising the amino acid sequence of SEQ ID NO: 52; h) an antibody HC comprising the amino acid sequence of SEQ ID NO: 53, and an antibody LC comprising the amino acid sequence of SEQ ID NO: 55; or i) Antibody HC comprising the amino acid sequence of SEQ ID NO:54, and antibody LC comprising the amino acid sequence of SEQ ID NO:55.

[0338] In another aspect, the present disclosure provides a combination comprising a first fusion protein and a second fusion protein, (a) a first fusion protein comprising: (i) an antigen binding protein that specifically binds to a first epitope on a urokinase plasminogen activator receptor (uPAR); (ii) an IL-12 p35 subunit or a variant thereof; (b) the second fusion protein is (i) an antigen binding protein that specifically binds to a second epitope on uPAR; (ii) IL-12 p40 subunit or a variant thereof.

[0339] In certain embodiments, the first epitope and the second epitope are the same.

[0340] In certain embodiments, the first epitope and the second epitope are different.

[0341] Functional part The proteins of the present disclosure (e.g., uPAR binding proteins, cytokines, and fusion proteins) can be further conjugated (i.e., linked, coupled, or fused) to functional moieties that confer a desired biological activity to the proteins of the present disclosure. Functional moieties include both protein functional moieties and non-protein functional moieties (e.g., PEG or polysaccharides). For example, but not limited to, a cytokine or variant thereof can be conjugated to the Fc region of an immunoglobulin or human serum albumin (HSA) via an optional linker for the purpose of extending the half-life of the cytokine or variant thereof (e.g., IL-12 p35-HSA or IL-12 p40-HSA conjugates).

[0342] In certain embodiments, the functional moiety is an Fc region (i.e., Fc domain) of an immunoglobulin. In certain embodiments, the Fc region can be linked to the proteins of the present disclosure (e.g., uPAR binding proteins, cytokines, and fusion proteins) via a single-chain linker as described in WO2016 / 100788 (hereby incorporated by reference). These linkers include a constant region of an immunoglobulin light chain (CL) linked to a CH1 constant region of an immunoglobulin heavy chain fused to the Fc region of an immunoglobulin (scCLCH1 or scCH1CL linker). For example, a fusion protein of the present disclosure (e.g., a cytokine linked to an anti-uPAR antigen binding protein) can be further linked to an Fc region of an immunoglobulin via a scCLCH1 or scCH1CL linker. This type of linker confers favorable properties to the fusion proteins of the present disclosure, such as improved biological activity and increased half-life.

[0343] Fusion proteins of the present disclosure may be further stabilized and their half-life increased in vivo by binding to serum albumin molecules that resist degradation and / or clearance or sequestration, such as human serum albumin (HSA), which are naturally occurring proteins that themselves have long half-lives in vivo.

[0344] Linker Any of the proteins described herein (e.g., uPAR binding proteins, cytokine proteins, and fusion proteins) can include one or more (e.g., two or more, three or more, four or more, five or more, six or more, or seven or more) linker moieties that link one or more (or all) of the polypeptides of a fusion protein, link one or more cytokine proteins, or link one or more uPAR binding proteins (e.g., linking the VH and VL domains of a uPAR antigen binding protein). In certain embodiments, the linker is an amino acid linker, which can be one amino acid long, but can be longer. For example, the linker peptide can be at least 2 (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, or at least 55 or more) amino acids in length. The linker peptide can contain any amino acid.

[0345] In certain embodiments, any one or more linkers present in the proteins of the present disclosure are selected from artificial flexible polypeptides comprising amino acids selected from Gly (G) and / or Ser (S). In certain embodiments, the linker has the general formula (GGGS (SEQ ID NO: 151))n or (GGGGS (SEQ ID NO: 152))n or (SGGSGGG (SEQ ID NO: 153)) n or (GGSGGSG (SEQ ID NO: 154)) nwherein n is an integer from 1 to 10. In certain embodiments, each linker is a polypeptide comprising about 1 to about 100 amino acids, for example, about 1 to 50 amino acids, about 1 to 25 amino acids, about 1 to 15 amino acids, about 1 to 10 amino acids, about 4 to 24 amino acids, about 5 to 20 amino acids, about 5 to 15 amino acids, and about 5 to 10 amino acids. In certain embodiments, the linker is (GGGGS (SEQ ID NO: 155))n, wherein n is 2 or 4. Any linker may further comprise an amino acid, such as, for example, Lys (K), Thr (T), Glu (E), and Asp (D).

[0346] In certain embodiments, the amino acid linker comprises (GGGGS)n (SEQ ID NO: 148), where n is an integer from 1 to 5. In certain embodiments, the amino acid linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 149) or GGSGGGGSGGGSGGGGSGGGGSGGGSGG (SEQ ID NO: 150). In certain embodiments, the amino acid linker comprises the amino acid sequence GGS.

[0347] In certain embodiments, the linker may comprise one or more mucin proteins or mucin domains of proteins (e.g., any protein encoded by a MUC gene (e.g., MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC11, MUC12, MUC13, MUC15, MUC16, MUC17, MUC19, MUC20, MUC21)). Mucin domain proteins and polypeptides contain extensive glycosylation, which allows mucin proteins and other polypeptides that contain mucin domains to behave structurally as a rigid random coil. The rod-like nature of the mucin domain allows for tight separation of the biologically active protein (e.g., cytokine) and the fusion partner (e.g., uPAR binding protein), making it less likely that activity will be lost with any of the fusion partners.

[0348] Such mucin domain polypeptides useful according to the present disclosure are described in WO2013 / 184939 and WO2013 / 184938, which are incorporated herein by reference. These linkers are useful for providing optimal spacing between the polypeptides of the fusion proteins of the present disclosure (e.g., between the ATF polypeptide and the cytokine polypeptide) or, for example, for increasing the half-life of the entire fusion protein, regardless of the position of the mucin domain in the fusion protein. For example, the mucin domain can be present at the N-terminus or C-terminus of the fusion protein. The mucin domain polypeptide linker can be further linked to an Fc region of an immunoglobulin polypeptide, which can also function to increase the half-life of the fusion protein of the present invention, as described in WO2013 / 184938.

[0349] Purification Tag Any of the proteins described herein (e.g., uPAR binding proteins, cytokine proteins, and fusion proteins) can include one or more (e.g., two or more, three or more, four or more, five or more, six or more, or seven or more) purification tags, which facilitate purification of the proteins described herein. In certain embodiments, the purification tag is an avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 240). In certain embodiments, the purification tag is a 6xHis tag (HHHHHH; SEQ ID NO: 241). Either or both of the avi tag and the 6xHis tag can be present in any order on a single protein. In certain embodiments, the purification tag is linked to the proteins described herein by a gly-ser linker. In certain embodiments, the gly-ser linker comprises GGS and / or GGSGGG (SEQ ID NO: 242). In certain embodiments, the purification is any one of GGSHHHHHHGGSGLNDIFEAQKIEWHE (SEQ ID NO: 243), GGSGGHHHHHHGGSGLNDIFEAQKIEWHE (SEQ ID NO: 244), and GGSGLNDIFEAQKIEWHEGGSHHHHHH (SEQ ID NO: 245).

[0350] Expression of antigen-binding proteins, cytokines, and fusion proteins In one embodiment, polynucleotides or nucleic acids are provided that encode the antigen binding proteins (e.g., anti-uPAR antigen binding proteins), cytokines or variants thereof (e.g., engineered IL-12 p35 or engineered IL-12 p40), or fusion proteins (e.g., anti-uPAR antigen binding protein-cytokine fusion proteins) disclosed herein. Methods of making the antigen binding proteins, cytokines, or fusion proteins, including expressing these polynucleotides, are also provided.

[0351] Polynucleotides encoding the antigen binding proteins, cytokines, or fusion proteins disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce desired amounts of the antigen binding protein or fusion protein. Thus, in certain aspects, the disclosure provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0352] The term "vector" or "expression vector" is used herein to mean a vector used in accordance with the present invention as a vehicle for introducing and expressing a desired gene in a cell. As known to those skilled in the art, such vectors can be easily selected from the group consisting of plasmids, phages, viruses and retroviruses. In general, vectors compatible with the present invention contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0353] For the purposes of the present invention, numerous expression vector systems may be employed. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retrovirus (e.g., RSV, MMTV, MOMLV, etc.), or SV40 virus. Others involve the use of polycistronic systems that contain internal ribosome binding sites. Additionally, cells in which the DNA has integrated into the chromosome may be selected by introducing one or more markers that allow for the selection of transfected host cells. Markers may confer prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequence to be expressed or may be introduced into the same cell by cotransformation. Additional elements may be required for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcription promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (e.g., human constant region genes) synthesized as discussed above.

[0354] In other embodiments, antigen binding proteins may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as heavy and light chains of antibodies, may be produced from a single polycistronic construct. Such systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are described in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of polypeptides disclosed in the present application.

[0355] More generally, once a vector or DNA sequence encoding an antibody or fragment thereof has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG "Mammalian Expression Vectors" Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Introduction of the plasmid into the host can be by electroporation. The transformed cells are grown under conditions appropriate for the production of the light and heavy chains and assayed for the synthesis of heavy and / or light chain proteins. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0356] As used herein, the term "transformation" is intended to be used in a broad sense to refer to the introduction of DNA into a recipient host cell, altering the genotype and resulting in a change in the recipient cell.

[0357] In this same sense, a "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. In describing the process for isolating a polypeptide from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody, unless otherwise indicated. In other words, recovery of the polypeptide from the "cells" can mean recovery from either centrifuged whole cells or from the cell culture fluid containing both the medium and the suspended cells.

[0358] In one embodiment, the host cell line used for antibody or fusion protein expression is of mammalian origin. One skilled in the art can determine the particular host cell line that is most suitable for the desired gene product to be expressed. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 that contains the SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney), and the like. In one embodiment, the cell line provides for altered glycosylation (e.g., defucosylation) of antibodies expressed from the cell line (e.g., PER.C6® (Crucell) or a FUT8 knockout CHO cell line (Potelligent® cells) (Biowa, Princeton, NJ)). Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from published literature.

[0359] In vitro production allows for scale-up to obtain large amounts of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in airlift reactors or continuous stirred reactors, or immobilized or entrapped cell cultures, for example in hollow fibers, microcapsules, agarose microbeads or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, for example, gel filtration, ion exchange chromatography, chromatography on DEAE cellulose and / or (immuno)affinity chromatography.

[0360] Genes encoding antigen-binding proteins featured in the present invention can also be expressed in non-mammalian cells such as bacteria or yeast or plant cells. In this regard, it will be appreciated that various unicellular non-mammalian microorganisms, such as bacteria, i.e., microorganisms capable of growing in culture or fermentation, can also be transformed. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, e.g., strains of Escherichia coli or Salmonella; Bacillaceae, e.g., Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that when expressed in bacteria, the protein can be part of inclusion bodies. The protein needs to be isolated, purified, and then assembled into a functional molecule.

[0361] In addition to prokaryotes, eukaryotic microorganisms may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms, although other strain members are commonly available. For expression in Saccharomyces, for example, the plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene, which provides a selection marker for mutant strains of yeast lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). Thus, the presence of the trp1 lesion, which is characteristic of the yeast host cell genome, provides an effective environment for detecting transformants by growth in the absence of tryptophan.

[0362] Methods of administering antigen-binding proteins, cytokines, and fusion proteins Methods of preparing and administering to a subject an antigen binding protein, cytokine, or fusion protein of the present disclosure, as well as a nucleic acid described herein, a vector described herein, a host cell described herein, or a composition described herein, are well known to or readily determined by those of skill in the art. The route of administration of an antigen binding protein of the present disclosure may be, for example, oral, parenteral, inhalation, or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration. In certain embodiments, the antigen binding protein or fusion protein is administered intravenously. The term intraocular as used herein includes, but is not limited to, subconjunctival, intravitreal, retrobulbar, or intracameral. The term topical as used herein includes, but is not limited to, administration by liquid or solution eye drops, emulsions (e.g., oil-in-water emulsions), suspensions, and ointments.

[0363] The dosage form may be an injectable solution, although all of these dosage forms are expressly contemplated to be within the scope of the present disclosure. Typically, suitable injectable pharmaceutical compositions may include buffers (e.g., acetate, phosphate or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin). However, in other methods consistent with the teachings herein, modified antibodies may be delivered directly to the site of harmful cell populations, thereby increasing the exposure of affected tissues to the therapeutic agent.

[0364] The effective amount of the composition of the present disclosure for treating the relevant conditions will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other medicaments administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Treatment dosages can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

[0365] As previously mentioned, the antigen binding proteins or fusion proteins, conjugates or recombinants thereof of the present disclosure may be administered in a pharma- ceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be understood that the antigen binding proteins of the present disclosure will be formulated to facilitate administration and promote stability of the active agent.

[0366] Pharmaceutical compositions according to the present disclosure typically include a pharma- ceutically acceptable, non-toxic, sterile carrier, such as saline, non-toxic buffers, preservatives, and the like. For purposes of this application, a pharma- ceutical effective amount of an antigen-binding protein shall mean an amount sufficient to achieve effective binding to an antigen and obtain a benefit, such as an amount sufficient to ameliorate the symptoms of a disease or disorder or to detect a substance or cell. In the case of tumor cells, the antigen-binding protein is typically capable of interacting with selected immunoreactive antigens on neoplasms or immunoreactive cells, increasing the killing of these cells. Of course, the pharmaceutical compositions of the present disclosure may be administered in a single dose or multiple doses to provide a pharma- ceutical effective amount of the modified binding polypeptide.

[0367] In accordance with the scope of the present disclosure, the antigen binding proteins of the present disclosure may be administered to humans or other animals in amounts sufficient to provide a therapeutic or prophylactic effect according to the aforementioned therapeutic methods. The antigen binding proteins of the present disclosure may be administered to such humans or other animals in conventional dosage forms prepared by combining the antigen binding proteins of the present disclosure with conventional pharma- ceutically acceptable carriers or diluents according to known techniques. Those skilled in the art will recognize that the form and characteristics of the pharma- ceutically acceptable carrier or diluent will be determined by the amount of active ingredient to be combined, the route of administration, and other well-known variables. Furthermore, those skilled in the art will appreciate that cocktails comprising one or more of the antigen binding proteins described in the present disclosure may prove to be particularly effective. Similarly, the nucleic acids described herein, the vectors described herein, the host cells described herein (particularly immune cells bearing CARs) or the compositions described herein may be administered to humans or other animals in amounts sufficient to provide a therapeutic or prophylactic effect according to the aforementioned therapeutic methods.

[0368] As used herein, "efficacy" or "in vivo efficacy" refers to the response to treatment with the pharmaceutical composition of the present disclosure using standard efficacy criteria, such as standard ophthalmic efficacy criteria. The success of treatment with the pharmaceutical composition of the present disclosure or in vivo efficacy refers to the efficacy of the composition for its intended purpose, i.e., the ability of the composition to produce the desired effect. In vivo efficacy can be monitored by standard methods established for a particular disease. In addition, various clinical chemistry parameters and other established standard methods specific to the disease may be used.

[0369] In some embodiments, the compounds and cells described herein are administered in combination with one or more different pharmaceutical compounds. In general, the therapeutic use of the compounds and cells described herein may be in combination with one or more therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser phototherapy, radiation therapy or vaccine therapy.

[0370] Treatment method The present disclosure describes a method for treating the disease or disorder associated with the expression of uPAR in patients by administering the antigen binding protein, cytokine, or fusion protein of the present disclosure.In certain embodiments, the disease or disorder associated with the expression of uPAR is cancer (e.g., uPAR-expressing cancer or uPAR-expressing tumor).

[0371] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition, uPAR binding protein, or fusion protein described herein.

[0372] In certain embodiments, the pharmaceutical composition or fusion protein is administered simultaneously with a cytokine or a variant thereof or a second fusion protein, where the second fusion protein comprises a different cytokine or variant thereof than the first fusion protein.

[0373] In certain embodiments, the pharmaceutical composition or fusion protein is administered sequentially with a cytokine or a variant thereof or a second fusion protein, where the second fusion protein comprises a different cytokine or variant thereof than the first fusion protein.

[0374] In certain embodiments, the cytokine or variant thereof comprises the IL-12 p35 subunit or the IL-12 p40 subunit.

[0375] In certain embodiments, the clearance rate of the fusion protein in the patient's tumor is slower than the clearance rate of the fusion protein in the patient's serum.

[0376] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a fusion protein and an unfused IL-12 subunit; (a) The fusion protein comprises: (i) an antigen-binding protein that specifically binds to the urokinase plasminogen activator receptor (uPAR); (ii) an IL-12 p35 subunit or a variant thereof, or an IL-12 p40 subunit or a variant thereof; (b) the non-fused IL-12 subunit comprises an IL-12 p35 subunit or a variant thereof, or an IL-12 p40 subunit or a variant thereof; Herein, methods are provided wherein when the fusion protein comprises the IL-12 p35 subunit or a variant thereof, the unfused IL-12 subunit comprises the IL-12 p40 subunit or a variant thereof, and when the fusion protein comprises the IL-12 p40 subunit or a variant thereof, the unfused IL-12 subunit comprises the IL-12 p35 subunit or a variant thereof.

[0377] In certain embodiments, the fusion protein and the unfused IL-12 subunit are administered sequentially.

[0378] In a specific embodiment, one or both of the fusion protein and the unfused IL-12 subunit are administered intravenously.

[0379] In certain embodiments, the fusion protein is administered first, followed by administration of the unfused IL-12 subunit after a sufficient amount of time has passed to allow the fusion protein to accumulate in the patient's tumor.

[0380] In certain embodiments, the fusion protein is administered first, followed by administration of the unfused IL-12 subunit after a sufficient amount of time has passed such that the concentration of the fusion protein has substantially decreased in the patient's serum.

[0381] In one aspect, the disclosure provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a first fusion protein and a second fusion protein; (a) a first fusion protein comprising: (i) an antigen binding protein that specifically binds to a first epitope on a urokinase plasminogen activator receptor (uPAR); (ii) an IL-12 p35 subunit or a variant thereof; (b) the second fusion protein is (i) an antigen binding protein that specifically binds to a second epitope on uPAR; (ii) an IL-12 p40 subunit or a variant thereof.

[0382] In certain embodiments, the first epitope and the second epitope are the same.

[0383] In certain embodiments, the first epitope and the second epitope are different.

[0384] In certain embodiments, the first fusion protein and the second fusion protein are administered sequentially.

[0385] In certain embodiments, one or both of the first and second fusion proteins are administered intravenously.

[0386] In certain embodiments, a first fusion protein is administered first, followed by administration of a second fusion protein after a sufficient amount of time has passed to allow the first fusion protein to accumulate in the patient's tumor.

[0387] In certain embodiments, a first fusion protein is administered first, followed by administration of a second fusion protein after a sufficient amount of time has passed for the concentration of the first fusion protein to be substantially reduced in the patient's serum.

[0388] In certain embodiments, the second fusion protein is administered first, followed by administration of the first fusion protein after a sufficient amount of time has passed to allow the second fusion protein to accumulate in the patient's tumor.

[0389] In certain embodiments, the second fusion protein is administered first, followed by the first fusion protein after a sufficient amount of time has passed for the concentration of the second fusion protein to be substantially reduced in the patient's serum.

[0390] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the embodiments will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0391] Example 1 - Generation and characterization of parent anti-uPAR antibodies NZBW mice were immunized by alternate injections of recombinant human uPAR and mouse uPAR extracellular domain proteins. Hybridomas were generated and screened against human and mouse uPAR by ELISA and uPAR-overexpressing HEK293 cells to identify antibodies with binding specificity to human and mouse uPAR. Top clones were further screened by ELISA for binding to 1) human uPAR with and without human uPA, 2) mouse uPAR with and without mouse uPA, and 3) ligand uPA-bound variants of uPAR (human uPAR variants that mimic the ligand-bound conformation), including human uPAR with the H47C / N259C mutation. The following amino acid sequences were used for screening: Human uPAR-His-Avi tag fusion LRCMQCKTNGDCRVEECALGQDLCRTTIVRLWEEGEELELVEKSCTHSEKTNRTLSYRTGLKITSLTEVVCGLDLCNQGNSGRAVTYSRSRYLECISCGSSDMSCERGRHQSLQCRSPEEQCLDVVTHWIQEGEEGRPKDDRHLRGCGYLPGCP GSNGFHNNDTFHFLKCCNTTKCNEGPILELENLPQNGRQCYSCKGNSTHGCSSEETFLIDCRGPMNQCLVATGTHEPKNQSYMVRGCATASMCQHAHLGDAFSMNHIDVSCCTKSGCNHPDLDVQYRGGSHHHHHHGGGSGLNDIFEAQKIEWHE Mouse uPAR-His-Avi tag fusion LQCMQCESNQSCLVEECALGQDLCRTTVLREWQDDRELEVVTRGCAHSEKTNRTMSYRMGSMIISLTETVCATNLCNRPRPGARGRAFPQGRYLECASCTSLDQSCERGREQSLQCRYPTEHCIEVVTLQSTERSLKDEDYTRGCGSLPG CPGTAGFHSNQTFHFLKCCNYTHCNGGPVLDLQSFPNGFQCYSCEGNNTLGCSSEEASLINCRGPMNQCLVATGLDVLGNRSYTVRGCATASWCQGSHVADSFPTHLNVSVSCCHGSGCNSPTGGSHHHHHHGGSGLNDIFEAQKIEWHE Human uPA-His-Avi tag fusion MYRMQLLSCIALSLALVTNSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCAGGSHHHHHHGGSGLNDIFEAQKIEWHE Mouse uPA-His-Avi tag fusion MYRMQLLSCIALSLALVTNSGSVLGAPDESNCGCQNGGVCVSYKYFSRIRRCSCPRKFQGEHCEIDASKTCYHGNGDSYRGKANTDTKGRPCLAWNAPAVLQKPYNAHRPDAISLGLGKHNYCRNPDNQKRPWCYVQIGLRQFVQECMVHDCGGSHHHHHHGGGSGLNDIFEAQKIEWHE

[0392] Antibodies designated M1, M5, M8, and M43 each bound to human uPAR independent of the ligand uPA and were therefore selected for further characterization.

[0393] The amino acid sequences of the heavy chain variable domain (VH) and light chain variable domain (VL) and the amino acid sequences of HCDR1 to 3 and LCDR1 to 3 of M1, M5, M8, and M43 are shown in Table 3 below. [Table 3-1] [Table 3-2]

[0394] The binding affinity of anti-uPAR antibodies M1, M5, M8, and M43 was determined. Binding affinity analysis was performed using the Octet platform based on Biolayer Interferometry (BLI) technology. Briefly, uPAR antibodies were immobilized by anti-mouse Fc (AMC) sensors. Each uPAR antibody was tested at a concentration of 10 μg / mL. Loading was histidine-tagged human uPAR (Sino#10925-H08H). A bare sensor was used as a control. The binding affinity of each antibody to human uPAR is shown in Table 4 below. [Table 4]

[0395] Additional binding affinity analysis was performed using the Octet platform with histidine-tagged human uPAR immobilized on the sensor at a concentration of 10 μg / mL. Loading was M1, M5, M8, and M43, respectively. A bare sensor was used as a control. The binding affinity of each antibody to human uPAR when immobilized is shown in Table 5 below. Each antibody showed a binding affinity of less than 1 pM, suggesting an avidity effect. [Table 5]

[0396] The uPAR binding epitope of each antibody was characterized using the Octet platform described above. Biotinylated human uPAR variants, uPAR DII-DIII domain or uPAR DIII domain were captured by SA sensor at 10 μg / ml for 180 seconds, followed by binding of M1, M5, M8, M43 or ATN568 at a concentration of 200 nM for 150 seconds. The following amino acid sequences were used for the DII-DIII and DIII domains: Human uPAR DII-DIII domain HisAvi tag MYRMQLLSCIALSLALVTNSGNSGRAVTYSRSRYLECISCGSSDMSCERGRHQSLQCRSPEEQCLDVVTHWIQEGEEGRPKDDRHLRGCGYLPGCPGSNGFHNNDTFHFLKCCNTTKCNEGPILE LENLPQNGRQCYSCKGNSTHGCSSEETFLIDCRGPMNQCLVATGTHEPKNQSYMVRGCATASMCQHAHLGDAFSMNHIDVSCCTKSGCNHPDLDVQYRGGSHHHHHHGGGSGLNDIFEAQKIEWHE Human uPAR DIII domain HisAvi tag MYRMQLLSCIALSLALVTNSGPILELENLPQNGRQCYSCKGNSTHGCSSEETFLIDCRGPMNQCLVATGTHEPKNQSYMVRGCATASMCQHAHLGDAFSMNHIDVSCCTKSGCNHPDLDVQYRGGSHHHHHHGGGSGLNDIFEAQKIEWHE

[0397] M1, M5, M8, and M43 bind to the DII-DIII domain of human uPAR, but M1 can also bind to DIII alone (Figure 1A, Figure 1B). Each of M1, M5, M8, and M43 was found to bind to uPAR regardless of the binding of the uPAR ligand uPA to uPAR. The anti-uPAR antibody ATN-658 was used as a comparison.

[0398] To test competitive binding of antibodies to uPA ligands, human uPA was captured by the SA sensor at 3.2 μg / ml (200 nM) for 45 s. Human recombinant protein uPAR-His was loaded at 100 nM for 180 s, followed by binding of M1, M5, M8, M43 or ATN568 at a concentration of 200 nM for 150 s.

[0399] Antibody competition assays were performed using the Octet platform. Antibody competition assays were performed by capturing biotinylated human uPAR on the SA sensor. M1, M5, M8, M43 and ATN-658 were then loaded as first ligands to bind to human uPAR at a predefined saturation concentration. Once the first ligand reached saturation, M1, M5, M8, M43 and ATN-568 were loaded as second ligands to compete with M1, M5, M8, M43 or ATN-658. The second ligand binding solution contained the same concentration of first preloaded ligand to prevent dissociation during binding of the second ligand. M1 was found to compete only with the comparative antibody ATN-658. M5 competed with M43. None of M1, M8 or M43 competed with each other for binding to human uPAR, indicating that the three antibodies have unique epitopes on human uPAR (i.e., the antibodies are non-competitive). The matrix competition assay data is shown in Table 6 below. [Table 6]

[0400] The antibodies were then tested for their ability to bind to human and mouse uPAR expressed on the surface of cells. Human HEK293 cells engineered to express human uPAR or mouse uPAR were dye-labeled and mixed with unlabeled parental HEK293 cells. The cells were incubated with 100 nM each of human and mouse uPA (uPAR ligands) for 30 minutes, then treated with titrations of uPAR antibodies for 30 minutes. Binding of uPAR antibodies was detected using a secondary antibody labeled with anti-mouse-IgG-phycoerythrin (PE). Mean fluorescence intensity (MFI) values ​​across the antibody concentrations tested are shown in Figures 2A and 2B. EC50 values ​​are shown in Table 7 below. The EC50 values ​​for cell binding without uPA ligand were similar to the experiments with uPA ligand. The EC50 binding values ​​of M1, M5, M8, and M43 were all superior to the comparable ATN-658. [Table 7]

[0401] Example 2 - Humanization of parent anti-uPAR antibodies The above anti-uPAR parent antibodies M1, M8, and M43 were subjected to humanization. First, various parameters of each antibody were determined, including 1) the percent natural human identity of each mouse antibody to the human antibody sequence, 2) regions of immunogenicity (e.g., T cell, B cell, and MHC-II antigenic epitopes), 3) antibody misfolding issues (e.g., aggregation and thermal stability), and 4) post-translational modifications (PTMs). PTMs include deamidation sites (e.g., Asn-Gly (NG), resulting in an asparagine to aspartic acid / isoaspartic acid change), isomerization sites (e.g., Asp-Gly (DG), Asp / Asp (DD), resulting in an aspartic acid to isoaspartic acid change), glycosylation sites (e.g., an Asn-X-Ser / Thr (NXS) amino acid motif, where "X" is any amino acid, e.g., NLS), oxidation sites (e.g., Met and / or Trp amino acids, which may be surface exposed and / or in the CDRs of an antibody), proteolysis sites (e.g., an Asp-Ser (DS) and / or Asp-Pro (DP) amino acid motif).

[0402] Humanized antibodies were then produced. The amino acid sequences of the heavy chain variable domain (VH) and light chain variable domain (VL) and the amino acid sequences of HCDR1-3 and LCDR1-3 are shown in Table 8 below. The full-length heavy chain (HC) and light chain (LC) amino acid sequences including the antibody constant regions are shown in Table 9 below. The amino acid sequences of the single chain variable (scFv) format antibodies are shown in Table 10 below. [Table 8-1] [Table 8-2] [Table 9-1] [Table 9-2] [Table 9-3] [Table 10-1] [Table 10-2]

[0403] The binding affinity of humanized anti-uPAR antibodies M1, M8, and M43 was determined. Binding affinity analysis was performed using the Octet platform described above. Briefly, uPAR antibodies were immobilized by anti-human Fc (AHC) sensor. Each uPAR antibody was tested at a concentration of 10 μg / mL. Loading was histidine-tagged human uPAR, starting at 50 nM, then titrated in 2-fold dilutions. The binding affinity of the tested humanized antibodies to human uPAR is shown in Table 11 below. Notably, the humanized antibodies retained similar binding affinity to human uPAR compared to the parent antibody. [Table 11]

[0404] Example 3 - Manipulation of Interleukin 12 (IL-12) IL-12 is a heterodimeric cytokine composed of p35 and p40 subunits, which associate to form IL-12p70. IL-12 is expressed by antigen-presenting cells and plays multiple roles in the immuno-oncology life cycle. IL-12 is a potent stimulator of immune responses, but toxicity issues have limited the clinical use of IL-12. Local delivery can reduce toxicity, but systemic delivery is difficult.

[0405] Engineering one or both of the p35 and p40 subunits offers several advantages. A set of "lock-and-key" mutations in each subunit can be added to modulate the affinity between the two subunits, enhancing specificity. For example, mutations introduced into the engineered subunits can decrease recognition of the wild-type subunit, further reducing toxicity. Paired charged residues at the interface between p35 and p40 can also be engineered to introduce additional specificity.

[0406] Cysteine ​​residue engineering of IL12p35 / IL12p40 As part of the initial optimization strategy, several point mutations in p35 and p40 were identified to improve stability and expression. An intermolecular disulfide bridge is formed between C74 of p35 and C177 of p40. A screen was performed for amino acid substitutions at C74 of p35 and C177 of p40 that would eliminate the disulfide bridge while retaining or improving the binding affinity between the two subunits. For p35, substitutions C74S, S(SS1), D, K, R, Q, W, and Y were made. For p40, substitutions C177S, H, M, F, Y, R, K, E, Q, I, W, and N were made. S(SS1) corresponds to the C15S, C88S, and C74S substitutions in p35. The C15S / C88S pair eliminates an internal disulfide bridge. Each p35 C74 variant was then tested against each of the p40 C177 variants. Binding affinities were measured using the Octet platform described above. Briefly, sensors were loaded with either p35 or p40 at 5-10 μg / mL. Capture compounds were provided at concentrations ranging from 500 nM to 7.8 nM. As shown in Figure 3, several p35 / p40 variant pairs exhibited high binding affinities.

[0407] Specific p35 / p40 variant pairs were selected for further binding affinity characterization. Specifically, p35 C74D, K, W, and Y variants were paired with either p40 C177S or C177F variants. Complete binding affinity data are shown in Tables 12 and 13 below. As shown below, each p35 / p40 variant pair exhibited high binding affinity. [Table 12] [Table 13]

[0408] In addition to the disulfide bridge substitutions mentioned above, a free thiol in p40 was also identified (C252). This site was also engineered to a C252S substitution to remove the free thiol.

[0409] IL12p35 / IL12p40 manipulation - improved stability Although p35 and p40 can be linked to fusion partners to improve stability and yield (see below), it would be advantageous to engineer the subunits to improve stability without a fusion partner, in particular p35, which is unstable by itself and relies significantly on a fusion partner or p40 for proper folding and expression.

[0410] Initially, the S27D and D188N mutations of p35 were identified through directed molecular evolution (see Leong et al. PNAS. 100(3):1163-1168. 2003). Substitutions of p35 are useful for increasing yield.

[0411] Several surface-exposed hydrophobic and flexible regions were identified in p35. In silico modeling and simulation approaches were used to identify potential amino acid mutations (i.e., substitutions, deletions, and / or insertions) that may improve the stability of p35. As a first pass, 85 single point mutations were ranked by delta-delta G (ddG, i.e., difference between delta G of WT and mutant p35), solubility, position-specific score matrix (pssm) score, and epitope prediction. This process attempted to introduce polar residues in place of solvent-exposed hydrophobic residues for better solubility and bulky hydrophobic residues internally for better core packing. Each individual mutant was tested for expression and stability by Western blot. After Western blot analysis, the protein was purified and tested by HPLC to determine % monomer and % aggregate. The point mutations tested by Western blot are shown below in Table 14. Sample IDs 70-90 were p35 mouse serum albumin (MSA) fusions. All of the p35-MSA fusions were expressed, but the sample ID numbers shown in bold italics (IDs 72, 75, 76, 80, 82, 83) showed the best expression of those tested. [Table 14-1] [Table 14-2]

[0412] Following Western blot analysis, sample IDs 69-90 (p35-MSA fusions) were tested by HPLC to determine % monomer and % aggregates. The results are shown in Table 15 below, showing that several p35 point mutants enjoy a greater monomer content compared to WT p35. [Table 15]

[0413] Results of the single point mutant screen revealed several point mutations that improved p35 expression and stability. In particular, the following single p35 mutants showed % monomer values ​​similar to or greater than those of the WT p35-MSA fusion: M12S, S27D, F39D, F39H, S44K, M111F, M111W, V114I, M119L, M145L, F150Y, L161D, L161Q.

[0414] The double mutant, Q35E / N28R, also exhibited % monomer values ​​that exceeded those of the WTp35-MSA fusion.

[0415] Double mutations are tested to further refine these parameters.

[0416] IL12p35 / IL12p40 fusion The IL-12 subunits p35 and p40 present several manufacturing challenges that must be overcome before IL-12 can be used as a viable therapeutic. p35 is poorly expressed. Expression can be improved by linking p35 to a fusion partner, such as an antibody or fragment thereof, or human serum albumin (HSA). p35 is also prone to aggregation. p40 presents similar challenges. p40 can be expressed without a fusion partner, but the purified composition is often contaminated with p40 homodimers (approximately 15-20%). p40 is also prone to dimerization and aggregation over time at 37°C.

[0417] The stability of the p35 and p40 subunits at 37°C was tested using various fusion partners. p40 was tested using an antibody Fab fusion partner and a full-length IgG fusion partner in which two p40 subunits were linked to each CH3 domain of the IgG molecule. The Fab and full-length antibodies were derived from the anti-uPAR antibody described above. p35 was tested using a mouse serum albumin (MSA) fusion partner and a CH1-hinge-CH2-CH3 antibody fragment fusion partner. Single-chain IL-12 (scIL-12), in which the p35 and p40 subunits are linked by an amino acid linker, was also tested. Exemplary IL-12 subunit fusion proteins are shown in Figure 4. Each fusion protein was tested by HPLC analysis to measure aggregate and dimer formation at 37°C at 0 hours, 24 hours, 48 ​​hours, 1 week, and 2 weeks. The results for each fusion protein are shown below in Tables 16-21. These results demonstrate that linking the p35 and p40 subunits to various fusion partners can reduce aggregate and dimer formation. [Table 16] [Table 17] [Table 18] [Table 19] [Table 20] [Table 21]

[0418] The binding affinity of p35-MSA and p40-fab fusion proteins to each other was then tested over time at 37° C. Using the Octet platform described above, biotinylated p35-MSA was immobilized at concentrations ranging from 50 nM to 0.78 nM and p40-fab was loaded at concentrations of 10-20 μg / mL. The binding affinity data at 0, 4, 24, and 47 hours are reported below in Table 22. These results show that the affinity of p35 / p40 remains high even after 47 hours at 37° C. [Table 22]

[0419] Example 4 - Split IL-12 Activity The therapeutic benefit of IL-12 is achieved, at least in part, by allowing the p35 and p40 subunits to heterodimerize to form IL-12p70. To that end, the heterodimerization ability of each subunit with different fusion partners was tested. The fusion protein combinations tested are shown in Table 23 below. [Table 23]

[0420] The above combinations were tested in a cell-based in vitro IL-12 activity assay. Briefly, frozen T cells (cultured with rhIL-2+CD3 / CD28 for 9 days prior to the start of the assay) were thawed, seeded at 300,000 cells / well, and incubated at 37°C for 1 h. IL-12 subunits were added to T cells at a 1:1 ratio (final starting concentration of each molecule was 250 nM). Phosphorylated Stat4 (pStat4) and IFN-gamma were then detected. For the pStat4 detection assay, cells and IL-12 subunits were incubated at 37°C for 45 min. For the IFN-gamma detection assay, cells and IL-12 subunits were incubated at 37°C for 24 h, after which the supernatants were harvested.

[0421] Each combination was tested for its ability to induce Stat4 phosphorylation in CD8+ (Figure 6A) and CD4+ (Figure 6B) T cells. Briefly, cells from each sample were fixed and incubated with fluorescent phycoerythrin (PE)-labeled anti-pStat4 antibody. Fluorescence was then detected. EC50 values ​​and EC50 fold change relative to scIL-12 are shown below in Table 24 for CD8+ T cells and Table 25 for CD4+ T cells. [Table 24] [Table 25]

[0422] Each combination was also tested for its ability to induce IFN gamma production in cells (Figure 7). The MSD T-Plex cytokine assay system was utilized. The MSD assay is an ELISA-based assay, where the MSD plates are pre-coated with a capture antibody against IFN gamma. After incubation of samples on the plate, a labeled detection antibody that also binds IFN gamma was utilized for chemiluminescent detection. The EC50 values ​​and fold change in EC50 relative to scIL-12 are shown below in Table 26. [Table 26]

[0423] Example 5 - Anti-uPAR / IL-12 subunit fusions To enhance the therapeutic efficacy of IL-12 while reducing toxicity issues, IL-12 subunits (p35 and p40) were linked to the parent anti-uPAR antibody described above. The uPAR antibody-IL-12 subunit fusion can deliver the subunits to the target uPAR-expressing tissue or cell (e.g., tumor tissue or cell), thereby ensuring that the potent IL-12 activity is restored only at the target site. To achieve this goal, the heterodimerization ability of each subunit with different uPAR antibody fusion partners was tested. The fusion protein combinations tested and the binding affinity between p35 and p40 are shown in Table 27 below. The binding affinity of the uPAR antibody fusions to uPAR (uPAR-his and uPAR-Fc) is also shown below. [Table 27]

[0424] Selected fusion proteins were then tested in an in vivo mouse tumor model to demonstrate tumor and serum pharmacokinetics by PET / CT imaging of radiolabeled tumor-targeted IL-12 subunits p40 (Figure 5A) and p35 (Figure 5B). Tumor-targeted subunits were measured over time in tumors and blood of mice to determine % of maximum exposure. Tumor-targeted p40 was a fusion of anti-uPAR antibody M37 with p40 in Fab format. Tumor-targeted p35 was a fusion of anti-uPAR antibody M37 with p35 in KiH format. Briefly, CT26 tumor-bearing BALB / c mice were injected with zirconium-89 ( 89 Zr)-labeled fusion protein was administered by intravenous injection (tail vein) in 200 μL with a molar mass of 15.5-16.9 nmol / kg (target: 16.4 nmol / kg) and a radiation dose of 182-192 μCi. Mice were then imaged by PET / CT at 4 h, 24 h, 48 h, 72 h, and 168 h after injection. At each imaging time point, blood was collected for gamma counting.

[0425] Tumor clearance of targeted IL-12 subunits was slower than serum clearance, promoting tumor-specific assembly.

[0426] The above fusion proteins were also tested in an in vivo mouse tumor model to demonstrate tumor and liver accumulation. Three anti-uPAR antibody fusion proteins were used as tumor targeting compounds and compared to non-targeting scIL-12 or p35-MSA fusion proteins. The three targeting fusion proteins were Fab-p40), IgG-p40, and p35-KiH. Each compound was radiolabeled to follow tissue accumulation in mice. The assay was performed as follows: 89 The same experiment was carried out using Zr-labeled fusion protein. As shown in Figure 8A, the targeted compound accumulates in the tumor and then begins to disappear over time. The unlabeled compound does not accumulate. Figure 8B shows the accumulation in the liver over time, indicating the clearance progression of the compound. The targeted compound has a slower rate of clearance and shows better retention compared to the untargeted compound.

[0427] The fusions were then tested in a humanized mouse model to test the in vivo activity of sequentially administered human IL-12 subunits. Immunodeficient NCG mice were administered 1 × 10 7The mice were humanized by injection of 100 human PBMCs. After 14 days, the mice were treated with PBS (vehicle), scIL-12, or two doses of split IL-12 molecules. For split IL-12 molecules, p35-MSA was administered together with p40-Fab. scIL-12 was administered at 0.24 mg / kg, whereas split IL-12 molecules were administered at 0.6 mg / kg (2.5 times the dose of scIL-12) and 6 mg / kg (25 times the dose of scIL-12), respectively. The interval between injections was less than 5 minutes. Serum was then collected from the mice at various time points for serum pharmacokinetic and pharmacodynamic (serum IFN gamma concentration) analysis. The data show that the split fusion molecules are functional in vivo. The data also show similar pharmacodynamic responses between single chain and split molecules, allowing the serum and tumor activity of these treatments to be distinguished in the following experiments (Figure 9). Notably, a 2.5-fold higher dose of scIL-12 reduced IFN-gamma production, indicating that off-target toxicity issues are mitigated by using split IL-12 fusions. This data further demonstrates the in vivo assembly capability of split IL-12 molecules.

[0428] Additional fusion proteins were tested with the possibility of dual targeting (i.e., antibody-p35 fusion and antibody-p40 fusion, each antibody binding to a different epitope on the same target protein). IL-12 subunits were bound to antibody variants M1 and M8, which bind to human uPAR. In this assay, IL-12p70 complexes are assembled by sequentially adding various elements to human uPAR-positive HEK cells, followed by washing. uPAR-expressing HEK cells were then incubated with CD8+ T cells, and pSTAT4 activity in the T cells was determined. M37 antibody was used as a non-targeting control. This antibody binds to mouse uPAR and does not cross-react with human uPAR. scIL-12 was used as a non-targeting single compound. scIL12-M8 Fab fusion was used as a targeting single compound. M37-p40-Fab and M37-p35-KiH were used as non-targeting negative controls. M8-p40-Fab and M37-p35-KiH were used in combination as a single targeting combination (i.e., only one of the IL-12 subunits is targeted to uPAR). M8-p40-Fab and M1-p35-KiH-Fab were used in combination as a dual targeting combination (i.e., both IL-12 subunits p35 and p40 are targeted to uPAR). Briefly, the assay was performed according to the following protocol: 1) 50,000 uPAR-expressing HEK cells were seeded into each well of a 96-well U-bottom low attachment / binding plate (Corning product no. 4520). 2) The plates were centrifuged and the cells were resuspended in 100 μL of the titration of the first molecule. 3) The cells and the first molecule were incubated at 4° C. for 30 minutes. 4) The cells were centrifuged and washed twice. 5) The cells were resuspended in 100 μL of the titration solution of the second molecule. 6) The cells and molecules were incubated at 4°C for 30 minutes. 7) The cells were centrifuged and washed three times. 8) Human T cells that had been previously frozen (left to stand at 37°C for 1 hour) were added to the wells at 100,000 cells / well. 9) The plate was placed in an incubator (37°C) for 1 hour. 10) The plate was centrifuged, washed and the downstream staining protocol was started.

[0429] After a T cell incubation step, pSTAT4 activity in the T cells was determined.

[0430] The non-targeted subunits or non-targeted scIL-12 showed reduced activity compared to the targeted subunits. The single targeted combination showed improved activity compared to all non-targeted approaches. Finally, the dual targeted subunits showed improved activity compared to the individually targeted pairs, regained most of the activity compared to targeted scIL-12, and enjoyed reduced systemic toxicity (Figure 10).

[0431] The above fusion proteins were made with parent anti-uPAR antibodies. Similar fusion proteins were also made with humanized anti-uPAR antibodies. The amino acid sequences of the M1 fusions are shown in Table 28 below. The amino acid sequences of the M8 fusions are shown in Table 29 below. The amino acid sequences of the M43 fusions are shown in Table 30 below. [Table 28-1] [Table 28-2] [Table 28-3] [Table 28-4] [Table 28-5] [Table 28-6] [Table 28-7] [Table 28-8]

Table 28-9

Table 28-10

Table 28-11

Table 28-12

Table 28-13

Table 28-14

Table 28-15

Table 28-16

Table 28-17

Table 29-1

Table 29-2

Table 29-3

Table 29-4

Table 29-5

Table 29-6

Table 29-7

Table 29-8

Table 29-9

Table 29-10

Table 29-11

Table 29-12

Table 29-13

Table 29-14

Table 30-1

Table 30-2

Table 30-3

Table 30-4

Table 30-5

Table 30-6

Table 30-7

Table 30-8

Table 30-9

Table 30-10

[0432] Example 6 - Additional uPAR targeting fusion proteins Design of the fusion protein of Example 6 Fusion protein constructs designed and / or synthesized according to Example 6 are shown in Table 31. Some of these amino acid sequences shown in Table 31 are used as controls in the following experimental examples. For testing in mouse models and mouse cell lines, sequences with mouse proteins or human proteins modified for better binding in mouse experiments were included. Any of the constructs using mouse proteins or human proteins modified for mouse experiments can be replaced with fully human proteins.

[0433] For expression in mammalian cells, N-terminal leader sequences were added to some of the fusion protein constructs shown in Table 31. 6xHis and FLAG tags and an avidin polypeptide were also added to some of the fusion protein constructs shown in Table 31 to facilitate purification during recombinant procedures.

[0434] It is understood that the constructs in Table 31 of the fusion proteins of Example 6 can be modified, for example, in amino acid composition or the length of the linker molecule between the polypeptide molecules, without losing the desired biological activity. The order of fusion of various polypeptides can be modified, for example, the uPAR targeting domain region can be at the N-terminus or C-terminus of the cytokine or immune checkpoint modulator portion of the fusion protein, or other fusion proteins such as Fc fusion proteins or human serum albumin (HAS) fusion proteins can be added to the constructs in Table 31 via optional linkers. The abbreviations in Table 31 include single chain (sc), circularly permuted (cp), and scrambled sequence (ss). [Table 31-1] [Table 31-2] [Table 31-3] [Table 31-4] [Table 31-5] [Table 31-6] [Table 31-7] [Table 31-8] [Table 31-9] [Table 31-10] [Table 31-11] [Table 31-12] [Table 31-13] [Table 31-14] [Table 31-15] [Table 31-16] [Table 31-17]

[0435] Example 6 Recombinant Procedure, Protein Expression and Protein Purification Gene synthesis and subcloning into mammalian expression vectors Gene synthesis and subcloning for expression of the designed constructs was performed using standard molecular biology techniques.

[0436] Protein expression All proteins were expressed using the EXPI293™ Expression System Kit (ThermoFisher Scientific) according to the manufacturer's protocol.

[0437] Protein purification After harvesting the expression culture supernatant, the expressed proteins were captured depending on the nature of the fusion protein. Proteins containing IgG Fc (mouse or human) were captured on a Protein A column and the column was washed with up to 5 column volumes of PBS. Proteins were eluted from the column by lowering the pH of the running buffer and neutralized directly with Tris buffer at pH 8. The purified proteins were then dialyzed overnight against PBS. For proteins containing hexahistidine (6xHis) fusions, proteins in the supernatant were captured on Ni-NTA Sepharose resin and eluted with increasing concentrations of imidazole. The purified proteins were then dialyzed overnight against PBS.

[0438] Kinetic Measurement of Binding of IL-2 and IL-15 Based Fusion Proteins to Human or Mouse uPAR - Example 6 Recombinant mouse uPAR-Fc (R&D Systems, Cat. No. 531-PA-100) was captured on a modified BIACORE™ CM5 sensor chip using a human antibody capture kit (GE Healthcare, Cat. No. BR100839) according to the manufacturer's protocol. Test molecules were passed over the chip at a flow rate of 30 μl / min for 2 min in an increasing concentration range. Analyte dissociation was measured for 10 min. The chip was regenerated by flowing 3M MgCl2 for 30 s. The resulting sensorgrams were analyzed using the instrument-specific software to calculate the binding affinity of the constructs. The results are shown in Table 32.

[0439] Recombinant human uPAR his (R&D Systems, 807-UK-100 / CF) was biotinylated and then captured on a BIACORE™ streptavidin sensor chip according to the manufacturer's protocol. The molecule of interest, also called analyte, was passed over the chip in a stepwise increasing concentration range at a flow rate of 30 μl / min for 3 min. Dissociation of the analyte was measured for 20 min. The chip was regenerated by flowing 3M MgCl2 for 30 s. The resulting sensorgrams were analyzed using the instrument's native software to calculate the binding affinity of the constructs. The results are shown in Table 32.

[0440] Overall, fusions of human-based IL-2 or IL-15 with uPA-ATF bound tightly to human uPAR in the range of 0.59-2.2 nM (see SEQ ID NOs: 98, 99 and 100 in Table 32). These fusion molecules bound with slightly lower potency compared to the control molecule SEQ ID NO: 20 (human uPA-ATF; 0.24 nM). Fusions of IL-2 and IL-15 with mouse uPA-ATF bound to mouse uPAR with similar potency (0.14-0.24 nM) as SEQ ID NO: 104 (mouse uPA-ATF; 0.14 nM). Furthermore, SEQ ID NO: 110, in which human cpIL-2:IL-2Rα is fused to human uPA-ATF incorporating the N22Y, W30R point mutations, had a K of 0.12 nM. D These data indicate that fusions of IL-2 and IL-15 based sequences with a uPAR targeting moiety (in this case uPA-ATF fused at either the N- or C-terminus) retain strong binding to uPAR in vitro. [Table 32]

[0441] In Vitro Efficacy of Molecules Designed to Activate the IL-2 Receptor on Human T Cells - Example 6 To evaluate the potency of molecules containing human sequence-based fusions of IL-2 or IL-15, HH cells (ATCC® CRL-2105™) were incubated with increasing concentrations of SEQ ID NOs: 98, 99, 100, 110, 94, 106, and 107 for 30 minutes. After incubation, cells were washed, lysed, and STAT5 phosphorylation was detected using the STAT5 alpha / beta (Phospho)[pY694 / pY699] Multispecies InstantOne™ ELISA Kit (ThermoFisher Scientific, Cat. No. 85-86112-11) according to the manufacturer's protocol. STAT5 phosphorylation is a receptor-proximal signaling event induced by IL-2 / IL-15 receptor activation. Data are shown in Table 33.

[0442] The non-targeting parent circularly permuted cpIL-2:IL2Rα fusion protein, SEQ ID NO:94, has an EC of 1.1 nM in the HH pSTAT5 assay. 50 The cpIL-2:IL2Rα fusion proteins containing a C-terminal uPAR targeting domain, i.e., SEQ ID NOs: 99, 106 and 110, were equally potent, with EC values ​​of 1.5 nM, 1.6 nM and 2.1 nM, respectively. 50 The cpIL-2:IL2Rα fusion protein containing the N-terminal uPAR targeting domain, SEQ ID NO: 98, inhibited EC 50 The non-targeting parent hIL-15Ra(sushi):cpIL-15 fusion protein SEQ ID NO:95 was more potent than SEQ ID NO:94 in the HH pSTAT5 assay, with an EC of 0.039 nM. 50 The variant of SEQ ID NO:95 with a targeting domain at the N-terminus was also more potent than SEQ ID NO:94, and the EC 50were 0.18 nM and 0.099 nM, respectively. In summary, the targeted variants of SEQ ID NO:94 with a C-terminal uPAR targeting domain and SEQ ID NO:95 with an N-terminal targeting domain are all as potent as their non-targeting parent molecules, suggesting that fusion to the uPAR targeting motif does not sterically inhibit / interfere with binding of the cytokine domain to its cognate receptor. [Table 33]

[0443] In Vitro Efficacy of Molecules Designed to Activate the IL-2 Receptor on Primary Mouse Splenocytes - Example 6 To examine the potency of molecules containing mouse sequence-based fusions of IL-2 or IL-15, splenocytes were harvested from wild type mouse spleens and incubated for 30 minutes with increasing concentrations of SEQ ID NO:96, mouse Fc:mouse IL-15Ra:cpIL-15, SEQ ID NO:101, SEQ ID NO:102 and SEQ ID NO:105. Cells were then fixed and stained with fluorescently labeled antibodies against surface and intracellular markers to identify mouse NK cells, CD8 T cells and CD4+CD25+Foxp3+ regulatory T cells (T reg ) was detected using standard flow cytometry techniques. The results are shown in Table 34.

[0444] Recombinant wild-type IL-2 (rIL-2) selectively stimulates cells expressing the high-affinity IL-2 receptor, which in normal mice and healthy humans primarily stimulates T reg At fairly high concentrations, rIL-2 can also activate a subset of NK cells and memory CD8 T cells by expressing the intermediate affinity IL-2 receptor complex. The circularly permuted murine cpIL-2:IL2Rα fusion protein, SEQ ID NO:96, was engineered to be more potent against cells expressing the intermediate affinity receptor compared to rIL-2. This is due to the fact that T regpSTAT5 induction in CD8 T cells, NK cells, and memory CD8 T cells was comparable, whereas EC 50 The murine cpIL-2:IL2Rα fusion protein with a C-terminal uPAR targeting domain, i.e., SEQ ID NO: 101, has a T reg , NK cells, and memory CD8 T cells, and EC 50 were 16.5 nM, 19.5 nM, and 27.3 nM, respectively. Similarly, the murine cpIL-2:IL2Rα fusion protein with an N-terminal uPAR targeting domain, i.e., SEQ ID NO: 105, reg EC of 21.9 nM, 34.9 nM, and 38.0 nM for CD8 T cells, NK cells, and memory CD8 T cells, respectively. 50 In contrast to rIL-2, rmIL-15 inhibits T cells because its affinity for the IL-2 / IL-15 receptor complex is not enhanced by the presence of CD25. reg This is because mouse Fc:mouse IL-15Ra:cpIL-15 does not selectively activate T reg pSTAT5 induction in CD8 T cells, NK cells, and memory CD8 T cells was comparable, whereas EC 50 The murine IL-15Ra(sushi):cpIL-15 fusion protein with an N-terminal uPAR targeting domain, i.e., SEQ ID NO: 102, has a T reg , NK cells, and memory CD8 T cells, and showed similar potency against EC 50 were 1.7 nM, 0.15 nM, and 0.50 nM, respectively. As with the uPAR-targeted variants of SEQ ID NO: 94 and SEQ ID NO: 95, all uPAR-targeted variants of the mouse orthologues were as potent as their non-targeted parent molecules, demonstrating that fusions to uPAR targeting motifs are effective in inhibiting primary mouse T reg , suggesting that it does not sterically inhibit / interfere with binding of the cytokine domain to its cognate receptor expressed on NK cells and memory CD8 T cells. [Table 34]

[0445] Binding of IL-2 or IL-15 based molecules to mouse tumor cell lines expressing uPAR - Example 6 The mouse allograft tumor lines 4T1, CT26, EMT6 (all the former obtained from ATCC®) and MC38 (obtained from NCI) were shown to express mouse uPAR on the cell surface by staining with fluorescently labeled anti-mouse uPAR (CD87) antibody as detected by flow cytometry. Adherent cells were lifted with CORNING® CELLSTRIPPER® Solution (cat. no. 25056CI), washed, and then incubated with increasing concentrations of the test molecules (see Table 35). After incubation for 60 minutes at 4°C, cells were stained with fluorescently labeled anti-His antibody and analyzed using standard flow cytometry techniques. Mean fluorescence intensity was plotted against the concentration of test substance, and EC 50 Values ​​were calculated using four-parameter logistic curve fitting. [Table 35]

[0446] Pharmacokinetics of SEQ ID NO:94 and SEQ ID NO:110 in MC38 tumor-bearing mice - Example 6 To determine the ratio of protein circulating in serum to tumor, single-dose PK experiments were performed in female MC38 tumor-bearing C57BL / 6 mice. Mice were dosed when tumor size was in the range of 200-400 mm3. Mice were subcutaneously dosed with either 30 μg SEQ ID NO: 94 or 75 μg SEQ ID NO: 110 at 0 hours, then terminal bleeds were taken at 0.5, 2, 4, 8, and 24 hours post-dose and processed into serum from individual animals. Tumors from the same animals were also harvested and snap frozen for downstream concentration analysis. Serum samples were analyzed by a human IL-2 specific MSD (Meso-Scale Discovery) assay. Tumor samples were homogenized and concentrations were measured by the same detection assay. Pharmacokinetic parameters were determined using a non-compartmental model in WinNonlin (Phoenix). Results are shown in Table 36.

[0447] Comparing the tumor to systemic exposure ratio (T / S) for both Cmax and AUC, uPAR-targeted IL-2, SEQ ID NO:110, showed a greater tumor penetration ratio compared to the non-targeted control molecule, SEQ ID NO:94, which showed a similar ratio of tumor to serum exposure. [Table 36]

[0448] In Vitro Efficacy of Molecules Designed to Activate the IL-10 Receptor - Example 6 STAT3 phosphorylation is a receptor-proximal signaling event in response to IL-10 receptor binding and activation. To examine the potency of molecules containing fusions of IL-10, human peripheral blood mononuclear cells were incubated with increasing concentrations of scIL-10:CL:CH1:Fc (scIL10 3aa linker);=SEQ ID NO: 136, which is equivalent to scIL-10:CL:CH1:Fc+5AA linker+huPA-ATF) for 1 hour. Cells were then fixed and stained with fluorescently labeled antibodies against surface and intracellular markers, and STAT3 phosphorylation was detected in mouse B cells, monocytes, CD4 and CD8 T cells using standard flow cytometry techniques. Data are shown in Table 37.

[0449] Both molecules have very similar potencies to each other across a range of cell types, confirming that fusion of the IL-10 molecule with the uPAR targeting moiety (in this case human uPA-ATF) does not affect the molecule's ability to bind and activate the IL-10 receptor expressed on human immune cells. [Table 37]

[0450] In Vitro Efficacy of uPAR Targeting Molecules Designed to Activate the IL-12 Receptor - Example 6 HEK-BLUE™ IL-12 cells (InvivoGen) are human embryonic kidney cells specifically designed for in vitro detection of bioactive IL-12 by monitoring IL-12-induced activation of the NF-κB / AP-1 pathway. This cell line expresses an inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a STAT4 inducible promoter. HEK-Blue IL-1β cells were plated at 50,000 cells / well in DMEM medium containing 4.5 g / L glucose and 2 mM L-glu and 10% heat-inactivated FBS (Gibco). Cells were incubated with various concentrations of IL-12 (R&D Systems, Cat. No. 219-IL-005 / CF) or SEQ ID NO: 141 for 20 hours at 37° C., 5% CO2. SEAP production was detected by adding QUANTI-Blue™ (InvivoGen) and incubating for 3 hours at 37° C., 5% CO 2 , then reading at 630 nm on a plate reader.

[0451] A single chain human IL-12 construct fused to the human uPA-ATF construct of SEQ ID NO:141 has similar potency (27 pM) compared to wild type human IL-12 (18 pM), suggesting that fusion of IL-12 to uPA-ATF does not affect the ability of the IL-12 portion of the molecule to bind and activate the IL-12 receptor.

[0452] In vitro efficacy of uPAR targeting molecules designed to activate the human 4-1BB receptor - Example 6 Fusion proteins containing the 4-1BB ligand were assayed using the 4-1BB Bioassay Kit from Promega. The assay uses 4-1BB effector cells that stably express 4-1BB and luciferase downstream of a response element. Luciferase expression is induced upon ligand binding to the receptor. The assay was performed according to the manufacturer's protocol. Dose-response curves were generated by incubating increasing concentrations of the test molecules and measuring the light produced from the expressed luciferase. The data are shown in Table 38.

[0453] Recombinant human 4-1BB ligand monomer (R&D Systems catalog number 2295-4L-025 / CF) showed no activity in the assay, suggesting that uncrosslinked 4-1BBL monomer is unable to activate 4-1BB since 4-1BBL normally functions as a trimer. Linked 4-1BBL trimers (SEQ ID NO: 130 and SEQ ID NO: 131) fused to human uPA-ATF at either the N- or C-terminus had an EC of 0.15 nM. 50 A stronger EC 50 (and a higher maximal signal). These results suggest that fusion with the human uPAR targeting domain uPA-ATF may result in a 2- to 3-fold stronger activity in signaling through 4-1BB, possibly due to an avidity effect resulting from strong binding to uPAR on the cell surface. Nevertheless, the fusion protein of 4-1BBL and uPA-ATF can functionally bind to 4-1BB and signal through 4-1BB. [Table 38]

[0454] In vitro efficacy of molecules designed to activate GM-CSF receptors on human monocytes - Example 6 To assess the potency of molecules containing human sequence-based fusions of GM-CSF, U937 cells (ATCC® CRL-1593.2™) were incubated for 30 minutes with increasing concentrations of recombinant human GM-CSF (R&D Systems, Catalog No. 215-GM-050 / CF), SEQ ID NO: 138, or SEQ ID NO: 103. After incubation, cells were fixed, permeabilized, stained intracellularly with a fluorescently labeled antibody specific for pSTAT5, and analyzed using standard flow cytometry techniques. The data are shown in Table 39.

[0455] Recombinant human GM-CSF (rhGM-CSF) has an EC of 18.6 pM in the U937 pSTAT5 assay. 50 As shown in Fig. 1, human uPAR targeting domain was fused to the C-terminus of GM-CSF, SEQ ID NO: 138. SEQ ID NO: 138 was approximately 20-fold more potent in inducing STAT5 phosphorylation and was associated with an EC 50 was 1.28 pM. Co-incubation of SEQ ID NO: 138 with increasing concentrations of the human uPAR targeting domain alone, SEQ ID NO: 103, reduced potency similar to that of rhGM-CSF alone. These results suggest that the fusion of the uPAR targeting motif to GM-CSF does not adversely affect the ability of the fusion protein to induce STAT5 phosphorylation as a result of receptor binding, but rather appears to enhance its activity. [Table 39]

[0456] In vivo pharmacokinetics of exemplary fusion proteins Certain human anti-uPAR-IL-12 subunit fusion proteins were analyzed in vivo in mouse tumor models. Specifically, the levels of the test fusion proteins were detected in the serum and tumor tissue of mice at several time points after administration. The fusion proteins used in this section are shown in Table 40 below. Experimental details are outlined in Table 41 below. [Table 40-1] [Table 40-2] [Table 40-3] [Table 40-4] [Table 41]

[0457] The model used in the above study was a SC RKO human colorectal cancer xenograft model in female NOD / SCID mice. To generate the tumor model, each mouse was inoculated subcutaneously with RKO tumor cells. The tumor size was approximately 250 mm 3 The tumors were used for randomization when they reached the final dose. Tumor and serum samples were collected 0.5, 1, 4, 24, 48, and 72 hours after the last dose. Meso Scale Discovery (MSD) assays were used to detect p35, p40, or p70 in the samples according to the manufacture's protocol.

[0458] As shown in Figure 11, the fusion protein tested in each group was detected in tumor tissue at all test time points, and the level was relatively stable. The data indicates that the fusion protein is productively accumulated in tumor tissue. As shown in Figure 12, in certain groups, the fusion protein level in serum gradually decreases over time, indicating that the fusion protein is rapidly excreted from the system, except for the fusion protein in target tumor tissue.

Claims

1. A kit comprising a first pharmaceutical composition comprising a first fusion protein and a second pharmaceutical composition comprising a second fusion protein, (a) the first fusion protein comprises: (i) an antigen-binding domain that specifically binds to a first epitope on a urokinase plasminogen activator receptor (uPAR), comprising a heavy chain variable domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 41, 20 and 21, respectively, and a light chain variable domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 22-24, respectively; and (ii) an IL-12 p35 subunit comprising SEQ ID NO:81, optionally wherein said IL-12 p35 subunit comprises one or more of the amino acid substitutions of SEQ ID NO:81 selected from the group consisting of: M12S, S27D, N28R, Q35E, F39D, F39H, S44K, C74K, C74D, C74W, C74Y, C74Q, M111F, M111W, V114I, M119L, M145L, F150Y, L161D, L161Q, and D188N; and (b) the second fusion protein comprises: (i) an antigen-binding domain that specifically binds to a second epitope on uPAR, the antigen-binding domain comprising a heavy chain variable domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 3-5, respectively, and a light chain variable domain comprising CDR1, CDR2 and CDR3 of SEQ ID NOs: 6-8, respectively; and (ii) an IL-12 p40 subunit comprising SEQ ID NO:84, optionally wherein said IL-12 p40 subunit comprises one or two of the following amino acid substitutions in SEQ ID NO:84: C177S, C177F, C177M, C177H, C177I, C177Q, and C252S; Including the kit.

2. The kit described in claim 1, wherein the antigen-binding domain that specifically binds to a first epitope on uPAR comprises a heavy chain variable domain comprising SEQ ID NO: 37 and a light chain variable domain comprising SEQ ID NO:

40.

3. A kit described in claim 1 or 2, wherein the antigen-binding domain that specifically binds to a second epitope on uPAR comprises a heavy chain variable domain comprising SEQ ID NO: 34 and a light chain variable domain comprising SEQ ID NO:

35.

4. The kit described in claim 3, wherein the light chain variable domain of the antigen binding domain that specifically binds to a second epitope on uPAR further comprises a light chain constant domain.

5. The kit described in claim 4, wherein the light chain constant domain comprises a lambda light chain constant domain.

6. The kit of claim 1, wherein the IL-12 p35 subunit comprises the following amino acid substitutions in SEQ ID NO:81: S27D, C74K and D188N.

7. The kit of claim 6, wherein the IL-12 p35 subunit comprises the sequence of SEQ ID NO:

83.

8. The kit of claim 1, wherein the IL-12 p40 subunit comprises the following amino acid substitutions in SEQ ID NO:84: C177S and C252S.

9. The kit of claim 8, wherein the IL-12 p40 subunit comprises the sequence of SEQ ID NO:

87.

10. The kit of any one of claims 1 to 9, wherein the first fusion protein further comprises a linker disposed between the antigen-binding domain that specifically binds to a first epitope on uPAR and the IL-12 p35 subunit, optionally wherein the linker comprises (GGGGS)n (SEQ ID NO: 148), where N=2.

11. The kit described in claim 10, wherein the first fusion protein further comprises a second antigen-binding domain that specifically binds to a first epitope on uPAR.

12. The kit described in claim 11, wherein the first fusion protein comprises the sequence of SEQ ID NO:

213.

13. A second fusion protein comprising a first polypeptide and a second polypeptide, the first polypeptide comprising a heavy chain variable domain of an antigen binding domain that specifically binds to a second epitope on uPAR fused to an IL-12 p40 subunit, a linker disposed between the heavy chain variable domain of the antigen binding domain that specifically binds to the second epitope on uPAR and the IL-12 p40 subunit; and 13. The kit of claim 1, wherein the second polypeptide comprises a light chain variable domain of an antigen binding domain that specifically binds to a second epitope on uPAR fused to a lambda light chain constant domain.

14. The kit described in claim 13, wherein the first polypeptide has the sequence of SEQ ID NO: 179 and the second polypeptide has the sequence of SEQ ID NO:

159.

15. The kit of claim 13 or 14, wherein the linker comprises (GGGGS)n (sequence number 148), where N=2.

16. A kit according to any one of claims 1 to 15 for use in a method for treating cancer in a patient in need thereof.

17. A kit described in any one of claims 1 to 15 for use in a method for treating or preventing a disease or disorder associated with uPAR activity or expression in a patient in need thereof.