Compositions of protein complexes and methods of use thereof

Abstract

Provided herein are protein complexes comprising a sensor domain and a therapeutic domain linked by a linker, and methods of use thereof. In aspects of the disclosure, the activity of the therapeutic domain comprises a dependence on the sensor domain binding to a target marker.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on November 14, 2022, is named 51177-046WO2_Sequence_Listing_11_14_22 and is 634,137 bytes in size. [Background technology]

[0002] background Interleukin 2 (IL-2) is a potent cytokine that is toxic when administered systemically. There is a need for versions of IL-2 that can be delivered systemically but controlled to have therapeutic activity against a competent subset of T cells. Summary of the Invention

[0003] overview In some aspects, the disclosure provides a conjugate comprising (a) a therapeutic domain comprising an IL-2 peptide; and (b) a sensor domain comprising an antibody, the sensor domain configured to bind PD-1 and IL-2 in a mutually exclusive manner. In some embodiments, the conjugate further comprises a linker linking the therapeutic domain to the sensor domain. In some embodiments, the sensor domain is configured to (i) bind IL-2 in the absence of PD-1, and (ii) not bind IL-2 in the presence of PD-1. In some embodiments, the antibody is an antibody fragment or an antibody derivative. In some embodiments, the sensor domain comprises a single dual binding antibody (DBA) configured to bind PD-1 and IL-2. In some embodiments, the DBA is selected from the group consisting of SEQ ID NOs: 11-20, 154-156, 168-173, 114-119, 415, 421, 433, 439, 445, 451, 457, 463, 469, 475, 481, 487, 493, 499, 505, 511, 517, 523, 529, 535, 541, 547, 553, 559, 565, 571, 577, 583, 589, 595, 601, 607, 613, 619, 625, 631, 637, 643, 649, 655, 661, or or 667.In some embodiments, the DBA comprises a heavy chain CDR1, CDR2, or CDR3 comprising a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences listed in Table 3, Table 7, Table 8, or Table 19. In some embodiments, the DBA comprises a sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of the sequences listed in Table 18. H or V LIn some embodiments, the complex comprises an Fc domain. In some embodiments, the Fc domain is derived from an IgG. In some embodiments, the Fc domain is a homodimer. In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises (a) a first polypeptide comprising a knob mutation and (b) a second polypeptide comprising a hole mutation. In some embodiments, the knob mutation or the hole mutation comprises any one of the following residue pairs relative to an IgG: 366 and 407, 405 and 394, or 407 and 366. In some embodiments, the knob mutation comprises an arginine residue, a phenylalanine residue, a tyrosine residue, or a tryptophan residue, and the hole mutation comprises an alanine residue, a serine residue, a threonine residue, or a valine residue. In some embodiments, the complex comprises a sensor domain comprising a full-length DBA, and an IL-2 peptide is linked to the N-terminus of the heavy chain of the full-length DBA, or an IL-2 peptide is linked to the N-terminus of the light chain of the full-length DBA. In some embodiments, the complex comprises a sensor domain comprising a full-length DBA, and an IL-2 peptide is linked to the C-terminus of the heavy chain of the full-length DBA. H ]-[C H ]-[hinge]-Fc-C, and a first polypeptide of N-[V L ]-[C L ]-C, or (b) a second polypeptide by N-[V H ]-[C H ]-[hinge]-Fc-C, and a first polypeptide consisting of N-[IL-2]-[linker]-[V L ]-[C L and a second polypeptide by the formula: [linker]-C, wherein N- represents the peptide N-terminus, C- represents the peptide C-terminus, and V H indicates the heavy chain variable domain of the above DBA, and C H indicates the immunoglobulin heavy chain constant domain, and V Lrepresents the light chain variable domain of the above DBA, [hinge] represents the hinge region of an immunoglobulin, Fc represents the Fc region of an immunoglobulin, and CL represents the light chain constant domain of an immunoglobulin. In some embodiments, the complex comprises any one of AF003345, AF003243, AF003246, AF003247, AF003341, AF003644, AF003651, AF003657, or AF003934. In some embodiments, the complex comprises (a) N-[IL-2]-[linker]-[V H ]-[C H ]-[hinge]-Fc[knob]-C, and a first polypeptide with N-[V L ]-[C L ]-C, and a second polypeptide by N-[V H ]-[C H ]-[hinge]-Fc[hole]-C; or (b) a third polypeptide consisting of N-[IL-2]-[linker]-[V H ]-[C H ]-[hinge]-Fc[hole]-C, and a first polypeptide of N-[V L ]-[C L ]-C, and a second polypeptide by N-[V H ]-[C H and a third polypeptide consisting of: Fc[knob]-[hinge]-Fc[knob]-C, wherein N- represents the N-terminus of the peptide, C- represents the C-terminus of the peptide, and [linker] represents the linker as defined above; H indicates the heavy chain variable domain of the above DBA, and C H indicates the immunoglobulin heavy chain constant domain, and V L represents the light chain variable domain of the above DBA, [hinge] represents the hinge region of an immunoglobulin, Fc[knob] represents the Fc of an immunoglobulin containing a knob mutation, Fc[hole] represents the Fc region of an immunoglobulin containing a hole mutation, and C Lrepresents an immunoglobulin light chain constant domain. In some embodiments, the knob or hole mutation comprises a mutation in any one of the following residue pairs for IgG: 366 and 407, 405 and 394, or 407 and 366. In some embodiments, the complex comprises any one of AF003229, AF003230, AF003232, AF003740, AF003747, AF003749, AF003753, AF003945, AF003947, AF003951, AF003952, AF003953, AF003955, AF003956, or AF003941. In some embodiments, the complex comprises (a) N-[IL-2]-[linker]-[V H ]-[C H ]-[hinge]-Fc-[scFv]-C, and (b) a first polypeptide comprising N-[V L ]-[C L and a second polypeptide by the formula: [linker]-C, wherein N- represents the peptide N-terminus, C- represents the peptide C-terminus, and V H represents the heavy chain variable domain of an anti-PD-1 monospecific antibody; H indicates the immunoglobulin heavy chain constant domain, and V L represents the light chain variable domain of an anti-PD-1 monospecific antibody, [hinge] represents the hinge region of an immunoglobulin, Fc represents the Fc region of an immunoglobulin, and C L represents the light chain constant domain of an immunoglobulin, and [scFv] represents the V H Domains and V L In some embodiments, the scFv comprises the N-[V H ]-[Linker 2]-[V L In some embodiments, the DBA is oriented according to the formula: H Domains and V LThe scFv containing the domains are (a) any one of Vs AB002022_2B07v1, AB002328_2B07v4, AB002360_7A04v1, AB002413_2A11v3, AB002342_2B07v5, AB002345_2B07v6, or AB002365_7A04v2. H V containing a sequence with at least 80% identity to the domain H domain, or (b) any one of AB002022_2B07v1, AB002328_2B07v4, AB002360_7A04v1, AB002413_2A11v3, AB002342_2B07v5, AB002345_2B07v6 or AB002365_7A04v2 L V containing a sequence with at least 80% identity to the domain L In some embodiments, the DBA includes a domain. H Domain and L The above scFv containing domains comprise (a) any one of the heavy chain CDRs of AB002022_2B07v1, AB002328_2B07v4, AB002360_7A04v1, AB002413_2A11v3, AB002342_2B07v5, AB002345_2B07v6, or AB002365_7A04v2, or (b) any one of the light chain CDRs of AB002022_2B07v1, AB002328_2B07v4, AB002360_7A04v1, AB002413_2A11v3, AB002342_2B07v5, AB002345_2B07v6, or AB002365_7A04v2. In some embodiments, the complex comprises any one of AF003864, AF003871, AF003872, AF003913, AF003918, AF003923, AF003927, AF004502, AF004503, AF004504, AF004505, AF004892, or AF004893. In some embodiments, the complex comprises (a) N-[V H ]-[C H ]-[hinge]-Fc[knob]-[linker]-[IL-2]-C, and a first polypeptide of N-[V L ]-[CL ]-C, and a second polypeptide by N-[V H ]-[C H ]-[hinge]-Fc[hole]-[linker]-[scFv]-C; or (b) a third polypeptide of N-[V H ]-[C H ]-[hinge]-Fc[hole]-[linker]-[IL-2]-C, and a first polypeptide of N-[V L ]-[C L ]-C, and a second polypeptide by N-[V H ]-[C H and a polypeptide of Dow3 consisting of: Fc[knob]-[hinge]-Fc[knob]-[linker]-[scFv]-C, wherein N- represents the N-terminus of the peptide, C- represents the C-terminus of the peptide, and [linker] represents the linker as above; V H indicates the heavy chain variable domain of the above DBA, and C H indicates the heavy chain constant domain of an immunoglobulin, VL represents the light chain variable domain of the above DBA, [hinge] represents the hinge region of an immunoglobulin, Fc[knob] represents the Fc of an immunoglobulin containing a knob mutation, Fc[hole] represents the Fc region of an immunoglobulin containing a hole mutation, C L represents an immunoglobulin light chain constant domain, and [scFv] represents an scFv of the DBA. In some embodiments, the complex comprises any one of AF004693, AF004695, AF004696, AF005416, AF005418, or AF005419. In some embodiments, the complex comprises (a) N-[V H ]-[C H ]-[het-hinge]-Fc[knob]-[linker]-[IL-2]-C, and a first polypeptide of N-[V L ]-[C L ]-C, and a second polypeptide by N-[V H ]-[C H ]-[het-hinge]-Fc[hole]-C, or (b) a third polypeptide consisting of N-[V H ]-[C H]-[het-hinge]-Fc[hole]-[linker]-[IL-2]-C, and a first polypeptide of N-[V L ]-[C L ]-C, and a second polypeptide by N-[V H ]-[C H and a third polypeptide by: V[het-hinge]-Fc[knob]-C, wherein N- represents the N-terminus of the peptide, C- represents the C-terminus of the peptide, and [linker] represents the linker as defined above; H indicates the heavy chain variable domain of the above DBA, and C H indicates the immunoglobulin heavy chain constant domain, and V L represents the light chain variable domain of the DBA, [het hinge] represents a hinge region heterologous to the Fc region, Fc[knob] represents the Fc of an immunoglobulin containing a knob mutation, Fc[hole] represents the Fc region of an immunoglobulin containing a hole mutation, and C Lrepresents an immunoglobulin light chain constant domain. In some embodiments, the hinge region heterologous to the Fc region is (a) a hinge region from an IgG3 antibody, or (b) a G4S-based linker. In some embodiments, the conjugate comprises AF003632 or AF003634. In some embodiments, the IL-2 peptide comprises a wild-type human IL-2 peptide. In some embodiments, the IL-2 peptide comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or substantially 100% sequence identity to human IL-2. In some embodiments, the IL-2 peptide comprises at least one mutation of R38, K43, E61, F42, Y45, L72, T3, or C125 of human IL-2.In some embodiments, the complex is selected from the group consisting of AF003232, AF003243, AF003246, AF003247, AF003341, AF003345, AF003632, AF003634, AF003644, AF003651, AF003652, AF003653, AF003657, AF003740, AF003744, AF003747, AF003749, AF003753, AF003864, AF003873, AF003876, AF003877, AF003913, AF003918, AF003923, AF003927, AF003930, AF003931, AF003933, AF003934, AF003935, AF003941, AF003945, AF003946, A F003947, AF003948, AF003951, AF003952, AF003953, AF003955, AF003956, AF004262, AF004265, A F004273, AF004276, AF004284, AF004287, AF004295, AF004298, AF004385, AF004386, AF004387, AF Contains one of the following: 004388, AF004389, AF004404, AF004405, AF004413, AF004414, AF004415, AF004416, AF004504, AF004505, AF004693, AF004695, AF004696, AF004771, AF004773, AF004892, or AF004893.

[0004] In some aspects, the present disclosure provides a method of enhancing T cell reactivity against a xenogeneic cell, comprising administering to a subject in need thereof any of the conjugates described herein. In some embodiments, the xenogeneic cell is a cancer cell.

[0005] In some embodiments, the present disclosure provides a method of treating a subject in need of a conjugate according to any one of claims 1 to 32, comprising administering to a subject in need thereof a conjugate according to any one of claims 1 to 32. In some embodiments, administering comprises intravenous, intramuscular, or subcutaneous administration. In some embodiments, the subject in need thereof has cancer. In some embodiments, the therapeutic domain treats a subject in need thereof. In some embodiments, the subject in need thereof is a mammal. In some embodiments, the subject in need thereof is a human.

[0006] In some embodiments, the disclosure provides compositions comprising a recombinant nucleic acid encoding any of the complexes described herein. In some embodiments, the disclosure provides host cells comprising any of the recombinant nucleic acids encoding any of the complexes described herein. In some aspects, the present disclosure provides a pharmaceutical composition comprising any of the conjugates described herein and a pharma- ceutically acceptable excipient.

[0007] In various embodiments, the present disclosure provides a conjugate comprising: a) a therapeutic domain; b) a linker; and c) a sensor domain, wherein the therapeutic domain is an IL-2 agonist, and the therapeutic domain is linked to the sensor domain by the linker, and the sensor domain is a dual binding antibody (DBA) capable of binding to the therapeutic domain (the IL-2 agonist domain) and a marker, and the marker is PD-1.

[0008] In some embodiments, the sensor domain is bound to the therapeutic domain in the absence of the marker. In some embodiments, the therapeutic domain is blocked from binding to the sensor domain when the sensor domain binds to the marker. In some embodiments, the activity of the therapeutic domain is decreased when the therapeutic domain binds to the sensor domain. In some embodiments, the therapeutic domain can exhibit therapeutic activity when the sensor domain binds to the marker. In some embodiments, the therapeutic domain is therapeutically active when the sensor domain binds to the marker.

[0009] In some embodiments, the sensor domain comprises an antibody. In some embodiments, the antibody is an antibody fragment or an antibody derivative. In some embodiments, the complex comprises an Fc domain. In some embodiments, the complex comprises a domain that improves kinetic properties. In some embodiments, the complex comprises two heavy chains and two light chains.

[0010] In some embodiments, the complex comprises two therapeutic domains. In some embodiments, the complex comprises two sensor domains. In some embodiments, the complex is a regulated therapeutic protein. In some embodiments, the antibody or antibody fragment comprises an IgG, a single domain antibody fragment, a nanobody, or a single chain variable fragment (scFv).

[0011] In some embodiments, the therapeutic domain is an IL-2 receptor agonist. In some embodiments, the IL-2 receptor agonist is IL-2, IL-15, or a variant or fusion thereof. In some embodiments, the therapeutic domain binds to the sensor domain.

[0012] In some embodiments, the linker is a polypeptide linker. In some embodiments, the linker comprises 2-200 amino acids in length. In some embodiments, the linker is attached to the heavy chain of the sensor domain, attached to the light chain of the sensor domain, fused to the N-terminus of the sensor domain, or fused to the C-terminus of the sensor domain. In some embodiments, the linker is attached to the heavy chain of the therapeutic domain, attached to the light chain of the therapeutic domain, fused to the N-terminus of the therapeutic domain, or fused to the C-terminus of the therapeutic domain.

[0013] In some embodiments, the activity of the therapeutic domain is decreased upon binding to the sensor domain. In some embodiments, the therapeutic domain is inactive when bound to the sensor domain. In some embodiments, the sensor domain blocks the activity of the therapeutic domain when bound to the therapeutic domain. In some embodiments, the therapeutic domain is active when the sensor domain is bound to a marker. In some embodiments, the affinity of the sensor domain for the marker is equal to or greater than the affinity of the sensor domain for the therapeutic domain.

[0014] In some embodiments, the affinity of the sensor domain for the marker is at least 2-fold, 5-fold, 10-fold, 100-fold, 1000-fold, 10000-fold, or 100000-fold greater than the affinity of the sensor domain for the therapeutic domain.

[0015] In some embodiments, the sensor domain is an antibody or a fragment thereof. In some embodiments, the sensor domain comprises one or both of the antigen binding domains of a bispecific antibody. In some embodiments, the bispecific antibody comprises a first antigen binding domain capable of binding to a therapeutic domain and capable of binding to a marker, and a second antigen binding domain capable of binding to a marker. In some embodiments, the bispecific antibody comprises a single therapeutic domain.

[0016] In some embodiments, the sensor bispecific antibody binds to IL-2 and PD-1. In some embodiments, the IL-2 receptor agonist is IL-2, IL-15, or a variant or fusion thereof.

[0017] In some embodiments, the sensor domain comprises a complementarity determining region (CDR) selected from Table 3, Table 7, Table 8 or Table 19. In some embodiments, the sensor domain is selected from Table 8 or Table 18. In some embodiments, the complex is selected from Table 15 or Table 2A. In some embodiments, the sensor domain comprises a complementarity determining region having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any of the complementarity determining regions selected from Table 3, Table 7, Table 8 or Table 19 ... H or V L V having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any of H or V LIn some embodiments, the sensor domain comprises a complementarity determining region having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 1-20, 142-173 or 238-252. In some embodiments, the sensor domain comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 21-27, 31-39 or 127-141. In some embodiments, the protein complex has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to SEQ ID NO:175, SEQ ID NO:181 to SEQ ID NO:182, SEQ ID NO:195 to SEQ ID NO:205 to SEQ ID NO:206, SEQ ID NO:210 to SEQ ID NO:212, SEQ ID NO:220 to SEQ ID NO:223, SEQ ID NO:226 to SEQ ID NO:231, SEQ ID NO:259 to SEQ ID NO:261, SEQ ID NO:266 to SEQ ID NO:289 to SEQ ID NO:293, or fragments thereof.

[0018] In various embodiments, the disclosure provides a method comprising administering any of the above complexes to a subject in need thereof. In various embodiments, the disclosure provides a method of treating a subject in need of any of the above complexes, comprising administering any of the above complexes to a subject in need thereof. In some embodiments, the administering comprises intravenous, intramuscular, or subcutaneous administration. In some embodiments, the subject in need thereof has cancer. In some embodiments, the therapeutic domain treats a subject in need thereof. In some embodiments, the subject in need thereof is a mammal. In some embodiments, the subject in need thereof is a human.

[0019] In some embodiments, the present disclosure provides IL-2 conjugates that can be delivered systemically yet exhibit reduced systemic toxicity.

[0020] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference herein. [Brief description of the drawings]

[0021] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0022] [Figure 1A-1B]1A and 1B show schematic diagrams of protein complexes of the present disclosure. FIG. 1A shows an exemplary dual binding protein complex in an inactive state. The protein complex has a sensor domain and a therapeutic domain. The sensor domain and the therapeutic domain are linked by a linker. The sensor domain is shown bound to the therapeutic domain, rendering the therapeutic domain inactive. FIG. 1B shows an exemplary dual binding protein complex in an active state. The protein complex has a sensor domain and a therapeutic domain. The sensor domain and the therapeutic domain are linked by a linker. The sensor domain is shown bound to a marker, rendering the therapeutic domain active.

[0023] [Diagram 2] Figure 2 shows a schematic diagram of a protein complex of the present disclosure comprising one or more sensor domains and one or more therapeutic domains. Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, and 2I show exemplary schematic diagrams of the configuration of protein complexes detailed in the Examples.

[0024] [Diagram 3] FIG. 3 shows that IL-2 signaling by five exemplary PD-1 / IL-2 DBA-cytokine protein complexes (2_A08, 2_A11, 2_B05, 2_B07, and 7_A04, SEQ ID NOs:67-68, 69-70, 71-72, 73-74, and 75-76, respectively) is reduced compared to the control IL-2-anti-HER2 protein complex (SEQ ID NOs:65-66).

[0025] [Figure 4A-4F]Figure 4A, Figure 4B, Figure 4C, Figure 4D, Figure 4E and Figure 4F provide the IL-2 activity of protein complexes containing the structures shown in Figure 2B in wells coated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth of 630 nm signal from HEK-Blue™ IL-2 reporter cells. Figures 4A-4C provide the IL-2 activity of three different PD-1 / IL-2 DBA-IL-2 complexes. Figure 4D provides the activity of an anti-PD-1 antibody-IL-2 complex. Figure 4E provides the activity of an anti-Her-2 antibody-IL-2 complex. Figure 4F provides the activity of an anti-IL-2 antibody-IL-2 complex.

[0026] [Diagram 5] Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E, Figure 5F, Figure 5G and Figure 5H provide the IL-2 activity of protein complexes containing the structures shown in Figure 2H in wells coated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth of 630 nm signal from HEK-Blue™ IL-2 reporter cells. Figures 5B-5D provide the results for two PD-1 / IL-2 DBA complexes containing an anti-PD-1 domain in the Fab arm and a PD-1 / IL-2 DBA scFv on the Fc arm. Figures 5A, Figure 5C and Figure 5E-5H provide the results for the control protein complexes.

[0027] [Figure 6] FIG. 6 provides the percent reduction in serum concentration in the blood of wild-type mice of the PD-1 / IL-2 DBA-cytokine complex ("2B07 IL-2 mut") and two control complexes.

[0028] [Figure 7A-7D] Figures 7A, 7B, 7C, and 7D provide CD8+ T cell and NK cell counts in blood and spleen tissue collected from wild-type mice five days after treatment with the PD-1 / IL-2 DBA-cytokine complex ("2B07 IL-2 mut") and two control complexes.

[0029] [Figure 8] Figure 8 provides tumor volume measurements as a function of days after tumor cell implantation in mice that received various intravenous doses of PD-1 / IL-2 DBA-IL-2 complexes, PD-1 / IL-2 DBA complexes lacking IL-2, or isotype control.

[0030] [Figure 9] FIG. 9 provides IL-2RBG binding to a symmetric complex that includes the structure shown in FIG. 2E.

[0031] [Figure 10] FIG. 10 provides IL-2RBG binding to a symmetric complex containing the structure shown in FIG. 2B.

[0032] [Figure 11] Figure 11 shows plots demonstrating in vitro IL-2RBG binding by PD-1 / IL-2 DBA-cytokine complexes and uncontrolled control complexes, comparing complexes made with two different forms of IL-2, including WT IL-2 and IL-2 3x. Figure 11A shows IL-2RBG binding to a complex with wild-type IL-2, and Figure 11B shows IL-2RBG binding to a complex with the IL-2 3x mutant.

[0033] [Figure 12A-12B] Figure 12A, Figure 12B, Figure 13A, Figure 13B, Figure 14A, Figure 14B, Figure 15 and Figure 16 show plots showing IL-2 activity of IL-2 linked protein complexes including the structures shown in Figure 2E, Figure 2B, Figure 2H, Figure 2G and Figure 2I, respectively, in cells treated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth in 630 nm signal from HEK-Blue™ IL-2 reporter cells, an engineered human kidney cell line that produces a detectable color change upon activation of its IL-2 receptor. [Figure 13A-13B]Figure 12A, Figure 12B, Figure 13A, Figure 13B, Figure 14A, Figure 14B, Figure 15 and Figure 16 show plots showing IL-2 activity of IL-2 linked protein complexes including the structures shown in Figure 2E, Figure 2B, Figure 2H, Figure 2G and Figure 2I, respectively, in cells treated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth in 630 nm signal from HEK-Blue™ IL-2 reporter cells, an engineered human kidney cell line that produces a detectable color change upon activation of its IL-2 receptor. [Figure 14A-14B] Figure 12A, Figure 12B, Figure 13A, Figure 13B, Figure 14A, Figure 14B, Figure 15 and Figure 16 show plots showing IL-2 activity of IL-2 linked protein complexes including the structures shown in Figure 2E, Figure 2B, Figure 2H, Figure 2G and Figure 2I, respectively, in cells treated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth in 630 nm signal from HEK-Blue™ IL-2 reporter cells, an engineered human kidney cell line that produces a detectable color change upon activation of its IL-2 receptor. [Figure 15] Figure 12A, Figure 12B, Figure 13A, Figure 13B, Figure 14A, Figure 14B, Figure 15 and Figure 16 show plots showing IL-2 activity of IL-2 linked protein complexes including the structures shown in Figure 2E, Figure 2B, Figure 2H, Figure 2G and Figure 2I, respectively, in cells treated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth in 630 nm signal from HEK-Blue™ IL-2 reporter cells, an engineered human kidney cell line that produces a detectable color change upon activation of its IL-2 receptor. [Figure 16]Figure 12A, Figure 12B, Figure 13A, Figure 13B, Figure 14A, Figure 14B, Figure 15 and Figure 16 show plots showing IL-2 activity of IL-2 linked protein complexes including the structures shown in Figure 2E, Figure 2B, Figure 2H, Figure 2G and Figure 2I, respectively, in cells treated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth in 630 nm signal from HEK-Blue™ IL-2 reporter cells, an engineered human kidney cell line that produces a detectable color change upon activation of its IL-2 receptor.

[0034] [Figure 17] Figure 17 is a plot showing the IL-2 activity of an IL-2 linked protein complex containing the structure shown in Figure 2H with a human Fc domain in cells treated with PD-1-Fc or an IgG1 control protein. Activity was measured as the increase in 630 nm signal from HEK-Blue™ IL-2 reporter cells (an engineered human kidney cell line that produces a detectable color change upon activation of its IL-2 receptor).

[0035] [Figure 18] FIG. 18 is a plot showing IL-2 activity in a protein complex containing the structure shown in FIG. 2B with the IL-2 3x variant.

[0036] [Figures 19A-19C] Figure 19A, Figure 19B, and Figure 19C show plots showing the IL-2 activity of IL-2-3x linked protein complexes containing the structure shown in Figure 2H with three different anti-PD-1 Fab arms (nivolumab, 4C10, and Knd, respectively) on cells plated in wells coated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth in 630 nm signal from HEK-Blue™ IL-2 reporter cells (an engineered human kidney cell line that produces a detectable color change upon activation of its IL-2 receptor).

[0037] [Figure 20]Figure 20 shows a plot depicting the IL-2 activity of IL-2 linked protein complexes containing the structures shown in Figure 2B with linkers of various lengths. The complexes were tested in wells coated with PD-1-Fc or IgG1 control protein. Activity was measured as the growth in 630 nm signal from HEK-Blue™ IL-2 reporter cells (an engineered human kidney cell line that produces a detectable color change upon activation of its IL-2 receptor).

[0038] [Figure 21A-21B] Figures 21A and 21B show plots showing PD-1-dependent induction of STAT5 phosphorylation by PD-1 / IL-2 DBA-cytokine complexes in human primary CD8+ T cells as measured by flow cytometry.

[0039] [Fig. 22A-22B] Figures 22A and 22B show plots showing PD-1 / IL-2 DBA-cytokine complex regulation of human T cell activation in a mixed lymphocyte reaction as assessed by granzyme B release.

[0040] [Figure 23A-23B] Figure 23A and Figure 23B show plots showing PD-1 / IL-2 DBA-cytokine complex modulation of anti-tumor immunity in a syngeneic tumor model. 500,000 MC38 tumor cells were implanted subcutaneously into human PD-1 knock-in mice, the mice were treated intravenously with the complex, and tumor volume was assessed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description The present disclosure provides compositions of protein complexes and methods of using the same. Interleukin 2 therapeutics are often unfeasible due to systemic on-target toxicity. Provided herein are protein complexes that specifically exhibit therapeutic effects on PD-1 positive cells, specifically antigen-experienced T cells. Furthermore, the protein complexes of the present disclosure are self-regulated, remaining inactive in the absence of PD-1 and activating upon binding to PD-1. The protein complexes disclosed herein may include a sensor domain (e.g., an antibody, Fab or scFv) linked to an IL-2 receptor agonist (therapeutic domain) via a linker. The sensor domain may be a dual-binding antibody with affinity for the therapeutic domain and PD-1 such that PD-1 and the therapeutic domain compete for binding to the sensor domain. In the absence of PD-1, the sensor domain binds to the therapeutic domain, preventing the therapeutic domain from exerting activity on the IL-2 receptor. When the sensor domain is bound to PD-1, the therapeutic domain is unbound and can exert activity. In some embodiments, the control of IL-2 receptor agonist activity by the complex may be reversible, i.e., when the sensor domain dissociates from PD-1, the sensor domain may bind to the therapeutic domain, and the therapeutic domain may not be able to exert activity again. Thus, the protein complex of the present disclosure includes a sensor domain that controls the IL-2 receptor agonist domain in the presence of PD-1, and binds to PD-1 to activate the IL-2 receptor agonist domain. Various structures and compositions of the protein complex, including pharmaceutical formulations, are disclosed herein. Also provided herein is a method of treating a subject in need thereof by administering the protein complex to the subject.

[0042] As used herein, "sensor domain" generally refers to a dual binding antibody capable of binding to PD-1 and to an IL-2 receptor agonist.

[0043] As used herein, a "therapeutic domain" generally refers to an IL-2 receptor agonist. Non-limiting examples of therapeutic domains include IL-2, IL-15, or any other molecule that acts on the IL-2 receptor in a manner similar to IL-2.

[0044] As used herein, "marker" generally refers to the PD-1 protein.

[0045] As used herein, "antibody" generally refers to an antibody, an antibody derivative, or a fragment(s) thereof that contains some or all of an antibody variable domain.

[0046] The term "recombinant nucleic acid" generally refers to a synthetic nucleic acid having a nucleotide sequence that does not occur in nature. Recombinant nucleic acids may be synthesized in a laboratory. Recombinant nucleic acids are prepared using recombinant DNA technology by using enzymatic modification of DNA, such as enzymatic restriction digestion, ligation, and DNA cloning. As used herein, recombinant nucleic acids may be DNA or RNA. Recombinant DNA may be transcribed in vitro to produce messenger RNA (mRNA), which may be isolated, purified, and used to transfect cells. Recombinant nucleic acids may encode proteins or polypeptides. Recombinant nucleic acids may be incorporated into living cells under appropriate conditions and expressed within the living cells. As used herein, "expression" of a nucleic acid usually refers to transcription and / or translation of the nucleic acid. The product of nucleic acid expression is usually a protein, but may also be mRNA. Detection of mRNA encoded by a recombinant nucleic acid in a cell that has incorporated the recombinant nucleic acid is considered positive evidence that the nucleic acid is "expressed" in the cell.

[0047] As used herein, the term "therapeutic domain" generally refers to a protein domain that has minimal sequence features to activate a given therapeutic activity in a cell or organism. When a therapeutic domain is a ligand of a ligand-receptor pair, the ligand has minimal sequence and / or structural features that allow it to bind to or activate the receptor.

[0048] The process of inserting or incorporating nucleic acids into cells can be by transformation, transfection or transduction. Transformation is the process of uptake of foreign nucleic acids by bacterial cells. This process is adapted for the propagation of plasmid DNA, protein production, and other uses. Transformation introduces recombinant plasmid DNA into competent bacterial cells that take up extracellular DNA from the environment. Some bacterial species are naturally competent under certain environmental conditions, while competence is artificially induced in laboratory settings. Transfection is the forced introduction of small molecules, such as DNA, RNA, or antibodies, into eukaryotic cells. Just to confuse life, "transfection" also refers to the introduction of bacteriophage into bacterial cells. "Transduction" is most often used to describe the introduction of recombinant viral vector particles into target cells, while "infection" refers to the natural infection of humans or animals with wild-type viruses.

[0049] Protein complexes The present disclosure provides a complex that can autoregulate IL-2 receptor agonist activity. The protein complex of the present disclosure may include a dual-binding antibody having affinity for PD-1 and affinity for an IL-2 receptor agonist ("sensor domain") and an IL-2 receptor agonist ("therapeutic domain"). The sensor domain and therapeutic domain may be linked by a linker. The sensor domain may control the activity of the therapeutic domain. Controlling the activity of the therapeutic domain may include binding of the sensor domain to the therapeutic domain, preventing the therapeutic domain from exerting activity on the IL-2 receptor. Controlling the activity of the therapeutic domain may further include unbinding or releasing of the therapeutic domain by the sensor domain upon binding of the sensor domain to PD-1. Thus, the protein complex of the present disclosure is an excellent drug candidate because the sensor domain-dependent activity of the IL-2 receptor agonist allows for cell-specific activity even upon systemic administration of the protein complex. Compared to the IL-2 receptor agonist administered alone, the protein complex of the present disclosure exhibits controlled therapeutic activity. As a result, compared to free IL-2 receptor agonist administered alone, the protein complexes of the present disclosure exhibit reduced systemic on-target toxicity.

[0050] The protein complex of the present disclosure may have an Fc region. The protein complex of the present disclosure may have a domain that improves kinetic properties. For example, the protein complex of the present disclosure may be further coupled to a half-life extender, such as an Fc region, albumin, PEG, or another zwitterionic polymer. The protein complex of the present disclosure may have two heavy chains and two light chains. The protein complex of the present disclosure may have two heavy chains and one light chain. The protein complex of the present disclosure may include multiple sensor domains and multiple therapeutic domains. For example, the protein complex of the present disclosure may include two sensor domains and two therapeutic domains, all of which are linked, and the two therapeutic domains are bound to the two sensor domains. In some embodiments, the protein complex of the present disclosure may include two sensor domains and one therapeutic domain, all of which are linked, and the therapeutic domain may be bound to both sensor domains or only one of the two sensor domains.

[0051] In some embodiments, PD-1 can be a surface protein, such as a cell surface protein, hi some embodiments, PD-1 can be expressed on antigen-experienced T cells.

[0052] In some embodiments, the IL-2 receptor agonist can be IL-2, a variant of IL-2, or a truncated form of IL-2. In some embodiments, the IL-2 receptor agonist can be IL-15, IL-15-sushi, a variant of IL-15, or a variant of IL-15-sushi. In some embodiments, the IL-2 receptor agonist can be an engineered or designed peptide that binds to the IL2 receptor beta and the IL-2 receptor gamma. In some embodiments, the IL-2 receptor agonist can be an antibody that binds to the IL2 receptor beta and the IL-2 receptor gamma.

[0053] In some embodiments, the binding of the sensor domain to the therapeutic domain versus the binding of the sensor domain to PD-1 is controlled by the relative affinity of the sensor domain to the therapeutic domain. In some embodiments, the sensor domain may have a lower dissociation constant (Kd) for PD-1 than the dissociation constant of the sensor domain to the therapeutic domain. Thus, the sensor may have a higher affinity (lower Kd) for PD-1 than the therapeutic domain. The sensor domain of the present disclosure may be engineered, for example by affinity maturation, to have a higher affinity (lower dissociation constant) for PD-1 than the therapeutic domain. In the absence of a marker, the sensor domain of the present disclosure may have a sufficiently high affinity for the therapeutic domain such that the therapeutic domain is bound by the sensor domain. In the presence of a marker, the affinity of the sensor domain for PD-1 is sufficiently high (low dissociation constant) such that PD-1 outcompetes the therapeutic domain for binding to the sensor domain. As a result, the equilibrium binding shifts from a state in which the sensor domain is bound to the IL-2 receptor agonist domain to a state in which the IL-2 receptor agonist domain is released and the sensor domain is bound to PD-1.

[0054] The sensor domain may have an affinity for PD-1 that is at least 2 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 5 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 10 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 15 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 20 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 25 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 30 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 35 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 40 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 45 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 50 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 60 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 70 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 80 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 90 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 100 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 150 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 200 times greater than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 250 times greater than the affinity for the therapeutic domain.The sensor domain may have an affinity for PD-1 that is at least 300 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 350 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 400 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 450 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 500 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 1000 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 10,000 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is at least 100,000 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 2 to 10 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 10-20 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 20-30 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 30-40 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 40-50 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 50-100 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 100-150 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 150-200 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 200-250 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 250-300 times higher than the affinity for the therapeutic domain. The sensor domain can have an affinity for PD-1 that is 300-350 times higher than its affinity for the therapeutic domain.The sensor domain may have an affinity for PD-1 that is 350-400 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 400-450 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 450-500 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 500-1000 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 10-80 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 30-70 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 40-60 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 20-50 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 10-1000 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 70-500 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 100-500 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 500-750 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 250-750 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 1000-100000 times higher than the affinity for the therapeutic domain. The sensor domain may have an affinity for PD-1 that is 2-100000 times higher than the affinity for the therapeutic domain.

[0055] A protein complex or fragment thereof of the present disclosure may comprise one or more complementarity determining regions (CDRs) having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or substantially 100% sequence identity to any one of the CDRs disclosed herein. A protein complex or fragment thereof of the present disclosure may comprise one or more heavy or light chain variable regions having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or substantially 100% sequence identity to any one of the heavy or light chain variable regions described herein. For example, a protein complex or fragment thereof of the present disclosure may comprise one or more CDRs having at least 80% sequence identity to any one of SEQ ID NOs: 1-20, 142-173, or 238-252. A protein complex or fragment thereof of the present disclosure may comprise one or more CDRs having at least 85% sequence identity to any one of SEQ ID NO:1 to SEQ ID NO:20, SEQ ID NO:142 to SEQ ID NO:173, or SEQ ID NO:238 to SEQ ID NO:252. A protein complex or fragment thereof of the present disclosure may comprise one or more CDRs having at least 90% sequence identity to any one of SEQ ID NO:1 to SEQ ID NO:20, SEQ ID NO:142 to SEQ ID NO:173, or SEQ ID NO:238 to SEQ ID NO:252. A protein complex or fragment thereof of the present disclosure may comprise one or more CDRs having at least 92% sequence identity to any one of SEQ ID NO:1 to SEQ ID NO:20, SEQ ID NO:142 to SEQ ID NO:173, or SEQ ID NO:238 to SEQ ID NO:252.The protein complex or fragment thereof of the present disclosure may comprise one or more CDRs having at least 95% sequence identity with any one of SEQ ID NO:1 to SEQ ID NO:20, SEQ ID NO:142 to 173, or SEQ ID NO:238 to 252. The protein complex or fragment thereof of the present disclosure may comprise one or more CDRs having at least 97% sequence identity with any one of SEQ ID NO:1 to SEQ ID NO:20, SEQ ID NO:142 to 173, or SEQ ID NO:238 to 252. The protein complex or fragment thereof of the present disclosure may comprise one or more CDRs having at least 99% sequence identity with any one of SEQ ID NO:1 to SEQ ID NO:20, SEQ ID NO:142 to 173, or SEQ ID NO:238 to 252. The protein complex or fragment thereof of the present disclosure may comprise one or more CDRs having any one of SEQ ID NO:1 to SEQ ID NO:20, SEQ ID NO:142 to 173, or SEQ ID NO:238 to 252.

[0056] The protein complex or a fragment thereof can have at least 80% sequence identity to any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to 175, SEQ ID NO:181 to 182, SEQ ID NO:195 to 196, SEQ ID NO:205 to 206, SEQ ID NO:210 to 212, SEQ ID NO:220 to 223, SEQ ID NO:226 to 231, SEQ ID NO:259 to 261, SEQ ID NO:266 to 282 or SEQ ID NO:289 to 293, or a fragment thereof. The protein complex can have at least 85% sequence identity to any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to 175, SEQ ID NO:181 to 182, SEQ ID NO:195 to 196, SEQ ID NO:205 to 206, SEQ ID NO:210 to 212, SEQ ID NO:220 to 223, SEQ ID NO:226 to 231, SEQ ID NO:259 to 261, SEQ ID NO:266 to 282 or SEQ ID NO:289 to 293, or a fragment thereof. The protein complex can have at least 90% sequence identity to any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to 175, SEQ ID NO:181 to 182, SEQ ID NO:195 to 196, SEQ ID NO:205 to 206, SEQ ID NO:210 to 212, SEQ ID NO:220 to 223, SEQ ID NO:226 to 231, SEQ ID NO:259 to 261, SEQ ID NO:266 to 282 or SEQ ID NO:289 to 293, or a fragment thereof. The protein complex can have at least 92% sequence identity to any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to 175, SEQ ID NO:181 to 182, SEQ ID NO:195 to 196, SEQ ID NO:205 to 206, SEQ ID NO:210 to 212, SEQ ID NO:220 to 223, SEQ ID NO:226 to 231, SEQ ID NO:259 to 261, SEQ ID NO:266 to 282 or SEQ ID NO:289 to 293, or a fragment thereof.The protein complex can have at least 95% sequence identity to any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to 175, SEQ ID NO:181 to 182, SEQ ID NO:195 to 196, SEQ ID NO:205 to 206, SEQ ID NO:210 to 212, SEQ ID NO:220 to 223, SEQ ID NO:226 to 231, SEQ ID NO:259 to 261, SEQ ID NO:266 to 282 or SEQ ID NO:289 to 293, or a fragment thereof. The protein complex can have at least 97% sequence identity to any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to 175, SEQ ID NO:181 to 182, SEQ ID NO:195 to 196, SEQ ID NO:205 to 206, SEQ ID NO:210 to 212, SEQ ID NO:220 to 223, SEQ ID NO:226 to 231, SEQ ID NO:259 to 261, SEQ ID NO:266 to 282 or SEQ ID NO:289 to 293, or a fragment thereof. The protein complex can have at least 99% sequence identity to any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to 175, SEQ ID NO:181 to 182, SEQ ID NO:195 to 196, SEQ ID NO:205 to 206, SEQ ID NO:210 to 212, SEQ ID NO:220 to 223, SEQ ID NO:226 to 231, SEQ ID NO:259 to 261, SEQ ID NO:266 to 282 or SEQ ID NO:289 to 293, or a fragment thereof. The protein complex is any one of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:80 to SEQ ID NO:112, SEQ ID NO:174 to 175, SEQ ID NO:181 to 182, SEQ ID NO:195 to 196, SEQ ID NO:205 to 206, SEQ ID NO:210 to 212, SEQ ID NO:220 to 223, SEQ ID NO:226 to 231, SEQ ID NO:259 to 261, SEQ ID NO:266 to 282 or SEQ ID NO:289 to 293, or a fragment thereof.

[0057] The protein complex of the present disclosure may have at least 95% sequence identity with any one of SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 80 to SEQ ID NO: 112, SEQ ID NO: 174 to SEQ ID NO: 175, SEQ ID NO: 181 to SEQ ID NO: 182, SEQ ID NO: 195 to SEQ ID NO: 205 to SEQ ID NO: 206, SEQ ID NO: 210 to SEQ ID NO: 212, SEQ ID NO: 220 to SEQ ID NO: 226 to SEQ ID NO: 231, SEQ ID NO: 259 to SEQ ID NO: 266 to SEQ ID NO: 282 or SEQ ID NO: 289 to SEQ ID NO: 293, or fragments thereof, and may have one or more CDRs having at least 80% sequence identity with any one of SEQ ID NO: 1 to SEQ ID NO: 20, SEQ ID NO: 142 to SEQ ID NO: 173, or SEQ ID NO: 238 to SEQ ID NO: 252. The protein complex of the present disclosure may have CDRs selected from SEQ ID NO: 1 to SEQ ID NO: 20, SEQ ID NO: 142 to SEQ ID NO: 173, or SEQ ID NO: 238 to SEQ ID NO: 252 arranged in any combination or order.

[0058] Any of the above fragments may retain a functional binding domain of the sensor or any functional therapeutic domain of the therapeutic agent. For example, a dual binding antibody protein complex may comprise a whole antibody or a fragment having a region of the antibody capable of binding to the marker and therapeutic domain. In the latter case, the fragment may be an scFv capable of binding to the marker and therapeutic domain. Exemplary sequences of protein complexes of the present disclosure are shown in Table 1 below. [Table 1] TIFF2024544757000002.tif250165TIFF2024544757000003.tif249165TIFF2024544757000004.tif248165TIFF2024544757000005.tif37165

[0059] A. Sensor Domain The protein complex of the present disclosure includes a sensor domain composed of a dual-binding antibody having affinity for PD-1 and affinity for an IL-2 receptor agonist. The sensor domain can be any protein capable of sensing the presence of a first moiety and modulating a second moiety, the first moiety being PD-1 and the second moiety being an IL-2 receptor agonist. For example, the present disclosure provides a sensor domain that can be an antibody or antibody fragment that can bind to a first moiety and bind to a second moiety to block activity, the first moiety being PD-1 and the second moiety being IL-2 or another IL-2 receptor agonist. In the absence of the first moiety, the sensor domain binds to the second moiety. When the first moiety is introduced into the system, the sensor domain binds to the first moiety and unbinds the second moiety. Thus, the binding and unbinding of the second moiety is reversible. The sensor domain inactivates or blocks the activity of the IL-2 receptor agonist domain by binding to it and preventing it from binding to its target (the IL-2 receptor). The sensor domain controls the IL-2 receptor agonist domain by releasing it to act on its target upon binding of PD-1.

[0060] In some embodiments, the sensor domain is a dual binding antibody. The dual binding antibody may be capable of binding to PD-1 and IL-2 receptor agonist domains. The dual binding antibody of the present disclosure may be selected or engineered to bind to PD-1 and therapeutic domains. The dual binding protein may have a higher affinity for PD-1 compared to the IL-2 receptor agonist domain. The dual binding protein may be affinity matured to have a higher affinity for PD-1 compared to the IL-2 receptor agonist domain.

[0061] In some embodiments, the sensor domain is an antibody. The sensor domain can also be a fragment of an antibody. An antibody fragment that corresponds to the sensor domain disclosed herein retains its ability to exhibit dual binding to both PD-1 and IL-2 receptor agonist domains. One or both domains of the bispecific antibody can be the sensor domain of the protein complex of the present disclosure. When a bispecific antibody is used, the bispecific antibody can include a first antigen-binding domain that can bind to the IL-2 receptor agonist domain and PD-1, and can also include a second antigen-binding domain that can bind to PD-1.

[0062] In some embodiments, the sensor domain is an anti-PD1 antibody or fragment thereof (eg, an scFv that binds to PD1 or PD-L1).

[0063] B. Therapeutic Domain The protein complex of the present disclosure includes a therapeutic domain composed of an IL-2 receptor agonist. The therapeutic domain of the present disclosure is linked to a sensor domain via a linker to form a protein complex. The therapeutic domain can exert a therapeutic activity by binding to the IL-2 receptor.

[0064] In some embodiments, the protein complexes of the present disclosure comprise a therapeutic domain comprising IL-2, or a variant or fusion of this cytokine. The therapeutic domain can also be a fragment of the moiety. The fragment retains the functional regions of the moiety necessary for binding to its target (e.g., the IL-2 receptor) and any functional regions necessary for activity.

[0065] In some embodiments, the protein complexes of the present disclosure comprise a therapeutic domain comprising IL-15, or a variant or fusion of this cytokine. The therapeutic domain can also be a fragment of the moiety. The fragment retains the functional regions of the moiety necessary for binding to its target (e.g., the IL-2 receptor) and any functional regions necessary for activity.

[0066] In some embodiments, the protein complexes of the present disclosure comprise a therapeutic domain comprising a peptide and an engineered protein or antibody capable of binding to IL-2 receptor beta and IL-2 receptor gamma. The therapeutic domain can also be a fragment of the moiety. The fragment retains the functional regions of the moiety necessary for binding to its target (e.g., IL-2 receptor) and any functional regions necessary for activity.

[0067] C. Linker The protein complex disclosed herein may include a linker. The linker may link two domains, such as a sensor domain and a therapeutic domain. Various linkers are consistent with the protein complex of the present disclosure. In some embodiments, the linker may be an amino acid linker or a chemical linker.

[0068] The linker may be a stable linker. For example, the linker may maintain the connection between the therapeutic domain and the sensor domain even when the sensor domain binds to the marker, thereby debinding the therapeutic domain from the sensor domain. For example, the sensor domain may debind the therapeutic domain, but the therapeutic domain may remain linked to the sensor domain via the linker. Examples of linkers consistent with this activity may include non-cleavable linkers.

[0069] The linker may be a flexible linker. A flexible linker is a linker of sufficient length to allow the therapeutic domain to bind to the IL-2 receptor once unbound from the sensor domain. The flexibility of the linker may affect the therapeutic effect. For example, when the sensor domain binds to PD-1 and the therapeutic domain unbound, the therapeutic domain needs to be able to encounter and bind to its target, the IL-2 receptor. If the linker is not flexible enough to allow the therapeutic domain to bind to the IL-2 receptor, the therapeutic efficacy may be reduced or absent. If the linker is flexible, the therapeutic domain may bind to the IL-2 receptor and exert high therapeutic efficacy. The flexibility of the linker may result from the length of the linker. For example, a short linker may sterically hinder the therapeutic domain from binding to the IL-2 receptor. A longer linker may allow the protein complex to be more flexible and allow the therapeutic domain to bind to the IL-2 receptor. In some embodiments, a linker that is too long may affect the ability of the sensor domain to bind to the therapeutic domain and inhibit activity in the absence of PD-1. In some embodiments, a linker that is too long may affect the stability of the protein therapeutic domain in vivo or the half-life of the protein therapeutic domain.

[0070] In some embodiments, the linker may be attached to the heavy chain of the sensor domain or the light chain of the sensor domain. The linker may be fused to the N-terminus or C-terminus of the sensor domain. In some embodiments, the linker may be fused to the N-terminus or C-terminus of the IL-2 receptor agonist domain. For example, the linker may be attached to the N-terminus or C-terminus of the scFV or ScFab.

[0071] Amino acid linkers. The amino acid linkers may include any amino acid residue. In some embodiments, preferred amino acid residues are entropically flexible amino acid residues. Preferred amino acid residues in the amino acid linkers of the present disclosure may include glycine and serine. Other preferred amino acid residues may include alanine, proline, threonine, and glutamic acid. In preferred embodiments, the amino acid linkers may include a length of 3 to 60 amino acid residues. In some embodiments, the amino acid linkers may include 20 amino acid residues. In some embodiments, the amino acid linkers may include 40 amino acid residues. In some embodiments, the amino acid linkers may include 60 amino acid residues. In some embodiments, the amino acid linkers may include 80 amino acid residues. The amino acid linkers may include at least 5 amino acid residues. The amino acid linkers may include at least 10 amino acid residues. The amino acid linkers may include at least 15 amino acid residues. The amino acid linkers may include at least 20 amino acid residues. The amino acid linkers may include at least 25 amino acid residues. The amino acid linker may comprise at least 30 amino acid residues. The amino acid linker may comprise at least 35 amino acid residues. The amino acid linker may comprise at least 40 amino acid residues. The amino acid linker may comprise at least 45 amino acid residues. The amino acid linker may comprise at least 50 amino acid residues. The amino acid linker may comprise at least 55 amino acid residues. The amino acid linker may comprise at least 60 amino acid residues. The amino acid linker may comprise at least 65 amino acid residues. The amino acid linker may comprise at least 70 amino acid residues. The amino acid linker may comprise at least 75 amino acid residues. The amino acid linker may comprise at least 80 amino acid residues. The amino acid linker may comprise at least 85 amino acid residues. The amino acid linker may comprise at least 90 amino acid residues. The amino acid linker may comprise at least 95 amino acid residues. The amino acid linker may comprise at least 100 amino acid residues.The amino acid linker may comprise at least 110 amino acid residues. The amino acid linker may comprise at least 120 amino acid residues. The amino acid linker may comprise at least 130 amino acid residues. The amino acid linker may comprise at least 140 amino acid residues. The amino acid linker may comprise at least 150 amino acid residues. The amino acid linker may comprise at least 160 amino acid residues. The amino acid linker may comprise at least 170 amino acid residues. The amino acid linker may comprise at least 180 amino acid residues. The amino acid linker may comprise at least 190 amino acid residues. The amino acid linker may comprise at least 200 amino acid residues. The amino acid linker may comprise at least 300 amino acid residues. The amino acid linker may comprise at least 400 amino acid residues. The amino acid linker may comprise at least 500 amino acid residues. The amino acid linker may comprise 5 to 10 amino acid residues. The amino acid linker may comprise 10 to 15 amino acid residues. The amino acid linker may contain 15 to 20 amino acid residues. The amino acid linker may contain 20 to 25 amino acid residues. The amino acid linker may contain 25 to 30 amino acid residues. The amino acid linker may contain 30 to 35 amino acid residues. The amino acid linker may contain 35 to 40 amino acid residues. The amino acid linker may contain 40 to 45 amino acid residues. The amino acid linker may contain 45 to 50 amino acid residues. The amino acid linker may contain 50 to 55 amino acid residues. The amino acid linker may contain 55 to 60 amino acid residues. The amino acid linker may contain 60 to 65 amino acid residues. The amino acid linker may contain 65 to 70 amino acid residues. The amino acid linker may contain 70 to 75 amino acid residues. The amino acid linker may contain 75 to 80 amino acid residues. The amino acid linker may contain 80 to 85 amino acid residues. The amino acid linker may comprise 85 to 90 amino acid residues. The amino acid linker may comprise 90 to 95 amino acid residues. The amino acid linker may comprise 95 to 100 amino acid residues. The amino acid linker may comprise 5 to 80 amino acid residues.The amino acid linker may contain 20 to 40 amino acid residues. The amino acid linker may contain 20 to 80 amino acid residues. The amino acid linker may contain 30 to 60 amino acid residues. The amino acid linker may contain 40 to 50 amino acid residues. The amino acid linker may contain 10 to 30 amino acid residues. The amino acid linker may contain 10 to 20 amino acid residues. The amino acid linker may contain 5 to 25 amino acid residues. The amino acid linker may contain 25 to 75 amino acid residues. The amino acid linker may contain 100 to 500 amino acid residues. The amino acid linker may contain 100 to 300 amino acid residues. The amino acid linker may contain 5 to 500 amino acid residues. The amino acid linker may contain 100 or less amino acid residues. The amino acid linker may contain 90 or less amino acid residues. The amino acid linker may contain 80 or less amino acid residues. The amino acid linker may comprise 70 or fewer amino acid residues. The amino acid linker may comprise 60 or fewer amino acid residues. The amino acid linker may comprise 50 or fewer amino acid residues. The amino acid linker may comprise 40 or fewer amino acid residues. The amino acid linker may comprise 30 or fewer amino acid residues. The amino acid linker may comprise 20 or fewer amino acid residues. The amino acid linker may comprise 10 or fewer amino acid residues. The amino acid linker may comprise 95 or fewer amino acid residues. The amino acid linker may comprise 90 or fewer amino acid residues. The amino acid linker may comprise 85 or fewer amino acid residues. The amino acid linker may comprise 80 or fewer amino acid residues. The amino acid linker may comprise 75 or fewer amino acid residues. The amino acid linker may comprise 70 or fewer amino acid residues. The amino acid linker may comprise 65 or fewer amino acid residues. The amino acid linker may comprise 60 or fewer amino acid residues. The amino acid linker may comprise 55 or fewer amino acid residues. The amino acid linker may comprise 50 or fewer amino acid residues. The amino acid linker may comprise 45 or fewer amino acid residues. The amino acid linker may comprise 40 or fewer amino acid residues. The amino acid linker may comprise 35 or fewer amino acid residues. The amino acid linker may comprise 30 or fewer amino acid residues.The amino acid linker may comprise 25 or fewer amino acid residues. The amino acid linker may comprise 20 or fewer amino acid residues. The amino acid linker may comprise 15 or fewer amino acid residues. The amino acid linker may comprise 10 or fewer amino acid residues. The amino acid linker may comprise 200 or fewer amino acid residues. The amino acid linker may comprise 300 or fewer amino acid residues. The amino acid linker may comprise 400 or fewer amino acid residues. The amino acid linker may comprise 500 or fewer amino acid residues.

[0072] Non-cleavable linkers. The non-cleavable linker may comprise a non-proteolytically cleavable peptide. The non-proteolytically cleavable peptide may be inactive against proteases present in a given sample or organism. For example, the peptide may be inactive against all human protease cleavage sequences, thereby providing a high degree of stability in humans and human samples. Such peptides may also provide secondary structures that render the protease cleavage site inactive or inaccessible to proteases. The non-cleavable linker of the present disclosure may provide a cleavage half-life of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, or at least 1 month in the presence of human proteases at 25°C in pH 7 buffer.

[0073] D. Protein Complex Structure The present disclosure provides a wide variety of protein complexes across a variety of structures. The protein complexes of the present disclosure may include an IL-2 receptor agonist domain and a sensor domain that are expressed as a single unit. The IL-2 receptor agonist domain may be expressed as an N-terminal extension of the sensor domain, may be expressed as a C-terminal extension of the sensor domain, or may be located within the sensor domain. For example, the protein complex may include, from N-terminus to C-terminus, an IL-2 receptor agonist domain, a peptide linker, an scFv domain, and optionally a tag, such as a peptide that includes a purification tag (e.g., V5 or myc tag) or a localization signal.

[0074] A protein complex may include multiple protein subunits. Multiple protein subunits (e.g., an IL-2 receptor agonist domain and a sensor domain, two sensor domains, or two subunits of a sensor domain) may be chemically or physically coupled after expression. Multiple protein subunits may include multiple sensor domains and / or therapeutic domains. A sensor domain and / or therapeutic domain may be composed of a single protein subunit, multiple protein subunits, or portions thereof. For example, a sensor domain may include an antibody Fab region that includes an immunoglobulin light chain and a portion of an immunoglobulin heavy chain.

[0075] Multiple protein subunits may include physical handles that facilitate their selective coupling. Physical handles may enable spontaneous, irreversible, and / or non-mediated (e.g., without the need for chaperone proteins or catalytic complexes) coupling between protein subunits, thereby enabling complexes and asymmetric protein complexes. For example, two different protein complex subunits expressed in a single Chinese Hamster Ovary (CHO) cell may include physical handles that spontaneously and irreversibly bind prior to cellular transport. Such physical handles may include "knob-into-hole" (KIH) constructs or charge-exchange constructs that include physical structures in which two protein subunits have mutual binding affinity and specificity. Such physical handles may include covalent bond pairs, such as multiple thiols, configured to form disulfide bonds. Physical handles may enable easy production of protein complexes that include identical or different domains.

[0076] A protein complex may contain two or more identical domains. An example of such a protein complex is provided in FIG. 2E, which shows an antibody (multi-sensor domain) bound to two IL-2 therapeutic domains. In this example, the protein complex contains two protein immunoglobulin light chain subunits and two immunoglobulin heavy chain subunits complexed to form a competent antibody. The two immunoglobulin heavy chain subunits contain an N-terminal linker coupled to an IL-2 therapeutic domain. Each immunoglobulin heavy chain is coupled to an immunoglobulin light chain such that the protein complex contains two Fab regions, each separately coupled to a therapeutic domain by a linker. A second example of such a protein complex is provided in FIG. 2D, which shows an antibody (multi-sensor domain) bound to two IL-2 therapeutic domains. In this example, the protein complex contains two protein immunoglobulin light chain subunits and two immunoglobulin heavy chain subunits complexed to form a competent antibody. The two immunoglobulin light chain subunits contain an N-terminal linker coupled to an IL-2 therapeutic domain. Each immunoglobulin heavy chain is coupled to an immunoglobulin light chain such that the protein complex contains two Fab regions, each separately coupled to a therapeutic domain by a linker. A third example of such a protein complex is provided in FIG. 2G, which shows an antibody bound to two IL-2 therapeutic domains and four sensor domains (multi-sensor domain). In this example, the protein complex contains two protein immunoglobulin light chain subunits and two immunoglobulin heavy chain subunits complexed to form a competent antibody. The two immunoglobulin heavy chain subunits contain an N-terminal linker coupled to the IL-2 therapeutic domain and a C-terminal linker coupled to a sensor domain (anti-PD-1 scFv domain) that does not target the therapeutic domain.Each immunoglobulin heavy chain is coupled to an immunoglobulin light chain such that the protein complex contains two Fab regions, each separately coupled to a therapeutic domain by a linker, and two Fc domains, each separately coupled to a sensor by a linker.

[0077] Although the above examples provide a symmetric protein complex with two identical sensor domains and two identical therapeutic domains, the protein complex may also include multiple different sensor domains and / or therapeutic domains. Such a protein complex may include an immunoglobulin unit with a first arm composed of a heavy-light chain pair and a second arm composed of an antibody fragment such as an scFv, scFab, VH or fragment thereof. In such a case, the heavy chain, antibody fragment or light chain may include an N-terminal extension with a linker and a therapeutic domain, as shown in Figure 2A, Figure 2C and Figure 2F, respectively. Alternatively, the heavy chain, antibody fragment or light chain may include a C-terminal extension with a linker and a therapeutic domain. The protein complex may also include a symmetric immunoglobulin unit with a single therapeutic domain. For example, the immunoglobulin unit may include an N-terminal linker and a therapeutic unit on a single heavy chain, as shown in Figure 2B. Alternatively, the immunoglobulin unit may include an N-terminal linker and a therapeutic unit on a single light chain. The immunoglobulin unit may also include a pair of antibody fragments attached to a single Fc region. The immunoglobulin unit may comprise a nanobody. The immunoglobulin unit may comprise a diabody.

[0078] The protein complex may include a flexible linker between the Fab arm and the Fc domain of the competent antibody such that the Fab sensor domain can bind to a therapeutic domain linked to the C-terminus of the Fc domain, as shown in Figure 5I. In this example, the protein complex includes two protein immunoglobulin light chain subunits and two immunoglobulin heavy chain subunits complexed to form a sensor antibody domain.

[0079] A protein complex may contain a sensor domain that does not target a therapeutic domain. Such a sensor domain may assist in target localization or enhance the binding of a separate sensor domain to PD-1. An example of a protein complex containing a sensor domain that does not target a therapeutic domain is shown in Figure 2H. This system includes a monospecific anti-PD-1 antibody, a first heavy chain containing a C-terminal linker coupled to a therapeutic domain, and a second heavy chain containing a C-terminal linker coupled to an IL-2 receptor agonist domain and a sensor domain with dual specificity for PD-1.

[0080] Protein complexes may include a range of sensor to therapeutic domain ratios. Protein complexes may include equal numbers of sensor domains and therapeutic domains, examples of which are provided by FIG. 2D and FIG. 2E, which show protein complexes with two sensor domains and two therapeutic domains. Protein complexes may include more sensor domains than therapeutic domains, such as the protein complexes of FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2F, and FIG. 2I, each of which includes two sensor domains and one therapeutic domain, or may include more sensor domains than therapeutic domains, such as FIG. 2G, which includes four sensor domains and two therapeutic domains, and FIG. 2H, which includes three sensor domains and one therapeutic domain. In such cases, the therapeutic domain may be capable of interacting with multiple sensor domains, or may be restricted to interacting with more than one sensor domain. The number of therapeutic domains with which a sensor domain may interact may depend on its linker. The linker may be short enough to prevent the therapeutic domain from interacting with the sensor domain, or may be long enough to allow the therapeutic domain to interact with multiple sensor domains.

[0081] In certain cases, the protein complex may include an antibody with an Fc-binding therapeutic domain and a sensor domain. As shown in FIG. 2H, the protein complex may include an antibody with a first heavy chain C-terminal extension that includes a linker and a therapeutic domain, and a second heavy chain C-terminal extension that includes a linker and a sensor domain. An antibody of this design may include a common target across its Fab and C-terminal extension sensor domains. For example, the antibody Fab region and the C-terminal extension sensor domain may each target the same epitope on PD-1. Conversely, an antibody of this design may include separate targets across its Fab region and C-terminal extension sensor domain. For example, the antibody Fab region and the C-terminal extension sensor domain may each target a different epitope on PD-1. As shown in FIG. 2I, a protein complex can include an antibody having a first heavy chain comprising a flexible linker of CH1 and CH2 domains (between the Fab arm and Fc domain) and a heavy chain C-terminal extension comprising a linker and a therapeutic domain, and a second heavy chain comprising a flexible linker of CH1 and CH2 domains (between the Fab arm and Fc domain) without a C-terminal extension.

[0082] In some embodiments, the amino acid in the protein complex described herein may comprise conservative substitution.Conservative substitution may comprise the replacement of one amino acid with a different amino acid having similar biochemical properties (e.g., charge, hydrophobicity and size).Examples of conservative substitution, as well as substitutions that may be preferred but are not necessarily preferred, are provided in Table 2 below. [Table 2]

[0083] In some embodiments, the present disclosure describes a recombinant nucleic acid encoding the protein complex disclosed herein. In some embodiments, the recombinant nucleic acid comprises a plasmid or vector encoding the entire protein complex. In some embodiments, the recombinant nucleic acid comprises a plasmid or vector encoding each of the therapeutic domain, the sensor domain, and the linker. In some embodiments, the recombinant nucleic acid comprises a plasmid or vector encoding any two of the therapeutic domain, the sensor domain, and the linker together.

[0084] Pharmaceutical preparations The protein complex or recombinant nucleic acid encoding the protein complex of the present disclosure can be formulated as a pharmaceutical composition. The pharmaceutical composition can include a pharmaceutically acceptable carrier or additive. As used herein, "pharmacologically acceptable" or "pharmacologically acceptable" includes molecular entities and compositions that do not cause adverse allergic reactions or other untoward reactions when administered to a subject, as appropriate. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active ingredients are also often incorporated into the composition.

[0085] Purpose The protein complexes of the present disclosure may be used for a variety of therapeutic applications. The protein complexes of the present disclosure may be used as therapeutic agents for administration to a subject in need thereof. The subject may be a human or a non-human mammal. The subject may have a disease. The disease may be cancer. Cancers include acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adolescent cancer; adrenal cortical carcinoma; AIDS-related cancer; Kaposi's sarcoma (soft tissue sarcoma); AIDS-related lymphoma (lymphoma); primary CNS lymphoma (lymphoma); anal cancer; appendix cancer - see gastrointestinal carcinoid tumors; astrocytoma, childhood (brain cancer); atypical teratoid / rhabdoid tumor, childhood, central nervous system (brain cancer); basal cell carcinoma of the skin - see skin cancer; bile duct cancer; bladder cancer; bone cancer (Ewing's sarcoma and osteosarcoma, as well as malignant fibrous histiocytoma);brain tumors;breast cancer;bronchial tumors (lung cancer);Burkitt's lymphoma - see non-Hodgkin's lymphoma;carcinoid tumors (gastrointestinal tract);cancer of unknown primary;cardiac (heart) tumors, childhood;central nervous system;atypical teratoid / rhabdoid tumors, childhood (brain cancer);medulloblastoma and other CNS embryonal tumors, childhood (brain tumors);germ cell tumors, childhood (brain tumors);primary CNS lymphoma;cervical cancer;childhood cancer;cancer of childhood, rare;cholangiocarcinoma - bile duct cancer cancer);chordoma, childhood (bone cancer);chronic lymphocytic leukemia (CLL);chronic myelogenous leukemia (CML);chronic myeloproliferative neoplasms;colorectal cancer;craniopharyngioma, childhood (brain cancer);cutaneous t-cell lymphoma - see lymphoma (mycosis fungoides and Sézary syndrome);ductal carcinoma in situ (DCIS) - see breast cancer;embryonal tumors, medulloblastoma and other central nervous system, childhood (brain cancer);endometrial carcinoma (uterine cancer);ependymoma, childhood (brain cancer);esophageal cancer;nasal neuroblastoma cystic cyst (head and neck cancer);Ewing's sarcoma (bone cancer);extracranial germ cell tumors, childhood;extragonadal germ cell tumors;eye cancer;intraocular melanoma;retinoblastoma;fallopian tube cancer;fibrous histiocytoma, malignant and osteosarcoma of bone;gallbladder cancer;gastric (stomach) cancer;gastrointestinal carcinoid tumors;gastrointestinal stromal tumors (GIST) (soft tissue sarcomas);germ cell tumors;pediatric central nervous system germ cell tumors (brain cancer);pediatric extracranial germ cell tumors;extragonadal germ cell tumors;ovarian germ cell tumors;testicular cancer;Gestational trophoblastic disease;Hairy cell leukemia;Head and neck cancer;Heart tumors, childhood;Hepatocellular (liver) cancer;Histiocytosis, Langerhans cell;Hodgkin's lymphoma;Hypopharyngeal cancer (head and neck cancer);Intraocular melanoma;Pancreatic islet cell tumors, pancreatic neuroendocrine tumors;Kaposi's sarcoma (soft tissue sarcoma);Kidney (renal cell) cancer;Langerhans cell histiocytosis;Laryngeal cancer (head and neck cancer);Leukemia;Lip and oral cavity cancer (head and neck cancer);Liver cancer;Lung cancer (non-small cell, small cell, pleuropulmonary blastoma, and tracheobronchial tumors);Lymphoma;Male breast cancer;Malignant fibrous histiocytoma of bone and osteosarcoma;Melanoma;Melanoma, intraocular ( eye);Merkel cell carcinoma (skin cancer);mesothelioma, malignant;metastatic cancer;occult primary metastatic squamous cell cervical cancer (head and neck cancer);midline canal carcinoma with Nat gene alterations;cancer of the mouth (head and neck cancer);multiple endocrine neoplasia syndrome;multiple myeloma / plasma cell neoplasm;mycosis fungoides (lymphoma);myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm;myeloid leukemia, chronic (CML);myeloid leukemia, acute (AML);myeloproliferative neoplasm, chronic;nasal and paranasal sinus cancer (head and neck cancer);nasopharyngeal carcinoma (head and neck cancer);neuroblastoma;non-Hodgkin's lymphoma;non-small cell lung cancer;oral cancer , lip and oral cavity and oropharyngeal cancer (head and neck cancer); osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; pancreatic cancer; pancreatic neuroendocrine tumors (islet cell tumors); papillomatosis (pediatric larynx); paraganglioma; paranasal sinus and nasal cancer (head and neck cancer); parathyroid cancer; penile cancer; pharyngeal cancer (head and neck cancer); pheochromocytoma; pituitary tumors; plasma cell neoplasm / multiple myeloma; pleuropulmonary blastoma (lung cancer); pregnancy and breast cancer; primary central nervous system (CNS) lymphoma; primary peritoneal cancer; prostate cancer; rectal cancer; recurrent cancer; renal cell (kidney) cancer; retinoblastoma; rhabdomyosarcoma, childhood ( soft tissue sarcoma);salivary gland cancer (head and neck cancer);sarcoma;pediatric rhabdomyosarcoma (soft tissue sarcoma);pediatric vascular tumor (soft tissue sarcoma);Ewing's sarcoma (bone cancer);Kaposi's sarcoma (soft tissue sarcoma);osteosarcoma (bone cancer);soft tissue sarcoma;uterine sarcoma;Sézary syndrome (lymphoma);skin cancer;small cell lung cancer;small intestine cancer;soft tissue sarcoma;squamous cell carcinoma of the skin - see skin cancer;occult primary metastatic squamous cell carcinoma of the neck (head and neck cancer);stomach (gastric) cancer;t-cell lymphoma, cutaneous - see lymphoma (mycosis fungoides and Sézary syndrome);testicular cancer;It may be cancer of the throat (head and neck cancer); nasopharyngeal cancer; oropharyngeal cancer; hypopharyngeal cancer; thymoma and thymic carcinoma; thyroid cancer; tracheobronchial tumors (lung cancer); transitional cell carcinoma of the renal pelvis and ureter (kidney (renal cell) cancer); carcinoma of unknown primary; rare cancers of childhood; ureter and renal pelvis, transitional cell carcinoma (kidney (renal cell) cancer); urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vascular tumors (soft tissue sarcomas); vulvar cancer; Wilms tumor and other childhood kidney tumors; or young adult cancer or any cancer mentioned at https: / / www.cancer.gov / types. ;

[0086] The protein complex may be administered as a pharmaceutical composition. The pharmaceutical composition of the present disclosure may be a combination of any of the protein complexes described herein with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or additives. The pharmaceutical composition facilitates administration of the protein complexes described herein to an organism. The pharmaceutical composition may be administered in a therapeutically effective amount as a pharmaceutical composition by various forms and routes, including, for example, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal, oral, inhalation, dermal, intraarticular, intrathecal, intranasal, and topical administration. The pharmaceutical composition may be administered in a local or systemic manner, for example, by injecting the protein complexes described herein directly into an organ, optionally with a depot.

[0087] Parenteral injections can be formulated for bolus injection or continuous infusion. Pharmaceutical compositions can be in a form suitable for parenteral injection as sterile suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulating agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the protein complexes described herein in water-soluble form. Suspensions of the protein complexes described herein can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Suspensions can also contain suitable stabilizers or agents that increase the solubility and / or reduce aggregation of such protein complexes described herein to allow for the preparation of highly concentrated solutions. Alternatively, the protein complexes described herein can be lyophilized or in powder form for reconstitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. In some embodiments, purified protein complexes are administered intravenously.Protein complexes of the present disclosure can have a serum half-life (e.g., as demonstrated herein, for example, in Example 7) long enough to allow for a dosing regimen that includes daily, every other day, twice a week, weekly, biweekly, or monthly administration frequency.Protein complexes of the present disclosure can have a serum half-life of at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 168 hours, at least 250 hours, at least 320 hours, or at least 400 hours.The serum half-life can be human serum half-life, mouse serum half-life, pig serum half-life, bovine serum half-life, dog serum half-life, cat serum half-life, or rabbit serum half-life.

[0088] The protein complexes of the present disclosure can be applied directly to organs or organ tissues or cells during surgical procedures or via percutaneous, subcutaneous, intramuscular, intratumoral, intrathecal, local or regional delivery. In some embodiments, the protein complexes can be applied directly to cancerous tissues (e.g., tumors). The protein complexes described herein can be administered locally and can be formulated into a variety of locally administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams and ointments. Such pharmaceutical compositions can contain solubilizers, stabilizers, tonicity agents, buffers and preservatives. The protein complexes can be expressed in Spirulina and delivered orally.

[0089] In carrying out the method of treatment or use provided herein, a therapeutically effective amount of the protein complex described herein is administered in a pharmaceutical composition to a subject suffering from cancer.In some embodiments, the subject is a mammal, such as a human.The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors.

[0090] The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, including additives and auxiliary agents that facilitate the processing of the active compound into a preparation that can be used pharma- ceutically. The formulation can vary depending on the selected route of administration. The pharmaceutical composition comprising the protein complex described herein can be produced, for example, by expressing the protein complex in a recombinant system, purifying the protein complex, lyophilizing, mixing, or dissolving the protein complex. The pharmaceutical composition can include at least one pharma- ceutical acceptable carrier, diluent, or additive, and the compound described herein as a free base or a pharma- ceutical acceptable salt form.

[0091] The preparation method of the protein complex described herein includes formulating the protein complex described herein with one or more inert pharmaceutically acceptable additives or carriers to form solid, semi-solid or liquid compositions.Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets and suppositories.These compositions may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.

[0092] Certain methods described herein include administering to a subject an intravenous pharmaceutical composition comprising the protein complex of the present disclosure, for example as described herein. The intravenous pharmaceutical composition of the protein complex includes any formulation suitable for administration to a subject by any intravenous method, including bolus, infusion over time, or any other intravenous method known in the art. In some embodiments, the infusion rate is such that the dose is administered over a period of less than 5 minutes, more than 5 minutes but less than 15 minutes, or more than 15 minutes. In other embodiments, the infusion rate is such that the dose is administered over a period of less than 5 minutes. In other embodiments, the infusion rate is such that the dose is administered over a period of more than 5 minutes and less than 15 minutes. In some other embodiments, the infusion rate is such that the dose is administered over a period of more than 15 minutes.

[0093] As used herein, "product" or "dosage form" refers to any solid, semi-solid, lyophilized, aqueous, liquid or frozen formulation or preparation used for administration. Upon administration, the release rate of the active moiety from the product is often greatly influenced by the additives that make up the product itself and / or the characteristics of the product. For example, enteric coats on tablets are designed to separate the tablet contents from the stomach contents, for example, to prevent gastric degradation, which often induces gastrointestinal discomfort or damage. According to currently accepted conventional understanding, systemic exposure of the active moiety is relatively insensitive to small formulation changes.

[0094] Non-limiting examples of pharma- ceutically acceptable excipients are described, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated by reference in its entirety.

[0095] The protein complexes of the present disclosure may be administered to a patient in an effective amount. As used herein, the term "effective amount" may refer to a sufficient amount of an agent or compound administered that relieves to some extent one or more of the symptoms of the disease or condition being treated. The result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired change in a biological system. Compositions containing such agents or compounds may be administered for preventive, enhancing, and / or therapeutic treatment. The appropriate "effective" amount in any individual case may be determined using techniques such as dose escalation studies.

[0096] The disclosed methods, compositions and kits can include methods for preventing, treating, arresting, reversing or ameliorating symptoms of a condition. Treatment can include treating a subject (e.g., an individual suffering from a disease or condition, a livestock, a wild animal or an experimental animal) with a protein complex of the disclosure. The protein complex of the disclosure can be administered to treat the disease of the subject. The subject can be a human. The subject can be a human; a non-human primate, such as a chimpanzee, or other ape or monkey species; a livestock animal, such as a cow, a horse, a sheep, a goat, a pig; a domestic animal, such as a rabbit, a dog, a cat; an experimental animal, including a rodent, such as a rat, a mouse, a guinea pig. The subject can be of any age. The subject can be, for example, an elderly adult, an adult, an adolescent, a prepubertal, a child, a toddler, an infant, or an intrauterine fetus.

[0097] Treatment can be provided to a subject before clinical onset of disease. Treatment can be provided to a subject after clinical onset of disease. Treatment can be provided to a subject 1 day, 1 week, 6 months, 12 months, or 2 years or more after clinical onset of disease. Treatment can be provided to a subject 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment can be provided to a subject less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of disease. Treatment can also include treating humans in clinical trials. Treatment can include administering a pharmaceutical composition to a subject, such as one or more pharmaceutical compositions described throughout this disclosure. Treatment can include once-daily dosing. Treatment may include delivering a protein complex of the present disclosure to a subject intravenously, subcutaneously, intramuscularly, via inhalation, dermal, intra-articular injection, oral, intrathecal, transdermal, intranasal, peritoneal routes, or directly onto or into the diseased tissue, e.g., via a topical, intra-articular injection route or an injectable application route.

[0098] In some embodiments, the present disclosure provides a method of treating cancer comprising administering to a subject in need thereof an effective amount of a protein complex of the present disclosure.

[0099] In some embodiments, the present disclosure provides a method of treating cancer comprising administering to a patient in need thereof an effective amount of a pharmaceutical composition comprising a protein complex of the present disclosure and a pharma- ceutically acceptable carrier.

[0100] kit The protein complexes of the present disclosure may be provided in various kits. In some embodiments, pharmaceutical compositions comprising the protein complexes of the present disclosure may be supplied as a kit. The kit may include a container containing the protein complex. The therapeutic protein complex may be provided in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted before injection. Alternatively, such kits may include a dry powder disperser, liquid aerosol generator, or nebulizer for administering the therapeutic protein complex. Such kits may further include written information regarding the application and use of the pharmaceutical composition.

[0101] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. As used in this specification and claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Any reference to "or" in this specification is intended to include "and / or" unless otherwise specified.

[0102] Whenever the terms "at least," "greater than," or "greater than or equal to" precede a first number in a series of two or more numerical values, the terms "at least," "greater than," or "greater than or equal to" apply to every number in the series. For example, 1, 2, or 3 or more is equal to 1 or more, 2 or more, or 3 or more.

[0103] Whenever the terms "no more than," "less than," "less than or equal to," or "at most" precede a first number in a series of two or more numerical values, the terms "no more than," "less than," "less than or equal to," or "at most" apply to each number in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0104] When values ​​are listed as ranges, such disclosure will be understood to include disclosure of all possible subranges within such ranges, as well as specific numerical values ​​falling within such ranges, whether or not a specific numerical value or specific subrange is explicitly listed.

[0105] [Table 2A] TIFF2024544757000008.tif252162TIFF2024544757000009.tif252162TIFF2024544757000010.tif252162TIFF2024544757000011.tif252162TIFF2024544757000012.tif252162TIFF2024544757000013.tif251162TIFF2024544757000014.tif247162TIFF2024544757000015.tif252162TIFF2024544757000016.tif252162TIFF2024544757000017.tif252162TIFF2024544757000018.tif247162TIFF2024544757000019.tif252162TIFF2024544757000020.tif251162TIFF2024544757000021.tif251162TIFF2024544757000022.tif251162TIFF2024544757000023.tif251162TIFF2024544757000024.tif252162TIFF2024544757000025.tif252162TIFF2024544757000026.tif252162TIFF2024544757000027.tif252162TIFF2024544757000028.tif247162TIFF2024544757000029.tif252162TIFF2024544757000030.tif246162TIFF2024544757000031.tif252162TIFF2024544757000032.tif251162TIFF2024544757000033.tif252162TIFF2024544757000034.tif251162TIFF2024544757000035.tif251162TIFF2024544757000036.tif251162TIFF2024544757000037.tif251162TIFF2024544757000038.tif252162TIFF2024544757000039.tif251162TIFF2024544757000040.tif252162TIFF2024544757000041.tif251162TIFF2024544757000042.tif246162TIFF2024544757000043.tif252162TIFF2024544 757000044.tif252162TIFF2024544757000045.tif252162TIFF2024544757000046.tif252162TIFF2024544757000047.tif188162. EXAMPLES

[0106] The following examples are illustrative, but not limiting, of the scope of the devices, methods, systems, and kits described herein.

[0107] Example 1 Isolation of a set of dual binding antibodies (DBAs) that bind human PD-1 and human IL-2 This example describes the isolation of a set of sensor domains of the disclosure, specifically DBAs that bind human PD-1 and human IL-2. The anti-PD-1 and anti-IL-2 DBAs were isolated from the Tumbler antibody phage display library (Distributed Bio, Inc.). An antibody phage display library was constructed to incorporate the heavy chain CDR1, heavy chain CDR2, and light chain diversity of the Superhuman 2.0 antibody library combined with 10 heavy chain CDR3 sequences from PD-1 binding antibodies (SEQ ID NO:11-SEQ ID NO:20). [Table 3]

[0108] This library was subjected to four rounds of selection with standard protocols. Briefly, the phage library was incubated with antigen, then captured on magnetic beads, washed on a Kingfisher magnetic particle processor, eluted from the magnetic beads, and amplified by passaging in E. coli. Round 1 was incubated with 50 nM human PD-1-His fusion (R&D Systems, product number 8986-PD) and captured with TRIS NTA biotin (Sigma-Aldrich product number 75543) and streptavidin magnetic beads. Round 2 was incubated with 100 nM biotinylated IL-2 (Creative Biomart, product number IL2-501H, biotinylated using standard protocols) and captured on streptavidin magnetic beads. Round 3 was incubated with 50 nM cynomolgus PD-1-Fc fusion (R&D Systems, product number 8578-PD) and captured on protein G magnetic beads. Round 4 was incubated with 50 nM biotinylated human IL-2 and captured on streptavidin magnetic beads. The final selection was plated as single colonies and 380 colonies were picked for Sanger sequencing. 151 unique clones were selected for expression. The scFv sequence of each clone was codon-optimized for E. coli expression and the corresponding DNA sequence was sent to Integrated DNA Technologies, Inc. (IDT) for synthesis as a gBlock with a T7 promoter, translation start site and T7 terminator. Proteins from each gBlock encoding scFv were expressed using the PURExpress In vitro Protein Synthesis Kit (New England Biolabs, Inc., product number E6800). PURExpress scFv proteins were used directly in HTRF binding assays and cell-based functional assays. Each scFv was tested for binding to PD-1 and human IL-2. Eighty-one antibodies showed dual binding activity against both PD-1 and IL-2, and a summary of the fluorescence signal values ​​of the binding curves is shown in Table 5.To examine the ability of the DBA binding domain to block IL-2 receptor binding, V5-tagged DBA scFvs were serially diluted in 384-well plates containing: Europium-labeled streptavidin, biotin-labeled IL-2 (Acro Biosystems, product number IL2-H82E4), IL-2 receptor beta (Fc-IL2RB) (Acro Biosystems, product number ILB-H5253), and APC-labeled anti-Fc antibody. Plates were incubated at room temperature for 2 hours and HTRF signals were read on an Envision (Perkin Elmer) as a measure of IL-2:IL2RB binding. Four scFvs (SEQ ID NO:31-SEQ ID NO:34) bound to PD-1, bound to IL-2, and blocked IL-2 binding to IL-2RB (Table 4). [Table 4] TIFF2024544757000050.tif52166 [Table 5] TIFF2024544757000052.tif247133TIFF2024544757000053.tif247133TIFF2024544757000054.tif249133TIFF2024544757000055.tif76133

[0109] Example 2 Double-binding antibody (DBA)-cytokine protein complexes

[0110] This example describes dual binding antibody (DBA)-cytokine protein complexes of the present disclosure. Various DBA-cytokine protein complexes of the present disclosure were designed to include a cytokine, a linker, and one or more dual binding antibody domains. A pictorial representation of an exemplary construct is shown in FIG. 5.

[0111] A series of DBA-cytokine protein complexes can be designed with two marker binding domains and one therapeutic domain. The DBAs used in this series, provided in Table 6 with sequences provided in Table 8, exhibit a range of affinities for the marker and therapeutic domains. Exemplary DBA complexes are provided in Tables 6, 9, and 10. [Table 6] TIFF2024544757000057.tif252167TIFF2024544757000058.tif183167 [Table 7] [Table 8] TIFF2024544757000061.tif13170 [Table 9] [Table 10] TIFF2024544757000064.tif251170TIFF2024544757000065.tif86168

[0112] Example 3 CD8 by PD-1 / IL-2 double-binding antibody (DBA) cytokine complex + Decreased T cell STAT5 phosphorylation This example describes the reduction of IL-2-mediated signaling by the addition of PD-1 / IL-2 DBA moieties to IL-2 molecules by fusion, as read out using CD8+ T cell STAT5 phosphorylation. Genes for the PD-1 / IL-2 DBAs shown in Table 11 were synthesized and expressed in HEK293 as IgG proteins with IL-2 fused to the N-terminus of the heavy or light chain via a linker (Genscript). Although only two of the antibodies blocked IL-2 binding to IL-2RB as scFv, over 30 antibodies were able to reduce IL-2 signaling with IL-2 domains linked in the format shown in Figures 2D and 2E. An exemplary set of these DBAs was selected for analysis and compared to the control anti-HER2-IL-2 immunocytokine (Table 11 and Figure 3). [Table 11] TIFF2024544757000067.tif252167TIFF2024544757000068.tif252167TIFF2024544757000069.tif105167

[0113] PD-1 / IL-2 DBA-cytokine complexes were serially diluted in complete RPMI (+10% FBS, 2 mM L-glutamine, sodium pyruvate) and added to a 96-well plate. 2 × 10 5Human PBMCs were added to each well and the plate was incubated at 37°C for 20 minutes. An equal volume of pre-warmed fixation buffer (Biolegend) was then added to each well and the plate was incubated at 37°C for 10 minutes. The cells were then fixed in pre-chilled Perm Buffer III (BD Biosciences) for 30 minutes at 4°C. The cells were washed with FACS wash buffer (PBS+2% FBS, 2mM EDTA) and stained with fluorophore-labeled antibodies against CD3, CD4, CD8 (BioLegend) and phospho-STAT5 (BD Biosciences) diluted 1:20 in FACS wash buffer. The cells were incubated for 1 hour at 4°C, washed with FACS wash buffer and analyzed on a SA3800 Spectral Analyzer. In the absence of PD-1, the PD-1 / IL-2 DBA / cytokine complex induced less STAT5 phosphorylation in T cells compared to the monospecific control anti-HER2 IL-2 immunocytokine (Figure 3).

[0114] Example 4 General Method: Generation of Complexes That Drive PD-1-Dependent IL-2 Activity in Human Cells This example describes a PD-1 / IL-2 protein complex for PD-1-dependent IL-2 activity in human cells in vitro and in vivo. The PD-1 / IL-2 protein complex includes a PD-1 sensor domain (e.g., an anti-PD-1 antibody or an anti-PD-1 scFv) linked to an IL-2 cytokine therapeutic domain via a linker, where the IL-2 cytokine is a therapeutic agent. In the absence of PD-1, the PD-1 sensor domain binds to the IL-2 therapeutic domain, rendering the IL-2 therapeutic inactive. In the presence of PD-1 (e.g., PD-1 is expressed on cells such as immune cells), the PD-1 sensor domain binds to PD-1, thereby unbinding the IL-2 therapeutic domain and allowing IL-2 to exhibit therapeutic activity.

[0115] The PD-1 / IL-2 protein complex is recombinantly expressed or chemically synthesized. The PD-1 / IL-2 protein complex is administered to human cells in vitro or to a mouse or human subject in vivo in need thereof. The human cells are cells that express PD-1. Administration to the mouse or human subject is intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal or mucosal. In the absence of PD-1, the IL-2 therapeutic domain remains bound to the PD-1 sensor domain and no therapeutic benefit is observed (e.g., cell activation in vitro and in the subject is unchanged). In the presence of PD-1, the PD-1 sensor domain binds to PD-1 and releases the binding of the IL-2 therapeutic domain. A therapeutic benefit is observed (e.g., cell activation is observed in vitro and in vivo in the subject). The subject has a disease. The disease is cancer. The cells may express PD-1 endogenously or after activation, or after introduction of a gene encoding PD-1. The therapeutic effect can be cell proliferation, differentiation, activation or induction of IL2 responsive genes. In vitro, when the cells are part of a mixture of cell types, any of these changes can be monitored for the responsive cell population in the mixture.

[0116] Example 5 PD-1 / IL-2 DBA cytokine complex induction of STAT5 phosphorylation in lymphoid cell lines This example describes PD-1 / IL-2 DBA-cytokine complex induction of STAT5 phosphorylation in lymphoid cell lines. To assess the dependency of PD-1 / IL-2 DBA-cytokine complex activity on binding to PD-1, PD-1 expression variants of IL-2 R+ T cell lines, such as Hut78 or Jurkat E6.1, are generated. PD-1+ and PD-1- variant cell lines are treated with titrated concentrations of the PD-1 / IL-2 DBA-cytokine complex of the present disclosure, and STAT5 phosphorylation is assessed by phospho-flow, TR-FRET, or other assays to measure IL-2 signaling.

[0117] A HEK 293 IL-2 reporter cell line is engineered to express PD-1. PD-1+ and PD-1- variant cell lines are treated with titrated concentrations of the PD-1 / IL-2 DBA-cytokine complex, and reporter activity is assessed as a measure of IL-2 signaling. The PD-1 / IL-2 DBA-cytokine complex shows high potency against the PD-1+ variant cell line.

[0118] Example 6 PD-1 / IL-2 DBA cytokine complex induction of STAT5 phosphorylation and other markers of activation, and proliferation in primary lymphocytes This example describes PD-1 / IL-2 DBA-cytokine complex induction of STAT5 phosphorylation and other markers of activation and proliferation in primary lymphocytes. PBMCs are labeled with a cell proliferation dye and incubated for 4 days with titrating concentrations of the disclosed PD-1 / IL-2 DBA-cytokine complex. PBMCs are stained with antibodies against immune cell phenotyping markers to distinguish CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) cells, as well as markers of cell activation such as CD25. Dye dilution of immune cell subsets is examined by flow cytometry as a measure of proliferation.

[0119] Total T cells were isolated from PBMCs using immunomagnetic negative selection (STEMCELL) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 72 hours to induce expression of PD-1. PD-1+ T cells are incubated with titrated concentrations of PD-1 / IL-2 DBA-cytokine complexes for 20 minutes. STAT5 phosphorylation is measured by flow cytometry in fixed and permeabilized T cells. In some experiments, PD-1 can be blocked on T cells with anti-PD-1 prior to treatment with PD-1 / IL-2 DBA-cytokine complexes to assess the dependence of PD-1 / IL-2 DBA-cytokine complex activity on binding to PD-1. PD-1 / IL-2 DBA-cytokine complexes induce minimal STAT5 phosphorylation when PD-1 is blocked, indicating activity that is conditional on their ability to bind PD-1.

[0120] Example 7 In vivo PD-1 / IL-2 DBA cytokine complex signaling in non-tumor peripheral tissues This example describes the pharmacokinetics of PD-1 / IL-2 DBA-cytokine complexes in the blood of wild-type mice and the signaling of the complexes in non-tumor peripheral tissues. The serum half-life and peripheral tissue activity of PD-1 / IL-2 DBA-cytokine complexes and appropriate uncontrolled controls, such as anti-PD-1, anti-HER2-IL-2, or anti-PD-1-IL-2, were measured in mice administered the complexes intravenously (iv). Blood, spleen, or both were collected at various time points after treatment and stained to identify CD8+ T cells and NK cells.

[0121] To examine the half-life of PD-1 / IL-2 DBA-cytokine complexes in circulation, wild-type C57BL / 6 mice received a single 2.5 milligram / kilogram intravenous dose of PD-1 / IL-2 DBA-cytokine complexes (2B07 IL-2 mut; SEQ ID NO:205-SEQ ID NO:206), anti-HER2 / IL-2-cytokine complexes (always-on IL-2 mut; SEQ ID NO:64 and SEQ ID NO:207) or anti-IL-2 / IL-2-cytokine complexes (always-off IL-2 mut; SEQ ID NO:208-SEQ ID NO:209) as outlined in Table 13. Mice were bled via the retro-orbital sinus 30 minutes, 4 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours and 168 hours after dosing. Blood was collected into serum separator tubes and isolated serum was frozen at -80°C until analysis. To determine serum levels of cytokine complexes, 96-well high-binding ELISA plates were coated with 1 μg / mL rabbit anti-hu IL-2 capture antibody (clone ab9618, Abcam) in carbonate-bicarbonate buffer overnight at 4° C. Plates were washed three times and blocked with SuperBlock blocking buffer (Thermo Scientific) for 1 h. Serum samples from various time points and treatment groups were diluted in SuperBlock, added to the plate, and incubated for 1 h. To detect cytokine complexes, plates were incubated with goat anti-mouse Fc-HRP (Jackson ImmunoResearch) at 1:5000 in SuperBlock for 1 h. Plates were then washed and developed with TMB substrate. Optical density (OD) was measured at 450 nm using an EnVision 2105 microplate reader (PerkinElmer). As shown in Figure 6, at all time points examined, the PD-1 / IL-2 DBA-cytokine complex was detected at serum concentrations similar to the anti-IL-2 / IL-2-cytokine complex. In contrast, the serum concentration of the uncontrolled anti-HER2 / IL-2-cytokine complex showed a greater decrease in serum concentration over time. [Table 12] TIFF2024544757000071.tif173165

[0122] To examine the activity of PD-1 / IL-2 DBA-cytokine complexes in peripheral tissues, wild-type C57BL / 6 mice received a single intravenous dose of 2.5 milligrams of PD-1 / IL-2 DBA-cytokine complexes (2B07 IL-2 mut; SEQ ID NO:205-SEQ ID NO:206), anti-HER2 / IL-2-cytokine complexes (always-on IL-2 mut; SEQ ID NO:64 and SEQ ID NO:207), anti-IL-2 / IL-2-cytokine complexes (always-off IL-2 mut; SEQ ID NO:208-SEQ ID NO:209) or PBS, as shown in Table 12. Prior to dosing, the presence of intact IL-2 within each IL-2 cytokine complex was confirmed by ELISA as a means of validating their potential biological activity. Five days after treatment, blood and spleens were collected and analyzed by flow cytometry to quantify the number of CD8+ T cells and NK cells per spleen and per microliter of blood. The PD-1 / IL-2 DBA-cytokine complex did not induce expansion of CD8 T cells or NK cells, whereas the HER2 / IL-2-cytokine complex induced expansion of peripheral CD8+ T cells and NK cells (Figures 7A-D).

[0123] Example 8 PD-1 / IL-2 DBA cytokine complex regulation of antitumor immunity in syngeneic tumor models This example describes PD-1 / IL-2 DBA-cytokine complex modulation of anti-tumor immunity in the MC38 syngeneic mouse tumor model. PD-1 / IL-2 DBA-cytokine complexes were evaluated for their ability to drive anti-tumor immunity in vivo. 500,000 MC38 tumor cells were implanted subcutaneously into human PD-1 knock-in mice (GenOway). Tumors were measured twice weekly and volume was calculated as (length x width x width / 2). Mice were randomized into treatment groups and were randomized until tumors reached approximately 100 mm 3Treatment began when tumors reached a volume of 100 μg / kg. Mice were treated intravenously with PD-1 / IL-2 DBA-cytokine complex (2B07 IL-2 mut; SEQ ID NOs: 210-212), PD-1 / IL-2 DBA lacking IL-2 (2B07; SEQ ID NOs: 212-213), or isotype control (SEQ ID NOs: 214-215) at the indicated doses of 5 or 0.5 milligrams / kilogram on days 7, 10, and 13 after tumor implantation, as shown in Table 13 below. The PD-1 / IL-2 DBA-cytokine complex demonstrated increased tumor growth inhibition compared to PD-1 / IL-2 DBA lacking IL-2 or the isotype control (FIG. 8). [Table 13] TIFF2024544757000073.tif205165

[0124] Example 9 PD-1 / IL-2 DBA cytokine complex regulation of antitumor immunity in a xenograft / human immune cell mixture model This example describes PD-1 / IL-2 DBA-cytokine complex modulation of anti-tumor immunity in a xenograft / human immune cell mixture model. A mixed system is used to examine the ability of PD-1 / IL-2 DBA-cytokine complex to drive anti-tumor immunity in an in vivo setting. Total human PBMCs or a combination of human T cells and monocyte-derived dendritic cells (moDCs) are mixed with human tumor cells (e.g., HPAC, A375, H441) at a 1:4 ratio and co-implanted subcutaneously into the flank of NSG mice. One day later, treatment with the PD-1 / IL-2 DBA-cytokine complex of the present disclosure or an appropriate uncontrolled control such as anti-PD-1, anti-HER2-IL-2, or anti-PD-1-IL-2 is initiated. Tumors are measured at least twice a week and the volume is calculated as (length x width x height / 2). The PD-1 / IL-2 DBA-cytokine complex exhibits increased anti-tumor efficacy compared to anti-PD-1 and anti-HER2-IL-2 and decreased extratumor activity compared to anti-PD-1-IL-2.

[0125] Example 10 General Methods: Characterization of Protein Complexes In Vitro and In Vivo

[0126] This example describes the evaluation of DBA-cytokine complexes for in vitro and in vivo stability. The protein complexes of the present disclosure are recombinantly expressed or chemically synthesized. The protein complexes include a sensor domain linked to a therapeutic domain. The linker is a peptide linker. The sensor domain can bind to a therapeutic domain and a marker. In the absence of the marker, the sensor domain binds to the therapeutic domain, rendering the therapeutic domain unable to bind to its target and unable to exert therapeutic activity. In the presence of the marker, the sensor domain binds to the marker, rendering the therapeutic domain free to bind to its target and exert therapeutic activity.

[0127] In vitro, protein complexes are tested for stability and functionality at baseline or after incubation in stress conditions, such as high temperature, pH change, oxidizing buffers or serum / plasma, using biophysical characterization methods to measure fragmentation, unfolding or aggregation, and / or methods to test changes in functional activity. In vivo, the pharmacokinetic properties of the protein are measured after administration in mammals (e.g., mice, rats or non-human primates), and distribution, clearance and degradation properties are measured. These measurements are used to engineer or select optimal therapeutic forms of DBA-protein complexes.

[0128] Example 11 Regulated IL-2 receptor signaling by the PD-1 / IL-2 double-binding antibody (DBA) cytokine complex This example describes PD-1-regulated IL-2 activity in HEK-Blue™ IL-2 reporter cells by PD-1 / IL-2 DBA-cytokine complexes. DBA-cytokine complexes and control antibody-cytokine complexes were produced in three formats shown in Figure 2E, Figure 2B, and Figure 2H by expression in mammalian cells using standard protocols. Wells of a 384-well ELISA plate were coated with a fixed concentration of PD-1-Fc or IgG1 control protein captured with anti-Fc antibody (Jackson ImmunoResearch, product number 109-005-098). Cytokine complexes were serially diluted 1:4 for eight points in growth medium from a starting concentration of 6 nM and incubated briefly before adding HEK-Blue™ IL-2 reporter cells.

[0129] Results of protein complexes containing the structure shown in Figure 2E are shown in Figures 9A-9D. As shown in Figure 2E, this symmetric format consists of one IL-2 linked to each antibody variable domain. The IL-2 activity of the PD-1 / IL-2 DBA-IL-2 complex AF4379 containing SEQ ID NOs: 174-175 had an EC50 of 31 pM in PD-1 coated wells versus 62 pM in IgG1 coated wells, as shown in Figure 9A, demonstrating PD-1 dependency. As shown in Figure 9D, the IL-2 activity of the antibody-cytokine complex AF4377 (anti-Her2 antibody) containing SEQ ID NOs: 64 and 176, and AF4378 (anti-IL-2 antibody) containing SEQ ID NOs: 177-178, was unchanged in the presence of PD-1 (shown in Figures 9B and 9C, respectively), whereas the IL-2 activity of the anti-PD-1 antibody AF4376 containing SEQ ID NOs: 179-180 was reduced in the presence of PD-1. The sequences of the protein complexes are summarized in Table 14 below. [Table 14] TIFF2024544757000075.tif252165TIFF2024544757000076.tif135165

[0130] Results for protein complexes containing the structure shown in Figure 2B are shown in Figures 4A-4F. This format consists of an asymmetric complex composed of two antibody domains with a single IL-2 linked to one of the domains. The IL-2 activity of the PD-1 / IL-2 DBA-IL-2 complexes AF4386 (containing SEQ ID NO:212 and SEQ ID NO:181-SEQ ID NO:182, results shown in Figure 4A), AF4387 (containing SEQ ID NO:183-SEQ ID NO:185, results shown in Figure 4B) and AF4389 (containing SEQ ID NO:186-SEQ ID NO:188, results shown in Figure 4C) had EC50s of 50 pM, 57 pM and 118 pM, respectively, in PD-1 coated wells and 1.79 nM, 419 pM and 1.67 nM, respectively, in IgG1 coated wells, demonstrating PD-1 dependency. The IL-2 activity of the anti-PD1 control protein AF4380 (SEQ ID NO: 180, comprising SEQ ID NO: 189-190, results shown in FIG. 4D), the anti-Her2 control protein AF4383 (SEQ ID NO: 64, comprising SEQ ID NO: 191-192, results shown in FIG. 4E) and the anti-IL-2 control protein AF4384 (SEQ ID NO: 178, comprising SEQ ID NO: 193-194, results shown in FIG. 4F) was unchanged. The sequences of the protein complexes are summarized in Table 15 below. [Table 15] TIFF2024544757000078.tif244160TIFF2024544757000079.tif245160TIFF2024544757000080.tif240160TIFF2024544757000081.tif22160

[0131] Results for protein complexes containing the structure shown in Figure 2H are shown in Figures 5A-5H. As shown in Figure 2H, these complexes are asymmetric and consist of two identical monospecific Fab arms with a single IL-2 attached to one Fc domain by a flexible linker and a single scFv attached to the other Fc domain by a flexible linker. The active PD-1 / IL-2 DBA complexes, AF4403 containing SEQ ID NOs: 180, 195, 199 and AF4404 containing SEQ ID NOs: 180, 196, 199, are composed of an anti-PD-1 domain in the Fab arm and a PD-1 / IL-2 DBA scFv on the Fc arm. The control antibody-cytokine complexes consist of a) an antibody-cytokine complex with an irrelevant antibody on the Fab arm with a DBA scFv on the Fc (AF4395 comprising SEQ ID NOs: 64, 197, 202 and AF4396 comprising SEQ ID NOs: 64, 198, 202), b) an antibody-cytokine complex with a non-DBA scFv on the Fc arm (AF4400 comprising SEQ ID NOs: 180, 199-200 and AF4401 comprising SEQ ID NOs: 180, 199, 201), and c) an antibody-cytokine complex with a non-DBA antibody in both the Fab and scFv domains (AF4392 comprising SEQ ID NOs: 64, 202-203 and AF4393 comprising SEQ ID NOs: 64, 202, 204). As shown in Figure 5B and Figure 5D, the IL-2 activity of DBA-cytokine complexes AF4403 and AF4404 had EC50 of 31 pM and 26 pM, respectively, in PD-1 coated wells and 62 pM and 64 pM, respectively, in control wells, demonstrating the PD-1 dependency of IL-2 activity. None of the above control proteins AF4395, AF4396, AF4400, AF4401, AF4392 and AF4393 showed a lower EC50 in PD-1 coated wells than in wells coated with IgG1 protein, as shown in Figure 5A, Figure 5C and Figure 5E-H. The sequences of the protein complexes are summarized in Table 16 below. [Table 16] TIFF2024544757000083.tif252165TIFF2024544757000084.tif252165TIFF2024544757000085.tif252165TIFF2024544757 000086.tif252165TIFF2024544757000087.tif252165TIFF2024544757000088.tif252165TIFF2024544757000089.tif71165

[0132] Example 12 Further dual-binding antibody binding to PD1 and IL2 Observing the effectiveness of dual binding antibodies (DBA) in previous examples, further dual binding antibody scFvs were generated (see Table 17, Table 18, and Table 19 below). This example describes the measurement of PD1 and IL2 binding to these dual binding antibody scFvs. The scFv DNA sequence of each clone was synthesized as a gBlock (Integrated DNA Technologies, Inc.) with a T7 promoter, a translation start site, coding sequences for the scFv sequence with cMyc and V5 tags, and a T7 terminator sequence. Proteins from each gBlock fragment were expressed using a cell-free transcription / translation system (Cosmo Bio USA, Inc., PUREfrex2.1, product number GFK-PF213 (with DS supplement), product number GFK-PF005). The scFv samples were subjected to ELISA analysis to detect binding of PD1 and IL2. In these experiments, wells of a 384-well plate were coated overnight at 4° with 1 μg / ml of anti-V5 antibody (Sv5-Pk1, BioRad). After washing, wells were blocked with SuperBlock (ThermoFisher, 37515) followed by the addition of saturating levels of scFv in SuperBlock. After washing, antigen was added and plates were incubated for 1 h. To detect PD1 binding, biotinylated recombinant PD1 was used (PD1-HisAvi, Acro Biosystems, PD1-H82E4). To detect IL2 binding, IL2 (R&D, 202-IL) was preincubated with biotinylated anti-IL2 mAb (mab202, biotinylated using standard methods) in a 2:1 ratio in SuperBlock buffer. Biotinylated antigen was detected using streptavidin-HRP using standard methods. Varying amounts of labeled test antigen were added to demonstrate binding and estimate the relative affinities of the different scFvs. Tables 17A-C show the EC50 values ​​for PD1 and IL2 binding for the three sets of scFvs, demonstrating the dual binding of these antibodies.

[0133] The data show that all antibodies in Tables 17A-C, except for the control antibodies AB000694, AB000719, and AB000880, are able to effectively bind to both PD1 and IL2. [Table 17A] [Table 17B] [Table 17C] [Table 18] TIFF2024544757000094.tif249157TIFF2024544757000095.tif249157TIFF2024544757000096.tif249157TIFF2024544757 000097.tif249157TIFF2024544757000098.tif249157TIFF2024544757000099.tif249157TIFF2024544757000100.tif24915 7TIFF2024544757000101.tif249157TIFF2024544757000102.tif249157TIFF2024544757000103.tif249157TIFF2024544757 000104.tif249157TIFF2024544757000105.tif249157TIFF2024544757000106.tif249157TIFF2024544757000107.tif24939 [Table 19] TIFF2024544757000109.tif249155TIFF2024544757000110.tif249155TIFF2024544757000111.tif249155TIFF2024544757000112.tif249142

[0134] Example 13 Regulated IL-2 receptor binding in vitro by PD-1 / IL-2 cytokine complexes containing an additional dual-binding antibody (DBA) domain This example describes PD-1-regulated IL-2 receptor binding by PD-1 / IL-2 DBA-cytokine complexes containing the additional DBA binding element described in Example 12. Anti-PD-1 / IL-2 DBA-cytokine complexes were analyzed along with appropriate uncontrolled controls such as anti-PD-1, anti-Her2, anti-PDL-1 or anti-IL-2 cytokine complexes. DBA-cytokine complexes and control antibody-cytokine complexes were produced in two formats: (1) "symmetric" immunocytokine (shown in FIG. 2E); or (2) "asymmetric" immunocytokine (shown in FIG. 2B), by expression in mammalian cells and purified using standard protocols. ELISA assays were performed with a constant amount of antibody-cytokine constructs coated onto each well probed with biotinylated IL-2 receptor beta gamma heterodimer Fc (IL-2RBG; Acro Cat. No. ILG-H5254) in the presence of varying amounts of PD-1-Fc or hIgG1-Fc. To perform the assay, 384-well ELISA plates were coated with anti-Fc antibody at 1 microgram / ml in 100 mM bicarbonate solution pH 9.0 overnight at 4°C and washed twice with SuperBlock. Antibody-cytokine complexes were then added to each well at a fixed concentration of 6 nM, incubated for 1 hour, and washed three times in PBS + 0.05% Tween 20 (PBST). Titrated concentrations of PD-1 Fc or IgG1 control Fc were added and incubated for 15 minutes, after which a fixed amount of 10 nM biotinylated IL-2RBG was added. Plates were incubated for an additional 30 minutes, washed, and biotinylated IL-2RBG detection was performed using streptavidin-HRP and standard ELISA protocols.

[0135] Symmetrical Design The results of protein complexes containing the structures shown in Figure 2E are shown in Figure 9. As shown in Figure 2E, this symmetric format is composed of one IL-2 linked to each antibody variable domain. As can be seen for the behavior of these constructs in Figure 9, IL-2RBG binding was increased in a dose-dependent manner by the addition of PD-1 Fc (but not by the addition of negative control hIgG1 Fc protein) for DB cytokine complexes AF3247, AF3644, AF3651, AF3652, AF3653, AF3657, AF3930, AF3931, AF3933, AF3934 and AF3935 (the sequences of the referenced peptides can be found in Table 2A) containing peptide IDs shown in Table 35 below. IL-2RBG binding to control monospecific antibody-cytokine complexes containing anti-Her2 (AF3243) and anti-IL-2 (AF3246) was not altered by the addition of PD-1 Fc protein. [Table 20]

[0136] Asymmetrical Design Results are shown in FIG. 10 for protein complexes containing the structures shown in FIG. 2B ("asymmetric" immunocytokine design) and described in Table 21 below. This format consists of an asymmetric complex composed of two antibody domains with a single IL-2 linked to one of the domains. IL-2RBG binding was increased in a dose-dependent manner by the addition of PD-1 Fc, but not by the addition of IgG1 control Fc protein to DBA-cytokine complexes AF3232, AF3740, AF3747, AF3749, AF3753, AF3945, AF3947, AF3951, AF3952, AF3953, AF3955 and AF3956 containing peptide IDs shown in Table 21 below (sequences of the referenced peptides can be found in Table 2A). IL-2RBG binding to the control monospecific antibody-cytokine complex anti-PDL-1 (AF3941) was not altered by the addition of PD-1 Fc protein. [Table 21]

[0137] Example 14 Inhibition of two types of IL-2 binding to IL-2RBG on the PD-1 / IL-2 cytokine complex in vitro This example describes two forms of IL-2 in PD-1 / IL-2 DBA-cytokine complexes that bind to IL-2RBG. Antibody-cytokine complexes in the format shown in Figure 2H were generated using wild-type (WT) IL-2 or IL-2 3x (an IL-2 variant with reduced binding to IL-2R alpha and R38D, K43E and E61R mutations, see e.g., Vazquez-Lombardi et al. Nat Commun. 8:15371 2017, which is incorporated herein by reference in its entirety). DBA-cytokine complexes and control antibody-cytokine complexes were produced by expression in mammalian cells and purified using standard protocols. To examine the ability of the DBA binding domain to block IL-2 binding to IL-2RBG, an ELISA assay was performed with a fixed amount of antibody-cytokine construct coated onto each well that was probed with biotinylated IL-2 receptor beta gamma heterodimer Fc (IL-2RBG; Acro Cat. No. ILG-H5254). In these experiments, 384-well ELISA plates were coated overnight at 4° C. with 1 microgram / ml of anti-Fc antibody (Jackson ImmunoResearch) in 100 mM bicarbonate solution pH 9.0 and washed twice with SuperBlock (ThermoFisher, 37515). The antibody-cytokine complex was then added to each well at a fixed concentration of 6 nM, incubated for 1 hour, and washed three times in PBS+0.05% Tween 20 (PBST). Titrated concentrations of biotinylated IL-2RBG were added and the plates were incubated for an additional 45 minutes. After washing, biotinylated IL-2RBG detection was performed using streptavidin-HRP and a standard ELISA protocol.

[0138] Results using the protein complexes listed in Table 22 below are shown in Figure 11. As shown in Figure 2H, these complexes are asymmetric and consist of two identical monospecific Fab arms with a single IL-2 (either WT or 3x) attached to one Fc domain by a flexible linker and a single scFv attached to the other Fc domain by a flexible linker. The antibody-cytokine complex contains an anti-PD-1 domain in the Fab arm and a PD-1 / IL-2 DBA scFv in the Fc arm. The control antibody-cytokine complex contains the same anti-PD-1 domain in the Fab arm and an anti-HER2 monospecific scFv in the Fc arm. In panel A of Figure 11, the antibody-cytokine complex contains the WT IL-2 form on the Fc domain and in panel B of Figure 11, the antibody-cytokine complex contains the 3x IL-2 form on the Fc. As shown in Figure 11, panel A, PD-1 / IL-2 DBA conjugates AF5418 and AF5419 have reduced IL-2RBG binding compared to the anti-Her2 non-DBA control conjugate AF5416, demonstrating the ability of the DBA domain to block receptor binding to WT IL-2. As shown in Figure 11, panel B, PD-1 / IL-2 DBA conjugates AF4695 and AF4696 have reduced IL-2RBG binding compared to the anti-Her2 non-DBA control conjugate AF4693, demonstrating the ability of the DBA domain to block receptor binding to IL-2 3x. [Table 22]

[0139] Example 15 Regulated IL-2 receptor signaling by PD-1 / IL-2 double-binding antibody (DBA) cytokine complexes in cellular assays This example describes PD-1-regulated IL-2 activity in HEK-Blue™ IL-2 reporter cells by PD-1 / IL-2 DBA-cytokine complexes. Anti-PD-1 / IL-2 DBA-cytokine complexes were analyzed along with appropriate uncontrolled controls such as anti-Her2, anti-PDL-1 or anti-IL-2 cytokine complexes. DBA-cytokine complexes and control antibody-cytokine complexes were produced in five formats shown in Figures 2B, 2D, 2E, 2G, 2H and 2I by expression in mammalian cells and purified using standard protocols. The peptide components of these complexes are outlined below in Table 23 (sequences of the individual components can be found in Table 2A). Cell-based reporter assays were performed for each of the five formats in the presence of various amounts of PD-1-Fc or hIgG1-Fc. [Table 23] TIFF2024544757000117.tif250167TIFF2024544757000118.tif42167

[0140] In the experiments shown in Figures 12A, 12B, 13A and 25, antibody-cytokine complexes were diluted with titrated concentrations of PD-1 Fc or IgG1 control Fc into wells of a 384-well TC-treated plate (Corning 3701) in complete DMEM (+10% FBS, 2 mM L-glutamine, sodium pyruvate) to a final concentration of 100 pM (a concentration previously shown to have a strong reporter signal for the always-on control, but little or no signal for the always-off control). After 15 minutes of incubation, HEK-Blue™ IL-2 reporter cells (12,500 cells) were added to each well and incubated overnight. Five microliters from each well were transferred to a new plate containing 45 microliters of QuantiBlue solution (Invivogen product no. rep-qbs). After 30 to 60 minutes, absorbance at 630 nm was determined using a Perkin-Elmer Envision.

[0141] In an alternative experimental format shown in Figure 13B, Figure 14A, Figure 14B, Figure 20 and Figure 22, wells of a 384-well ELISA plate were coated with a fixed concentration of PD-1-Fc or IgG1 Fc control protein captured with anti-Fc antibody (Jackson ImmunoResearch, product number 109-005-098). Cytokine complexes were serially diluted 1:4 for eight points in growth medium from a starting concentration of 6 nM and incubated briefly before addition of HEK-Blue™ IL-2 reporter cells.

[0142] The results of protein complexes containing the structure shown in Figure 2E are shown in Figure 12A and Figure 12B. As shown in Figure 2E, this symmetric format is composed of one IL-2 linked to each antibody heavy chain variable domain. IL-2 activity was increased in a dose-dependent manner by the addition of PD-1 Fc for DBA-cytokine complexes AF3247, AF3644, AF3651, AF3657 and AF3934, but not by the addition of hIgG1 Fc protein. IL-2 activity for the control anti-Her2 AF3243 and anti-IL-2 AF3246 monospecific antibody-cytokine complexes was not changed by the addition of PD-1 Fc protein. The symmetric format in Figure 12B is similar to the DBA-cytokine complex in Figure 12A, but IL-2 is conjugated to the heavy chain variable domain of AF3341 and the light chain variable domain of AF3345. Both symmetric formats show increased IL-2 activity upon addition of PD-1 Fc but not hIgG1 Fc.

[0143] Results for protein complexes containing the structure shown in Figure 2B are shown in Figures 13A and 13B. This format consists of an asymmetric complex composed of two antibody domains with a single IL-2 linked to one of the domains. In Figure 13A, IL-2 activity increased with the addition of PD-1 Fc in a dose-dependent manner, but not with the addition of IgG1 control Fc protein for DBA-cytokine complexes AF3232, AF3744 and AF3747. In Figure 13B, results are shown using asymmetric constructs in an alternative assay format in which PD-1 Fc or hIgG1 Fc is captured to the plate. DBA-cytokine complexes AF3946, AF3948, AF3952, AF3955 and AF3956 demonstrate increased IL-2 activity in wells coated with PD-1 compared to wells coated with hIgG1 Fc. The IL-2 activity of the control anti-PDL-1 monospecific antibody-cytokine complex AF3941 was not altered by the addition of PD-1 Fc protein.

[0144] The results for protein complexes containing the structure shown in Figure 2H are shown in Figures 14A and 14B. As shown in Figure 2H, these complexes are asymmetric and consist of two identical monospecific Fab arms with a single IL-2 attached to one Fc domain by a flexible linker and a single scFv attached to the other Fc domain by a flexible linker.

[0145] In Figure 14A, the PD-1 / IL-2 DBA complex is composed of an anti-PD-1 domain in the Fab arm (PD1-Nivolumab control) and a PD-1 / IL-2 DBA scFv in the Fc arm. The control antibody-cytokine complex is composed of the same anti-PD-1 domain in the Fab arm and a non-DBA scFv in the Fc arm. When titrated amounts of cytokine complex are added to cells, the PD-1 / IL-2 DBA-containing cytokine complexes AF4504 and AF4505 in the top graph show reduced reporter activation compared to equimolar amounts of the control anti-HER2 IL-2 immunocytokines AF4502 and AF4503. The same titrated amounts of cytokine complex were added to wells coated with PD-1 Fc or human IgG1 Fc control in the bottom graph. Both AF4504 and AF4505 demonstrate increased IL-2 activity in PD-1 coated wells compared to wells coated with human IgG1 Fc. The IL-2 activity of the control anti-HER2 IL-2 immunocytokines AF4502 and AF4503 was unchanged in wells coated with PD1 Fc compared to the hIgG1 Fc control. In Figure 14B, the PD-1 / IL-2 DBA complex is composed of a different anti-PD-1 domain (AB000881_PD1_control) in the Fab arm and a PD-1 / IL-2 DBA scFv on the Fc arm. The PD-1 / IL-2 DBA-containing cytokine complexes AF3913, AF3918, AF3923 and AF3927 demonstrate increased IL-2 activity in wells coated with PD-1 Fc compared to wells coated with hIgG1 Fc. The IL-2 activity for the anti-IL-2 non-DBA scFv control antibody-cytokine complex AF3864 was unchanged in wells coated with PD-1.

[0146] Results of protein complexes containing the structure shown in Figure 2G are shown in Figure 15. As shown in Figure 2G, this symmetric format is composed of one IL-2 linked to each antibody heavy chain variable domain of the Fab arm and one scFv attached to each Fc domain. The antibody-cytokine complex is composed of a PD-1 / IL-2 DBA in the Fab arm and an anti-PD-1 scFv (AB000880_PD1_4C10_control) on the Fc domain. The control antibody-cytokine complex is composed of the same anti-PD-1 domain in the Fc domain and a non-DBA in the Fab arm. AF3871 has a non-DBA anti-Her2 antibody on the Fab arm and AF3872 has a non-DBA anti-IL-2 antibody on the Fab arm. PD-1 / IL-2 DBA-containing complexes AF3873, AF3876 and AF3877 demonstrate increased IL-2 reporter activity in wells coated with PD-1 Fc compared to wells coated with hIgG1. IL-2 activity against two control antibody-cytokine conjugates, AF3871 and AF3872, was unchanged in wells coated with PD-1 Fc.

[0147] Results of protein complexes containing the structure shown in Figure 2I are shown in Figure 16. As shown in Figure 2I, these complexes are asymmetric, consisting of a PD-1 / IL-2 DBA in the Fab arm with a single IL-2 attached to one Fc domain by a flexible linker. The hinge region of the antibody is a hybrid of IgG1 and IgG3 hinge sequences with disulfide bridges removed to increase flexibility between the Fab arm and the IL-2 cytokine on the Fc domain. The control antibody-cytokine complex is composed of a monospecific anti-PD-1 domain in the Fab arm and the same IL-2 on the Fc domain. IL-2 activity was increased in a dose-dependent manner by the addition of PD-1 Fc, but not by the addition of hIgG1 Fc protein to the DBA-cytokine complex AF3634. The IL-2 activity of the control anti-PD-1 monospecific antibody-cytokine complex AF3632 was not altered by the addition of PD-1 Fc protein.

[0148] Results for protein complexes containing the structure shown in Figure 2H are shown in Figure 17. As shown in Figure 2H, and similar to the constructs in Figures 14A and 14B, these complexes are asymmetric and consist of two identical monospecific Fab arms with a single IL-2 attached to one Fc domain by a flexible linker and a single scFv attached to the other Fc domain by a flexible linker. Unlike the constructs in Figure 17, the Fc portion of Jijin is of human IgG1 isotype. The two constructs shown in Figure 17 consist of an anti-PD-1 domain (AB000881_PD1_control) in the Fab arm and a PD-1 / IL-2 DBA scFv on the Fc arm. Both PD-1 / IL2 DBA-containing cytokine complexes AF4892 and AF4893 demonstrate increased IL-2 activity in wells coated with PD-1 Fc compared to wells coated with mIgG2a Fc control.

[0149] Example 16 Regulated IL-2 3× receptor signaling by PD-1 / IL-2 3× dual binding antibody (DBA) cytokine complexes in vitro This example describes PD-1-regulated IL-2 3x (IL-2 variant with reduced binding to IL-2R alpha, Lombardi et al., 2017) activity in HEK-Blue™ IL-2 reporter cells by PD-1 / IL-2 3x DB-cytokine complexes. Anti-PD-1 / IL-2 3x DB-cytokine complexes were analyzed along with appropriate uncontrolled controls such as anti-Her2, anti-PD-1 or anti-IL-2 cytokine complexes. DB-cytokine complexes and control antibody-cytokine complexes were produced in two formats shown in Figure 2B and Figure 2H by expression in mammalian cells and purified using standard protocols. Cell-based reporter assays were performed for each of the two formats in the presence of plate-bound PD-1-Fc or hIgG1-Fc. 384-well ELISA plates (Corning 3700) were coated with 1 microgram / ml anti-Fc antibody (Jackson ImmunoResearch) in 100 mM bicarbonate solution pH 9.0 overnight at 4°C and washed twice with SuperBlock (ThermoFisher). PD1-Fc or IgG1 control was then added to each well at a constant concentration of 6 nM, incubated for 1 hour, and washed three times in PBS + 0.05% Tween 20 (PBST). Antibody-cytokine complexes were serially diluted 1:4 in 8 points from a starting concentration of 6 nM in complete DMEM (+ 10% FBS, 2 mM L-glutamine, sodium pyruvate). After 15 minutes of incubation, HEK-Blue™ IL-2 reporter cells (12,500 cells) were added to each well and incubated overnight. Five microliters from each well was transferred to a new plate containing 45 microliters of QuantiBlue solution (Invivogen product no. rep-qbs). After 30 to 60 minutes, absorbance at 630 nm was determined using a Perkin-Elmer Envision.

[0150] Results for protein complexes containing the structure shown in Figure 2B are shown in Figure 18. This format consists of an asymmetric complex composed of two antibody domains with a single IL-2 3x linked to one of the domains. DB-cytokine complexes AF4385, AF4386, AF4387, AF4388 and AF4389 demonstrate increased IL-2 3x activity in wells coated with PD-1 Fc compared to wells coated with hIgG1 Fc. IL-2 3x activity for the control anti-PD-1 monospecific antibody-cytokine complex AF4380 and anti-IL-2 monospecific antibody-cytokine complex AF4384 was not altered by the addition of PD-1 Fc protein.

[0151] The results of protein complexes containing the structure shown in Figure 2H are shown in Figures 19A, 19B, and 19C. As shown in Figure 2H, these complexes are asymmetric and consist of two identical monospecific Fab arms with a single IL-2 3x attached to one Fc domain by a flexible linker and a single scFv attached to the other Fc domain by a flexible linker.

[0152] In Figure 19A, the PD-1 / IL-2 DBA complex is composed of an anti-PD-1 domain (AB000694_nivo) in the Fab arm and a PD-1 / IL-2 DBA scFv in the Fc arm. The control antibody-cytokine complex is composed of the same anti-PD-1 domain in the Fab arm and a non-DBA scFv in the Fc arm. The PD-1 / IL-2 3xDBA containing cytokine complexes AF4404, AF4405, AF4695 and AF4696 demonstrate increased IL-2 3x activity in wells coated with PD-1 Fc compared to wells coated with hIgG1 Fc. The IL-2 3x activity for the anti-IL-2 non-DBA scFv control antibody-cytokine complex AF4401 and the anti-Her2 non-DBA scFv control antibody-cytokine complex AF4694 was unchanged in wells coated with PD-1 Fc. In Figure 19B, the PD-1 / IL-2 DBA complex is composed of a different anti-PD-1 domain (AB000880_PD1_R04_C10) in the Fab arm and a PD-1 / IL-2 DBA scFv on the Fc arm. The PD-1 / IL-2 3xDBA-containing cytokine complexes AF4413, AF4414, AF4415, and AF4416 demonstrate increased IL-2 3x activity in wells coated with PD-1 Fc compared to wells coated with hIgG1 Fc. The IL-2 3x activity for the anti-IL-2 non-DBA scFv control antibody-cytokine complex AF4412 was unchanged in wells coated with PD-1 Fc. In Figure 19C, the PD-1 / IL-2 DBA complex is composed of an anti-PD-1 domain in the Fab arm that does not block PDL-1 binding to PD-1, nor does it block nivolumab binding to PD-1. Both PD-1 / IL-2 DBA-containing conjugates AF4771 and AF4773 demonstrate increased IL-2 3x activity in wells coated with PD-1 Fc compared to wells coated with hIgG1 Fc. [Table 24] TIFF2024544757000120.tif146163

[0153] Example 17 Regulated IL-2 receptor signaling by PD-1 / IL-2 dual-binding antibody (DBA) cytokine complexes with variable linker lengths in cells This example describes PD-1-regulated IL-2 activity in a HEK-Blue™ IL-2 reporter cell model by PD-1 / IL-2 DB-cytokine complexes with various linker lengths. DB-cytokine complexes were produced in the format shown in FIG. 2B, where the glycine-serine (GS) linker connecting the IL-2 cytokine to the DBA domain was varied from 5 GS repeats to 25 GS repeats. DB-cytokine complexes were expressed in mammalian cells and purified using standard protocols. Cell-based reporter assays were performed in the presence of plate-bound PD-1-Fc or hIgG1-Fc. In this example, 384-well ELISA plates (Corning 3700) were coated overnight at 4° C. with 1 microgram / ml anti-Fc antibody (Jackson ImmunoResearch) in 100 mM bicarbonate solution pH 9.0 and washed twice with SuperBlock (ThermoFisher). PD1-Fc or IgG1 control was then added to each well at a constant concentration of 6 nM, incubated for 1 hour, and washed three times in PBS + 0.05% Tween 20 (PBST). Antibody-cytokine complexes were serially diluted 1:4 in 8 points from a starting concentration of 6 nM in complete DMEM (+ 10% FBS, 2 mM L-glutamine, sodium pyruvate). After 15 minutes of incubation, HEK-Blue™ IL-2 reporter cells (12,500 cells) were added to each well and incubated overnight. Five microliters from each well were transferred to a new plate containing 45 microliters of QuantiBlue solution (Invivogen product no. rep-qbs). After 30 to 60 minutes, absorbance at 630 nm was determined using a Perkin-Elmer Envision.

[0154] Results with the protein complexes shown in Figure 2B with various linker lengths are shown in Figure 20. Asymmetric complexes consist of two antibody domains with a single IL-2 linked to one of the domains. Various linker lengths were selected from GS5 to GS25 to test the linker length dependency on PD-1 regulation. Cytokine complexes containing the PD-1 / IL-2 DBA domain 2B07 variant in AF4262, AF4273, AF4284 and AF4295 all demonstrated increased IL-2 activity in wells coated with PD-1 Fc compared to wells coated with hIgG1 Fc. Similar results were observed in cytokine complexes containing the PD-1 / IL-2 DBA domain 7A04 variant in AF4265, AF4276, AF4287 and AF4298. These data demonstrate that PD-1 regulation is possible with multiple cytokine antibody linker lengths ranging from GS5 to GS25.

[0155] Example 18 PD-1-dependent induction of STAT5 phosphorylation by PD-1 / IL-2 DB-cytokine complexes in human primary CD8+ T cells This example describes PD-1 / IL-2 DB-cytokine complex induction of STAT5 phosphorylation in primary human CD8+ T cells. DB-cytokine complexes were produced in the format shown in Figure 2H. CD8+ T cells were isolated from human PBMCs using immunomagnetic negative selection (STEMCELL) and stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 72 hours to induce PD-1 expression. Stimulated CD8+ T cells were incubated with anti-PD-1 blocking antibody or isotype control antibody for 1 hour. Titrated concentrations of PD-1 / IL-2 DBA cytokine complex (PD-1 controlled IL-2) or anti-HER2 / IL-2 cytokine complex (always-on IL-2) were then added to the CD8+ T cells and incubated for 20 minutes at 37°C. CD8+ T cells were fixed with Perm Buffer III (BD Biosciences), washed, and stained with antibodies against CD8, CD45RA, CD45RO, and pSTAT5. STAT5 phosphorylation in CD45RA+ and CD45RO+ T cell populations was assessed by flow cytometry. The results of this experiment are shown in Figure 21A and Figure 21B. In CD8+CD45RA+ T cells, which are mostly PD-1 negative, the PD-1 / IL-2 DBA cytokine complex induces a lower frequency of STAT5 phosphorylation-positive CD8+ T cells compared to the uncontrolled anti-HER2 / IL-2 cytokine complex control. In CD8+CD45RO+ T cells, which are mostly PD-1 positive, the PD-1 / IL-2 DBA cytokine complex induces a similar frequency of STAT5 phosphorylation-positive CD8+ T cells compared to the uncontrolled anti-HER2 / IL-2 cytokine complex control. Furthermore, CD8+CD45RO+ T cells pretreated with anti-PD-1 blocking antibody had a lower frequency of STAT5 phosphorylation-positive cells after treatment with the PD-1 / IL-2 DBA cytokine complex, indicating the dependence of activity on PD-1 binding. Taken together, these data indicate that the PD-1 / IL-2 DBA cytokine complex exhibits reduced activity on PD-1-negative cells. In PD-1-positive cells, PD-1 / IL-2 DBA cytokine complex activity is reduced by PD-1 blockade.

[0156] Example 19 PD-1 / IL-2 DB-cytokine complex regulation of human T cell activation in the mixed lymphocyte reaction This example describes PD-1 / IL-2 DB-cytokine complex regulation of human CD4+ T cell activation in a mixed lymphocyte reaction (MLR) model. In this model, we evaluated the activation of T cells against foreign antigen-presenting cells and the ability of our immunocytokine constructs to regulate their activation. CD4+CD25- T cells were isolated from human PBMCs using immunomagnetic negative selection (STEMCELL) and labeled with CellTrace Violet proliferation dye (ThermoFisher) according to the manufacturer's protocol. To generate monocyte-derived dendritic cells (MDDCs), monocytes were isolated from PBMCs of different donors using immunomagnetic negative selection (STEMCELL) and cultured in the presence of GM-CSF (100ng / mL) and IL-4 (50ng / mL). The medium was changed after 3 days and MDDCs were collected on day 7. 10,000 MDDCs were added to each well of a 96-well round-bottom plate, followed by the addition of 50,000 proliferation dye-labeled CD4+ T cells. A dilution series of each immunocytokine complex was generated and added to the cell culture. After 5 days at 37°C, cells were stained with live / dead viability dye (ThermoFisher) and then incubated in fixation / permeabilization buffer (BD Biosciences) for 20 minutes at 4°C. Cells were then stained with fluorophore-conjugated antibodies against CD4 and Granzyme B (BD Bioscience), and the frequency of Granzyme B-expressing cells among proliferating CD4+ T cells was assessed by flow cytometry. The results of this experiment are shown in Figure 22A and Figure 22B. Although HER2-IL2 induced minimal specific activation of T cells (as expected, since T cells do not carry HER2 molecules), uncontrolled and controlled PD1-IL2 constructs were able to stimulate T cell activation, as indicated by dose-dependent Granzyme B expression. These data demonstrate the PD-1 binding-dependent activity of regulated PD1-IL2 complexes (through their ability to activate T cells). Moreover, the degree of granzyme B induction observed was comparable between regulated and unregulated PD1-IL2 complexes, indicating that the addition of a regulating moiety to IL2 does not reduce the activity of IL2 under appropriately permissive conditions.

[0157] Example 20 PD-1 / IL-2 DB-cytokine complex regulation of antitumor immunity in syngeneic tumor models This example describes PD-1 / IL-2 DB-cytokine complex modulation of anti-tumor immunity in the MC38 syngeneic mouse tumor model. PD-1 / IL-2 DB-cytokine complexes were evaluated for their ability to drive anti-tumor immunity in vivo. 500,000 MC38 tumor cells were implanted subcutaneously into human PD-1 knock-in mice (GenOway). Tumors were measured twice weekly and volume was calculated as (length x width x width / 2). Mice were randomized into treatment groups and were randomized until tumors reached approximately 100 mm 3 Treatment was initiated when tumors reached a volume of 1000 mg / kg. Mice were treated intravenously with PD-1 / IL-2 DB-cytokine complex, uncontrolled anti-PD1-IL2, anti-HER2-IL2, anti-PD-1, or anti-HER2 at 0.5 milligrams per kilogram of body weight on days 7, 10, and 13 after tumor implantation. The results of this experiment are shown in Figures 23A and 23B. The PD-1 / IL-2 DB-cytokine complex showed comparable tumor growth inhibition compared to uncontrolled PD1-IL2 and superior tumor growth inhibition compared to anti-PD1 and anti-HER antibodies.

[0158] Example 21 PD1-IL2 Augmentation of T Cell Bispecific Engager Activity (Hypothetical Example) PD1-regulated IL2 can be used to generate PD-1-regulated immunocytokines that enhance the activity of T cell bispecific antibodies (TCBs). In this example, PD1-IL2 TCBs can be generated in which PD1-IL2 DBA scFv is fused to the C-terminus of one heavy chain and an IL-2 variant is fused to the C-terminus of the opposing heavy chain of the TCB. The N-terminal variable region of PD1-IL2 TCB can be directed against CD3 and tumor-associated antigens such as PSMA, HER2, CD20, or against CD3 and an unrelated antigen. To evaluate PD1-IL2 TCB activity, human T cells are isolated from fresh PBMCs and co-cultured with tumor cell lines expressing various levels of tumor-associated antigens. Naked TCB or titrated concentrations of PD1-IL2 TCB are added to the T cell:tumor cell co-cultures. Tumor killing as well as T cell activation and cytokine production are evaluated at various time points.

[0159] Example 22 PD-1-regulated IL-2 activity targeting T cell-associated antigens (hypothetical example) PD-1 regulated immunocytokines can be generated in which PD1 regulated IL2 is targeted to T cells using any T cell marker. For example, PD1-IL2 DBA scFv can be fused to the C-terminus of one heavy chain, and IL-2 variants can be fused to the C-terminus of the opposing heavy chain of an antibody against a T cell expressed marker, including but not limited to CD28, CD28H, OX40, GITR, CD137, CD27, HVEM, CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, TIGIT, CD244, ICOS, CD40L, CD4, CD8, KLRG1, FasL and CD7. T cells can be activated under various conditions to induce expression of a given T cell marker. Then titrations of PD1-IL2 DB containing immunocytokines against the marker of interest or an unrelated marker can be added. STAT5 phosphorylation can then be evaluated as a measure of target IL-2 activity. In some experiments, blocking antibodies against the marker of interest may be added prior to treatment with the PD1-IL2 DB-containing immunocytokine to demonstrate specificity.

[0160] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and alternatives will occur to those skilled in the art without departing from the present disclosure. It will be understood that various alternatives to the embodiments of the present disclosure described herein may be used in practicing the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. A complex, (a) a therapeutic domain comprising an IL-2 peptide; (b) a linker; and (c) a sensor domain comprising an antibody, wherein the sensor domain is configured to bind to PD-1 and IL-2 in a mutually exclusive manner; and Including, The conjugate, wherein the therapeutic domain is linked to the sensor domain by the linker.

2. (a) the sensor domain is configured to (i) bind to IL-2 in the absence of PD-1, and (ii) not bind to IL-2 in the presence of PD-1. (b) the antibody is an antibody fragment or an antibody derivative; (c) the sensor domain comprises a single double binding antibody (DBA) configured to bind to PD-1 and IL-2; (d) the complex comprises an Fc domain, and / or (e) the IL-2 peptide comprises a wild-type human IL-2 peptide; The composite of claim 1.

3. The method of claim 2, wherein the sensor domain comprises a single DBA configured to bind to PD-1 and IL-2; (a) the DBA comprises a heavy chain complementarity determining region (CDR) 3 having at least 80% identity to any one of SEQ ID NOs: 11-20, 154-156, 168-173, 114-119, 415, 421, 433, 439, 445, 451, 457, 463, 469, 475, 481, 487, 493, 499, 505, 511, 517, 523, 529, 535, 541, 547, 553, 559, 565, 571, 577, 583, 589, 595, 601, 607, 613, 619, 625, 631, 637, 643, 649, 655, 661, or 667; (b) the DBA comprises a heavy chain CDR1, CDR2 or CDR3 comprising a sequence having at least 80% identity to any of the sequences listed in Table 3, Table 7, Table 8 and Table 19; and / or (c) the DBA comprises a heavy chain variable domain (VH) and / or a light chain variable domain (VL) comprising a sequence having at least 80% identity to any of the sequences listed in Table 18; The composite of claim 1.

4. The complex described in claim 2, wherein the complex comprises an Fc domain and the Fc domain is a homodimer or a heterodimer.

5. The complex described in claim 4, wherein the Fc domain is a heterodimer and comprises (a) a first polypeptide comprising a knob mutation and (b) a second polypeptide comprising a hole mutation. (a) the knob mutation and the hole mutation each comprise a mutation in any one of the following pairs of residues for IgG: (i) 366 and 407, (ii) 405 and 394, or (iii) 407 and 366; and / or (b) the knob mutation comprises an arginine, phenylalanine, tyrosine, or tryptophan residue, and the hole mutation comprises an alanine, serine, threonine, or valine residue; The complex of claim 5.

7. the complex comprises a sensor domain comprising a full-length DBA; (a) the IL-2 peptide is linked to the N-terminus of the heavy chain of the full-length DBA, or the IL-2 peptide is linked to the N-terminus of the light chain of the full-length DBA, or (b) the IL-2 peptide is linked to the C-terminus of the heavy chain of the full-length DBA; The composite of claim 1.

8. The complex is (a) N-[IL-2]-[linker]-[V Ｈ ]-[C Ｈ a first polypeptide consisting of: N-[V Ｌ ]-[C Ｌ ]-C, and a second polypeptide Contains, or (b) N-[V Ｈ ]-[C Ｈ a first polypeptide consisting of: N-[IL-2]-[linker]-[V Ｌ ]-[C Ｌ ]-C, and a second polypeptide Including, In the formula, N- represents the N-terminus of the peptide, C- represents the C-terminus of the peptide, [linker] represents the linker, and V Ｈ represents the heavy chain variable domain of the DBA, and C Ｈ indicates the immunoglobulin heavy chain constant domain, V Ｌ represents the light chain variable domain of the DBA, [hinge] represents the hinge region of an immunoglobulin, Fc represents the Fc region of an immunoglobulin, and C Ｌ The conjugate of claim 1 , wherein represents an immunoglobulin light chain constant domain.

9. 9. The conjugate of claim 8, wherein the conjugate comprises any one of AF003345, AF003243, AF003246, AF003247, AF003341, AF003644, AF003651, AF003657, or AF003934.

10. The complex is (a) N-[IL-2]-[linker]-[V Ｈ ]-[C Ｈ ]-[hinge]-Fc[knob]-C; and N-[V Ｌ ]-[C Ｌ ]-C, and N-[V Ｈ ]-[C Ｈ a third polypeptide consisting of: Contains, or (b) N-[IL-2]-[linker]-[V Ｈ ]-[C Ｈ a first polypeptide consisting of: N-[V Ｌ ]-[C Ｌ ]-C, and N-[V Ｈ ]-[C Ｈ a third polypeptide consisting of Fc[knob]-[hinge]-Fc[knob]-C; Including, In the formula, N- represents the N-terminus of the peptide, C- represents the C-terminus of the peptide, [linker] represents the linker, and V Ｈ represents the heavy chain variable domain of the DBA, and C Ｈ indicates the immunoglobulin heavy chain constant domain, V Ｌ represents the light chain variable domain of the DBA, [hinge] represents the hinge region of an immunoglobulin, Fc[knob] represents the Fc of an immunoglobulin containing a knob mutation, Fc[hole] represents the Fc region of an immunoglobulin containing a hole mutation, and C Ｌ The conjugate of claim 1 , wherein represents an immunoglobulin light chain constant domain. (a) the knob mutation and the hole mutation each comprise a mutation in any one of the following pairs of residues for IgG: (i) 366 and 407, (ii) 405 and 394, or (iii) 407 and 366; (b) the knob mutation comprises an arginine, phenylalanine, tyrosine, or tryptophan residue and the hole mutation comprises an alanine, serine, threonine, or valine residue; and / or (c) the complex comprises any one of AF003229, AF003230, AF003232, AF003740, AF003747, AF003749, AF003753, AF003945, AF003947, AF003951, AF003952, AF003953, AF003955, AF003956, or AF003941; The composite of claim 10.

12. The complex is (a) N-[IL-2]-[linker]-[V Ｈ ]-[C Ｈ a first polypeptide of the formula: (b) N-[V Ｌ ]-[C Ｌ ]-C, and a second polypeptide Contains, or In the formula, N- represents the N-terminus of the peptide, C- represents the C-terminus of the peptide, [linker] represents the linker, and V Ｈ indicates the heavy chain variable domain of an anti-PD-1 monospecific antibody, and C Ｈ indicates the immunoglobulin heavy chain constant domain, V Ｌ represents the light chain variable domain of an anti-PD-1 monospecific antibody, [hinge] represents the hinge region of an immunoglobulin, Fc represents the Fc region of an immunoglobulin, and C Ｌ represents the light chain constant domain of an immunoglobulin, and [scFv] represents the V of the DBA. Ｈ Domains and V Ｌ The complex of claim 1, which represents an scFv comprising a domain. (a) The scFv is N-[V Ｈ ]-[Linker 2]-[V Ｌ ]-C; (b) the scFv comprising the V H and V L domains of the DBA comprises: (i) a VH domain comprising a sequence having at least 80% identity to the VH domain of any one of AB002022_2B07v1, AB002328_2B07v4, AB002360_7A04v1, AB002413_2A11v3, AB002342_2B07v5, AB002345_2B07v6, and AB002365_7A04v2; and / or (ii) a V L domain comprising a sequence having at least 80% identity with the V L domain of any one of AB002022 2B07v1, AB002328 2B07v4, AB002360 7A04v1, AB002413 2A11v3, AB002342 2B07v5, AB002345 2B07v6, and AB002365 7A04v2. Including, (c) the scFv comprising the V H and V L domains of the DBA is (i) the heavy chain CDRs of any one of AB002022_2B07v1, AB002328_2B07v4, AB002360_7A04v1, AB002413_2A11v3, AB002342_2B07v5, AB002345_2B07v6, and AB002365_7A04v2; and / or (ii) the light chain CDRs of any one of AB002022_2B07v1, AB002328_2B07v4, AB002360_7A04v1, AB002413_2A11v3, AB002342_2B07v5, AB002345_2B07v6, and AB002365_7A04v2. and / or (d) the complex comprises any one of AF003864, AF003871, AF003872, AF003913, AF003918, AF003923, AF003927, AF004502, AF004503, AF004504, AF004505, AF004892, and AF004893; The composite of claim 12.

14. The complex is (a) N-[V Ｈ ]-[C Ｈ a first polypeptide consisting of: Fc[knob]-[hinge]-Fc[knob]-[linker]-[IL-2]-C; N-[V Ｌ ]-[C Ｌ ]-C, and N-[V Ｈ ]-[C Ｈ a third polypeptide consisting of Fc[hole]-[linker]-[scFv]-C; Contains, or (b) N-[V Ｈ ]-[C Ｈ a first polypeptide consisting of: Fc[hole]-[linker]-[IL-2]-C; N-[V Ｌ ]-[C Ｌ ]-C, and N-[V Ｈ ]-[C Ｈ a third polypeptide consisting of Fc[knob]-[hinge]-Fc[knob]-[linker]-[scFv]-C; Including, In the formula, N- represents the N-terminus of the peptide, C- represents the C-terminus of the peptide, [linker] represents the linker, and V Ｈ represents the heavy chain variable domain of the DBA, and C Ｈ indicates the immunoglobulin heavy chain constant domain, V Ｌ represents the light chain variable domain of the DBA, [hinge] represents the hinge region of an immunoglobulin, Fc[knob] represents the Fc of an immunoglobulin containing a knob mutation, Fc[hole] represents the Fc region of an immunoglobulin containing a hole mutation, and C Ｌ The complex of claim 1, wherein [scFv] represents the light chain constant domain of an immunoglobulin and [scFv] represents the scFv of the DBA.

15. 15. The conjugate of claim 14, wherein the conjugate comprises any one of AF004693, AF004695, AF004696, AF005416, AF005418 and AF005419.

16. The complex is (a) N-[V Ｈ ]-[C Ｈ a first polypeptide consisting of: Fc[knob]-[het-hinge]-Fc[knob]-[linker]-[IL-2]-C; N-[V Ｌ ]-[C Ｌ ]-C, and N-[V Ｈ ]-[C Ｈ a third polypeptide consisting of: Contains, or (b) N-[V Ｈ ]-[C Ｈ a first polypeptide consisting of: Fc[hole]-[het-hinge]-Fc[hole]-[linker]-[IL-2]-C; N-[V Ｌ ]-[C Ｌ ]-C, and N-[V Ｈ ]-[C Ｈ a third polypeptide consisting of: Including, In the formula, N- represents the N-terminus of the peptide, C- represents the C-terminus of the peptide, [linker] represents the linker, and V Ｈ represents the heavy chain variable domain of the DBA, and C Ｈ indicates the immunoglobulin heavy chain constant domain, V Ｌ represents the light chain variable domain of the DBA, [het hinge] represents a hinge region heterologous to the Fc region, Fc[knob] represents the Fc of an immunoglobulin containing a knob mutation, Fc[hole] represents the Fc region of an immunoglobulin containing a hole mutation, and C Ｌ The conjugate of claim 1 , wherein represents an immunoglobulin light chain constant domain. (a) the hinge region heterologous to the Fc region is (i) a hinge region derived from an IgG3 antibody, or (ii) a G4S-based linker; and / or (b) the complex comprises AF003632 or AF003634; The composite of claim 16.

18. 3. The conjugate of claim 2, wherein the IL-2 peptide comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or substantially 100% sequence identity to human IL-2.

19. The complex may be any of AF003232, AF003243, AF003246, AF003247, AF003341, AF003345, AF003632, AF003634, AF003644, AF003651, AF003652, AF003653, AF003657, AF003740, AF003744, AF003747, AF003749, AF00 03753, AF003864, AF003873, AF003876, AF003877, AF003913, AF003918, AF003923, AF003927, AF00 3930, AF003931, AF003933, AF003934, AF003935, AF003941, AF003945, AF003946, AF003947, AF003 948, AF003951, AF003952, AF003953, AF003955, AF003956, AF004262, AF004265, AF004273, AF004 276, AF004284, AF004287, AF004295, AF004298, AF004385, AF004386, AF004387, AF004388, AF0043 89, AF004404, AF004405, AF004413, AF004414, AF004415, AF004416, AF004504, AF004505, AF004693, AF004695, AF004696, AF004771, AF004773, AF004892, and AF004893.

20. A pharmaceutical for enhancing T cell reactivity against xenogeneic cells in a subject in need thereof, comprising the conjugate of any one of claims 1 to 19.

21. The pharmaceutical according to claim 20 , wherein the heterologous cells are cancer cells.

22. A pharmaceutical for treating a subject in need thereof, comprising a complex described in any one of claims 1 to 19.

23. (a) The pharmaceutical is formulated for intravenous, intramuscular, or subcutaneous administration; (b) the subject in need thereof has cancer; (c) the therapeutic domain treats the subject in need thereof; and / or (d) the subject in need of treatment is a mammal. The pharmaceutical composition of claim 22.

24. The method of claim 23, wherein the subject in need of treatment is a human.

25. A composition comprising a recombinant nucleic acid encoding the complex of any one of claims 1 to 19.

26. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 19 and a pharmaceutically acceptable additive.

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