PD-1-modulated IL-2 immune cytokine and its use

Abstract

The present invention provides PD-1 modulated IL-2 immunoconjugates and compositions thereof. The present invention also features polynucleotides, vectors, host cells, production methods, pharmaceutical compositions, methods for treating diseases or disorders such as cancer, compositions for related applications and uses, and kits for use with one or more of the methods.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in XML format, the entirety of which is incorporated herein by reference. The XML copy, created on 2 May 2024, is named "51177-047WO2_Sequence_Listing_5_2_24" and has a size of 197,852 bytes. [Background technology]

[0002] background Interleukin-2 (IL-2) is a potent cytokine that exhibits toxicity when administered systemically. While it can be delivered systemically, there is a need for a version of IL-2 that can be controlled to exhibit therapeutic activity against a specific subset of T cells. [Overview of the project]

[0003] overview The present invention provides, in particular, PD-1-regulated IL-2 immune complexes (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, and an anti-PD-1 antibody moiety that binds to PD-1) and / or antibodies (e.g., comprising two DBA moieties or two anti-PD-1 antibody moieties), compositions comprising the immune complexes and / or antibodies (e.g., pharmaceutical compositions), polynucleotides encoding the immune complexes and / or antibodies, vectors, host cells, methods of production, and methods and uses thereof.

[0004] In one embodiment, the present invention relates to an immune complex comprising: (a) a first binding domain comprising (i) an IL-2 polypeptide, (ii) a linker, and (iii) a double-binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the IL-2 polypeptide, linker, and DBA moiety are oriented as follows: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide, and -C represents the polypeptide) The first binding domain is connected at the C-terminus of (i) when the DBA portion is bound to the IL-2 polypeptide, the binding of the IL-2 polypeptide to its receptor is substantially blocked, and (ii) when DBA is bound to PD-1, the binding of the DBA portion to the IL-2 polypeptide is substantially blocked, and the IL-2 polypeptide can bind to the IL-2 receptor; and (b) the second binding domain comprises an anti-PD-1 antibody portion including VH and VL.

[0005] In some embodiments, the anti-PD-1 antibody portion does not substantially bind to the IL-2 polypeptide.

[0006] In some embodiments, the DBA portion comprises a Fab molecule. In some embodiments, the DBA portion comprises a Fab heavy chain comprising the VH and heavy chain constant domain 1 (CH1) of the DBA portion, and a Fab light chain comprising the VL and light chain constant domain (CL) of the DBA portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other. In some embodiments, the DBA portion is a conventional Fab molecule.

[0007] In some embodiments, the anti-PD-1 antibody moiety comprises a Fab molecule. In some embodiments, the anti-PD-1 antibody moiety comprises a Fab heavy chain comprising VH and CH1 of the anti-PD-1 antibody moiety and a Fab light chain comprising VL and CL of the anti-PD-1 antibody moiety, wherein VH of the Fab heavy chain and VL of the Fab light chain are substituted for each other, or CH1 of the Fab heavy chain and CL of the Fab light chain are substituted for each other.

[0008] In some embodiments, (a) the DBA portion is a conventional Fab molecule, and (b) the anti-PD-1 antibody portion is a Fab molecule comprising a Fab heavy chain containing VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other.

[0009] In some embodiments, the Fab molecule is such that (a) the DBA portion is a conventional Fab molecule, and (b) the anti-PD-1 antibody portion comprises a Fab heavy chain containing VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other.

[0010] In some embodiments, the anti-PD-1 antibody portion is a conventional Fab molecule.

[0011] In some embodiments, (a) the DBA portion is a Fab molecule comprising a Fab heavy chain containing VH and CH1 of the DBA portion and a Fab light chain containing VL and CL of the DBA portion, wherein VH of the Fab heavy chain and VL of the Fab light chain are substituted for each other, or CH1 of the Fab heavy chain and CL of the Fab light chain are substituted for each other, and (b) the anti-PD-1 antibody portion is a conventional Fab molecule.

[0012] In some embodiments, (a) the DBA portion is a Fab molecule comprising a Fab heavy chain containing VH and CH1 of the DBA portion and a Fab light chain containing VL and light chain CL of the DBA portion, wherein CH1 of the Fab heavy chain and CL of the Fab light chain are substituted for each other, and (b) the anti-PD-1 antibody portion is a conventional Fab molecule.

[0013] In some embodiments, the anti-PD-1 antibody portion is a single-stranded variable fragment (scFv).

[0014] In some embodiments, the anti-PD-1 antibody is an scFv in which VH and VL are connected in the following orientation: N-VH-VL-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide). In other embodiments, the anti-PD-1 antibody is an scFv in which VH and VL are connected in the following orientation: N-VL-VH-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide). In some embodiments, the VH and VL of the scFv are connected by a linker. In some embodiments, VH, VL, and the linker are connected in the following orientation: N-VH-linker-VL-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide). In another embodiment, the anti-PD-1 antibody is scFv in which VH and VL are connected in the following orientation: N-VL-linker-VH-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0015] In some embodiments, the immune complex further comprises an Fc domain containing a first subunit and a second subunit. In some embodiments, (a) the DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and / or (b) the anti-PD-1 antibody portion and the second subunit are connected in the following orientation: N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0016] In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the IgG Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain.

[0017] In some embodiments, the first subunit comprises one or more CH domains selected from a first CH2(CH21) domain and / or a first CH3(CH31) domain, and the second subunit comprises one or more CH domains selected from a second CH2(CH22) domain and / or a second CH3(CH32) domain. In some embodiments, at least one of the one or more CH domains is paired with another CH domain. In some embodiments, the CH31 domain and the CH32 domain each comprise a projection or cavity, and the projection or cavity of the CH31 domain can be positioned in the cavity or projection of the CH32 domain, respectively. In some embodiments, the CH31 domain and the CH32 domain meet at the interface between the projection and the cavity. In some embodiments, the CH21 domain and the CH22 domain each comprise a projection or cavity, and the projection or cavity of the CH21 domain can be positioned in the cavity or projection of the CH22 domain, respectively. In some embodiments, the CH21 and CH22 domains associate at the interface between the protrusion and the cavity.

[0018] In some embodiments, (a) the first subunit contains a tryptophan residue at position 366, and the second subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index), or (b) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (numbered according to the Kabat EU index). In some embodiments, the first subunit and / or the second subunit contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbered according to the Kabat EU index).

[0019] In some embodiments, the DBA moiety and the anti-PD-1 antibody moiety bind to different epitopes of PD-1. In some embodiments, the DBA moiety and the anti-PD-1 antibody moiety bind to the same epitope of PD-1.

[0020] In some embodiments, binding of the DBA portion to PD-1 inhibits the binding of PD-1 to PD-L1, and / or binding of the anti-PD-1 antibody portion to PD-1 inhibits the binding of PD-1 to PD-L1. In some embodiments, binding of the DBA portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, or binding of the anti-PD-1 antibody portion to PD-1 does not inhibit the binding of PD-1 to PD-L1. In some embodiments, (a) binding of the DBA portion to PD-1 inhibits the binding of PD-1 to PD-L1, and binding of the anti-PD-1 antibody portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, or (b) binding of the DBA portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, and binding of the anti-PD-1 antibody portion to PD-1 inhibits the binding of PD-1 to PD-L1. In some embodiments, binding of the DBA portion to PD-1 inhibits the binding of PD-1 to PD-L1, and binding of the anti-PD-1 antibody portion to PD-1 inhibits the binding of PD-1 to PD-L1. In some embodiments, binding of the DBA portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, and binding of the anti-PD-1 antibody portion to PD-1 does not inhibit the binding of PD-1 to PD-L1.

[0021] In some embodiments, the DBA portion comprises the following six complementarity-determining regions (CDRs): (a) CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and QQYNSFPVT (SEQ ID NO: 57). (b) CDR-L3 containing an amino acid sequence; (b) CDR-H1 containing the amino acid sequence of AYYIH (SEQ ID NO: 82), CDR-H2 containing the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 containing the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), CDR-L1 containing the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 containing the amino acid sequence of GASSLQS (SEQ ID NO: 74), and CDR-L3 containing the amino acid sequence of QESYTSSNT (SEQ ID NO: 75).

[0022] In some embodiments, the DBA portion is (a) a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 58; (b) a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 76; or (c) a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 91; or (d) a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 91.

[0023] In some embodiments, the DBA portion is (a) a VH containing the amino acid sequence of SEQ ID NO: 67 and a VL containing the amino acid sequence of SEQ ID NO: 58; (b) a VH containing the amino acid sequence of SEQ ID NO: 85 and a VL containing the amino acid sequence of SEQ ID NO: 76; or (c) a VH containing the amino acid sequence of SEQ ID NO: 111 and a VL containing the amino acid sequence of SEQ ID NO: 91; or (d) a VH containing the amino acid sequence of SEQ ID NO: 85 and a VL containing the amino acid sequence of SEQ ID NO: 91.

[0024] In some embodiments, the anti-PD-1 antibody portion comprises the following six CDRs: (a) CDR-H1 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21); (b) CDR-H1 containing the amino acid sequence of SYAMS (SEQ ID NO: 46), CDR-H2 containing the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), GEGYAGSSYFRASDI ( (c) CDR-H3 containing the amino acid sequence of sequence number 48), CDR-L1 containing the amino acid sequence of RASQSISSYLN (sequence number 37), CDR-L2 containing the amino acid sequence of TASSLQS (sequence number 38), and CDR-L3 containing the amino acid sequence of QQSYSTPLT (sequence number 39); or (c) CDR-H1 containing the amino acid sequence of SYWMS (sequence number 10), CDR-H2 containing the amino acid sequence of AISGSGGSRYYAESVKG (sequence number 11), CDR-H3 containing the amino acid sequence of SPLQWIDV (sequence number 12), CDR-L1 containing the amino acid sequence of RASQGISSWLA (sequence number 1), CDR-L2 containing the amino acid sequence of EASSLQS (sequence number 2), and CDR-L3 containing the amino acid sequence of QQANQFPFT (sequence number 3).

[0025] In some embodiments, the anti-PD-1 antibody portion includes (a) a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (b) a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 40; or (c) a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 13 and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 4.

[0026] In some embodiments, the anti-PD-1 antibody portion comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 31 and / or a VL containing the amino acid sequence of SEQ ID NO: 22, (b) a VH containing the amino acid sequence of SEQ ID NO: 49 and / or a VL containing the amino acid sequence of SEQ ID NO: 40, or (c) a VH containing the amino acid sequence of SEQ ID NO: 13 and / or a VL containing the amino acid sequence of SEQ ID NO: 4.

[0027] In some embodiments, (a)(i) the DBA portion includes CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and CDR-L3 containing the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57). (ii) The anti-PD-1 antibody portion consists of CDR-H1 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21). (b)(i) The DBA portion includes CDR-H1 containing the amino acid sequence of AYYIH (SEQ ID NO: 82), CDR-H2 containing the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 containing the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), CDR-L1 containing the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 containing the amino acid sequence of GASSLQS (SEQ ID NO: 74), and CDR-L3 containing the amino acid sequence of QESYTSSNT (SEQ ID NO: 75), and ( ii) The anti-PD-1 antibody portion comprises CDR-H1 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21);Or (c)(i) The DBA portion includes CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and CDR-L3 containing the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57), and (ii) The anti-PD-1 antibody portion includes CDR-H1 containing the amino acid sequence of SYAMS (SEQ ID NO: 46), CDR-H2 containing the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), CDR-H3 containing the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO: 48), CDR-L1 containing the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), CDR-L2 containing the amino acid sequence of TASSLQS (SEQ ID NO: 38), and CDR-L3 containing the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39).

[0028] In some embodiments, (a)(i) the DBA portion includes a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion includes a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (b)(i) the DBA portion includes a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103 and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion includes a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22 (c)(i) The DBA portion includes a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion includes a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (d)(i) The DBA portion includes a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion includes a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40.

[0029] In some embodiments, (a)(i) the DBA portion comprises VH containing the amino acid sequence of SEQ ID NO: 67 and VL containing the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises VH containing the amino acid sequence of SEQ ID NO: 31 and VL containing the amino acid sequence of SEQ ID NO: 22; (b)(i) the DBA portion comprises VH containing the amino acid sequence of SEQ ID NO: 103 and VL containing the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises VH containing the amino acid sequence of SEQ ID NO: 31 and VL containing the amino acid sequence of SEQ ID NO: 22 (c)(i) The DBA portion includes a VH containing the amino acid sequence of SEQ ID NO: 111 and a VL containing the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion includes a VH containing the amino acid sequence of SEQ ID NO: 31 and a VL containing the amino acid sequence of SEQ ID NO: 22; (d)(i) The DBA portion includes a VH containing the amino acid sequence of SEQ ID NO: 67 and a VL containing the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion includes a VH containing the amino acid sequence of SEQ ID NO: 49 and a VL containing the amino acid sequence of SEQ ID NO: 40.

[0030] In some embodiments, the linker is 5 to 30 amino acids long. In some embodiments, the linker is 20 amino acids long. In some embodiments, the linker is an amino acid sequence (G2SG2) x This includes x, where x is an integer from 1 to 6 (sequence keys 149 to 154). In some embodiments, x is 2 (sequence key 150) or 4 (sequence key 152).

[0031] In some embodiments, the IL-2 polypeptide is connected to the VH of the DBA portion via a linker. In some embodiments, the IL-2 polypeptide is connected to the VH of the DBA portion in the following orientation: N-[IL-2 polypeptide]-[linker]-[VH]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0032] In other embodiments, the IL-2 polypeptide is connected to the VL of the DBA portion via a linker. In some embodiments, the IL-2 polypeptide is connected to the VL of the DBA portion in the following orientation: N-[IL-2 polypeptide]-[linker]-[VL]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0033] In some embodiments, the IL-2 polypeptide is a mutant human IL-2 polypeptide containing an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, and / or an alanine residue at position 125 (numbered relative to the human IL-2 sequence of SEQ ID NO: 147). In some embodiments, the IL-2 polypeptide is a mutant human IL-2 polypeptide containing an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, an alanine residue at position 125, and / or a threonine residue at position 126 (numbered relative to the human IL-2 sequence of SEQ ID NO: 147). In some embodiments, the mutant human IL-2 polypeptide contains the amino acid sequence of SEQ ID NO: 146. In some embodiments, the mutant human IL-2 polypeptide contains the amino acid sequence of SEQ ID NO: 199. In some embodiments, PD-1 is human PD-1.

[0034] In one embodiment, the present invention relates to an immune complex comprising (a) a first binding domain comprising (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146, (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152, and (iii) a DBA moiety bound to the PD-1 and IL-2 polypeptides in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, and the IL-2 polypeptide, linker, and DBA moiety are linked in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and when the DBA moiety is bound to the IL-2 polypeptide, (b) a first binding domain in which the binding of the IL-2 polypeptide to its receptor is substantially blocked, and when the DBA portion is bound to PD-1, the binding of the DBA portion to the IL-2 polypeptide is blocked, and the IL-2 polypeptide can bind to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody portion, wherein the anti-PD-1 antibody portion is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL; (c) an Fc region comprising a first subunit and a second subunit, wherein (i) the first subunit comprises a tryptophan residue at position 366, and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (Kabat (i) numbered according to the EU index, or (ii) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (numbered according to the Kabat EU index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).(a) The DBA portion includes an Fc region (according to the EU index), and the DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the anti-PD-1 antibody portion and the second subunit are connected according to N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and (a) the DBA portion is a conventional Fab molecule, and the anti-PD-1 antibody portion is anti-PD-1 The present invention provides an immunocomplex comprising a Fab molecule comprising a Fab heavy chain containing VH and CH1 of the antibody portion and a Fab light chain containing VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or (b) a Fab molecule comprising a DBA portion comprising a Fab heavy chain containing VH and CH1 of the DBA portion and a Fab light chain containing VL of the DBA portion and CL of the light chain, wherein the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other, and the anti-PD-1 antibody portion is a conventional Fab molecule.

[0035] In one embodiment, the present invention relates to an immune complex comprising (a) a first binding domain comprising (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 199, (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152, and (iii) a DBA moiety bound to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, and the IL-2 polypeptide, linker, and DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and when the DBA moiety is bound to the IL-2 polypeptide, IL- (b) a first binding domain in which the binding of the IL-2 polypeptide to its receptor is substantially blocked, and when the DBA portion is bound to PD-1, the DBA portion is blocked from binding to the IL-2 polypeptide, and the IL-2 polypeptide can bind to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody portion, wherein the anti-PD-1 antibody portion is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL; (c) an Fc region comprising a first subunit and a second subunit, wherein the first subunit comprises a tryptophan residue at position 366, and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (Kabat (i) numbered according to the EU index, or (ii) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (numbered according to the Kabat EU index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).The present invention provides an immunocomplex comprising an Fc region including (according to the EU index), wherein the DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), the anti-PD-1 antibody portion and the second subunit are connected according to N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide, and the DBA portion is a conventional Fab molecule), and the anti-PD-1 antibody portion comprises a Fab heavy chain containing VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other.

[0036] In one embodiment, the present invention relates to an immune complex comprising: (a) a first binding domain comprising (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and (iii) a double-binding antibody (DBA) moiety that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and which is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, wherein the IL-2 polypeptide, linker, and DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the DBA moiety comprises IL-2 polypeptide (i) The first binding domain comprises (b) an anti-PD-1 antibody moiety, wherein the DBA moiety is bound to PD-1, the DBA moiety is bound to IL-2 polypeptide, and the IL-2 polypeptide can bind to its receptor. (i) The first subunit comprises a tryptophan residue at position 366, and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (Kabat (i) numbered according to the EU index, or (ii) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (numbered according to the Kabat EU index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).The immunocomplex is provided, comprising an Fc region including (according to the EU index), wherein the DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the anti-PD-1 antibody portion and the second subunit are connected according to N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0037] In some embodiments, the immune complex comprises: (a) a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (b) a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 104, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (c) a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 112, and a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 110 (d) a polypeptide, a third polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 108; (d) a first polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 124, a second polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 134, a third polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 128; (e) a first polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 124, a second polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 134, a third polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide containing at least 95% the amino acid sequence of SEQ ID NO: 120;(f) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120; (g) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 128. (h) A first polypeptide containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 197, a second polypeptide containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (i) A first polypeptide containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 198, a second polypeptide containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108.

[0038] In some embodiments, the immune complex comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (c) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (d) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128 Polypeptides; (e) a first polypeptide containing the amino acid sequence of SEQ ID NO: 124, a second polypeptide containing the amino acid sequence of SEQ ID NO: 134, a third polypeptide containing the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 120; (f) a first polypeptide containing the amino acid sequence of SEQ ID NO: 122, a second polypeptide containing the amino acid sequence of SEQ ID NO: 134, a third polypeptide containing the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 120; (g) a first polypeptide containing the amino acid sequence of SEQ ID NO: 122, a second polypeptide containing the amino acid sequence of SEQ ID NO: 134, a third polypeptide containing the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 128; (h) a first polypeptide containing the amino acid sequence of SEQ ID NO: 197, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 108;(i) A first polypeptide containing the amino acid sequence of SEQ ID NO: 198, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 108.

[0039] In one embodiment, the present invention provides an immune complex comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 96, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 108.

[0040] In one embodiment, the present invention provides an immune complex comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

[0041] In one embodiment, the present invention provides an immune complex comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128.

[0042] In one embodiment, the present invention provides an immune complex comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120.

[0043] In one embodiment, the present invention provides an immune complex comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 197, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 108.

[0044] In one embodiment, the present invention provides an immune complex comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 198, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO: 108.

[0045] In one embodiment, the present invention provides an isolated polynucleotide or a set of isolated polynucleotides encoding any of the immune complexes described herein.

[0046] In one embodiment, the present invention provides a vector or set of vectors comprising any one of the isolated polynucleotides described herein or any one of the sets of isolated polynucleotides.

[0047] In one embodiment, the present invention provides (i) any of the isolated polynucleotides or any set of isolated polynucleotides described herein, or (ii) a host cell or a set of host cells comprising a vector or a set of vectors described herein.

[0048] In one embodiment, the present invention provides a method for producing an immune complex, comprising the step of (a) culturing one of the host cells or one of the set of host cells described herein under conditions suitable for the expression of an immune complex. In some embodiments, the method further comprises recovering the immune complex.

[0049] In some embodiments, the host cell expresses a first binding domain and a second binding domain. In some embodiments, the first host cell expresses the first binding domain, and the second host cell expresses the second binding domain.

[0050] In some embodiments, the method further includes the step of recovering the first binding domain and the second binding domain. In some embodiments, the method further includes bringing the recovered first binding domain into contact with the recovered second binding domain.

[0051] In one embodiment, the present invention provides an immune complex produced by any one of the methods described herein.

[0052] In one embodiment, the present invention provides a pharmaceutical composition comprising one of the immune complexes described herein and a pharmaceutically acceptable carrier.

[0053] In one embodiment, the present invention provides any one of the immunocomplexes or pharmaceutical compositions described herein for use as a pharmaceutical.

[0054] In one embodiment, the present invention provides the use of any one of the immunocomplexes or pharmaceutical compositions described herein in the manufacture of a pharmaceutical product.

[0055] In one embodiment, the present invention provides any one of the immunocomplexes or pharmaceutical compositions described herein for use in the treatment of cancer in subjects requiring treatment for cancer.

[0056] In one embodiment, the present invention provides the use of any one of the immunocomplexes or pharmaceutical compositions described herein in the manufacture of a pharmaceutical for treating cancer in a subject requiring treatment for cancer.

[0057] In one embodiment, the present invention provides the use of any one of the immunocomplexes or pharmaceutical compositions described herein for treating cancer in subjects requiring treatment for cancer.

[0058] In one embodiment, the present invention provides a method for treating cancer in a subject, comprising administering an effective amount of one of the immune complexes or pharmaceutical compositions described herein to the subject.

[0059] In some aspects, the cancer is PD-1 positive.

[0060] In some embodiments, the immune complexes for use, pharmaceutical compositions for use, uses, or methods described herein further include administering additional therapeutic agents to a target. [Brief explanation of the drawing]

[0061] [Figure 1] The results of PD-1 / PD-L1 blockade bioassays for different PD-1 binding factors are shown. The 7G12 construct contains the anti-PD-1 moiety AB003058. The "continuously on" construct contains 0376 binding factors. The isotype is a negative control. [Figure 2] This describes the efficacy of PD-1-regulated IL-2 immune complexes as IL-2R signaling and their cis / trans signaling. PD-1-IL-2v: constitutively on PD-1-IL-2 construct (i.e., "constitutively on P1AE4422-14542"); FAP-IL-2V: non-targeted IL-2 polypeptide; AF5842: PD-1-regulated IL-2 immune complex. Preblock: preblocked by competing anti-PD-1. [Figure 3A-3B] This study demonstrates the efficacy of various immune complexes and cis / trans signaling in the activation of IL-2 receptor (IL-2R) signaling. Figure 3A is a graph showing the assay results. Figure 3B shows the structure and orientation of the tested molecules. Crescents / circles in the Fc region indicate knob-in-hole modifications. Filled circles indicate IL-2 polypeptides. Unpatterned VH / VL indicate the DBA portion. Patterned VH / VL indicate the anti-PD-1 antibody portion. bl.: Blocking. [Figure 4A-4B]This shows the efficacy of various immune complexes and cis / trans signaling in the activation of IL-2R signaling. Figure 4A is a graph showing the assay results. Figure 4B shows the structure and orientation of the molecules tested. Crescents / circles in the Fc region indicate knob-in-hole modifications. Filled circles indicate IL-2 polypeptides. Unpatterned VH / VL indicate the DBA moiety. Striped VH / VL indicate the anti-PD-1 antibody moiety. Checkered VH / VL indicate the non-blocking DBA moiety. For each pair of cylinders representing VH / VL or CH1 / CL: darker colored cylinders represent VH or CH1, and lighter colored cylinders represent CL or CH1. b or bl.: blocking; nb: non-blocking. [Figure 5A-5B] This shows the efficacy of various immune complexes and cis / trans signaling in the activation of IL-2R signaling. Figure 5A is a graph showing the assay results. Figure 5B shows the structure and orientation of the molecules tested. Crescents / circles in the Fc region indicate knob-in-hole modifications. Filled circles indicate IL-2 polypeptides. Unpatterned VH / VL indicate the DBA moiety. Striped VH / VL indicate the anti-PD-1 antibody moiety. Checkered VH / VL indicate the non-blocking DBA moiety. For each pair of cylinders representing VH / VL or CH1 / CL: darker colored cylinders represent VH or CH1, and lighter colored cylinders represent CL or CH1. P1AI7462 contains the scFV of the anti-PD-1 antibody moiety. b or bl.: blocking; nb: non-blocking. [Figure 6] This study demonstrates the efficacy of the PD-1-regulated IL-2 immune complex in activating IL-2R signaling and cis / trans signaling. bl.: Blocking [Figures 7A-7B] This shows the ability of various immune complexes to induce cytotoxic T cell effector function. Minimal MLR: Minimal mixed lymphocyte response. Figure 7A shows the assay. Figure 7B shows the assay results for different immune complex constructs. [Figure 8A-8B]This demonstrates the ability of the PD-1-regulated IL-2 immune complex to rescue conventional Tconv effector function from Treg suppression. Figure 8A shows the assay. Figure 8B shows the results of the rescue assay. Each symbol represents a separate donor, and the horizontal line shows the median with N=9 donors from three independent experiments. P was calculated using one-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figures 9A-9C] The efficacy of various immune complexes and cis / trans signaling in the activation of IL-2R signaling are shown. Figure 9A shows the assay. Figure 9B shows the structure and orientation of the molecules tested. Crescents / circles in the Fc region indicate knob-in-hole modifications. Filled circles indicate IL-2 polypeptides. Unpatterned VH / VL indicate the DBA moiety. Striped VH / VL indicate the anti-PD-1 antibody moiety. Checkered VH / VL indicate the non-blocking DBA moiety. For each pair of cylinders representing VH / VL or CH1 / CL: darker colored cylinders represent VH or CH1, and lighter colored cylinders represent CL or CH1. Figure 9C shows the assay results. In particular, compared to P1AI7440, P1AJ1837 differs only in having an extra Asp residue at the C-terminus of the DBA light chain. The same applies to P1AJ1838 (with one extra C-terminal Asp residue) and P1AI7441. b: blocking; nb: non-blocking; dei: deimmunization. [Figure 10A-10D]For example, the efficacy, cis / trans signaling, and leakiness of various immune complexes regarding IL-2R signaling against activated CD4 cells (Figure 10A), activated CD8 cells (Figure 10B), and NK cells (Figure 10C) are shown. Figure 10D shows the structure and orientation of the molecules tested. Crescents / circles in the Fc region indicate knob-in-hole modifications. Filled circles indicate IL-2 polypeptides. Unpatterned VH / VL indicates the DBA moiety. Striped VH / VL indicates the anti-PD-1 antibody moiety. Checkered VH / VL indicates the non-blocking DBA moiety. For each pair of cylinders representing VH / VL or CH1 / CL: darker colored cylinders represent VH or CH1, and lighter colored cylinders represent CL or CH1. B: blocking; nb: non-blocking; dei: deimmunization. [Figures 11A-11B] The results of a binding competition assay to determine whether the blocking DBA portion (Figure 11A) or non-blocking DBA portion (Figure 11A) of the PD-1 regulatory IL-2 immune complex compete for PD-1 binding with a blocking anti-PD-1 antibody partial arm or a non-blocking anti-PD-1 antibody partial arm are shown. B: blocking; nb: non-blocking; mat: affinity matured. [Figures 12A-12B] The results of an enzyme-linked immunosorbent assay (ELISA)-based assay to evaluate the switching behavior of PD-1-regulated IL-2 immune complexes are shown. Figure 12A shows the PD-1 concentration-dependent switching of the immune complexes. Figure 12B further provides IgG concentration-dependent negative controls for each immune complex tested. [Figure 13A-13M]The results of the HEK-Blue IL-2 reporter assay, showing PD-1 concentration-dependent and PD-1-regulated IL-2 immune complex concentration-dependent signals, are shown. Each panel shows results for different immune complexes. Figure 13A: Constant-on P1AE4422-14542 control; Figure 13B: P1AI7455; Figure 13C: P1AI7441; Figure 13D: P1AI7465; Figure 13E: P1AI7476; Figure 13F: P1AI7440; Figure 13G: P1AI7464; Figure 13H: P1AI7474; Figure 13I: P1AI7438; Figure 13J: P1AI7462; Figure 13K: P1AI7473; Figure 13L: P1AI7443; Figure 13M: P1AI7467. [Figure 14] This study demonstrates the in vivo efficacy of a PD-1-regulated IL-2 immune complex as a monotherapy in inhibiting pancreatic tumor growth in a syngeneic model using the mouse pancreatic Panc02-Fluc cell line. [Figure 15] Lung weight in mice treated with the vehicle, PD-1-modulated IL-2 immune complex, or pembrolizumab is shown as a metric for the adverse effects of the treatment. [Figures 16A-16B] The effects of PD-1-regulated IL-2 immune complexes on CD8+ T cell expansion in tumors (Figure 16A) and blood (Figure 16B) are shown. [Figures 17A-17B] Figure 17A illustrates the efficacy of PD-1-regulated IL-2 immune complexes and controls in activating IL-2R signaling, as well as cis / trans signaling. Figure 17A compares the 1+1 format of PD-1-reg-IL-2v, which has one DBA and one PD-1 binding factor with IL-2v bound to the DBA, with the asymmetric format of compound C, which has two DBAs with IL-2v bound to one DBA. Figure 17B compares PD-1-reg-IL-2v with PD-1-reg-IL-2vQ126T, which contains further mutations in IL-2v to further reduce binding affinity to IL-2Rb and thus increase the conditional activity of IL-2v toward PD-1 expression. Data are shown as mean ± SEM from four donors. [Figures 18A-18C]For example, the efficacy of various immune complexes for IL-2R signaling, cis / trans signaling, and leakiness are shown for activated CD4 cells (Figure 18A), activated CD8 cells (Figure 18B), and NK cells (Figure 18C). Data are shown mean ± SEM from two donors. [Figure 19] This study demonstrates IL-2 stimulation and subsequent SEAP secretion of JAK / STAT5 activation by different PD-1-regulated IL-2 immune complexes, as detected by the HEK-blue™ IL-2 reporter cell line. Activation of the reporter cell line was detected as a change in absorbance at 640 nm. [Figure 20] This study demonstrates the efficacy and cis / trans signaling of PD-1 regulated IL-2 immune complexes with 10-mer or 20-mer linkers between DBA and IL-2 polypeptides. Data are shown as mean ± SEM from four donors. [Modes for carrying out the invention]

[0062] Detailed explanation The present invention is at least in part based on the applicant's discovery that the PD-1-regulated IL-2 immune complex disclosed herein is unexpectedly advantageous for targeting IL-2-mediated cytotoxicity in a PD-1-rich cellular environment (e.g., cells expressing PD-1). IL-2 is a potent cytokine that exhibits toxicity when administered systemically. However, the PD-1-regulated IL-2 immune complex described herein enables targeted administration of IL-2, for example, for the treatment of cancer. In particular, the immune complex of the present invention exhibits remarkable sensitivity and discrimination for inducing IL-2 signaling in the presence versus absence of PD-1. Thus, the present invention provides an improved therapeutic option for targeted treatment of cancer.

[0063] The present invention is also at least in part based on the applicant's finding that the structure and orientation of the PD-1-regulated IL-2 immune complex disclosed herein exhibits superior therapeutic efficacy compared to other tested structures and orientations of immune complexes.

[0064] For example, an immune complex as disclosed herein may comprise a first binding domain containing an IL-2 polypeptide and a double-binding antibody (DBA) moiety that binds to the PD-1 and IL-2 polypeptides, for example, in a mutually exclusive manner, and a second binding domain containing an anti-PD-1 antibody. In some examples, the use of a monospecific anti-PD-1 antibody moiety in the second binding domain of the immune complex disclosed herein (e.g., as a targeted arm) may result in superior efficacy (e.g., with respect to antitumor activity) and safety compared to a construct containing only DBA and IL-2 polypeptides that bind to PD-1 (e.g., a bivalent construct containing two DBA moieties). For example, such a construct as disclosed herein may exhibit reduced leakiness in terms of IL-2 unmasking, resulting in reduced toxicity.

[0065] The present invention is also at least in part based on the applicant's finding that the structure and orientation of the PD-1-regulated IL-2 immune complex disclosed herein may enable improved manufacturability and ease of manufacture compared to other tested structures and orientations of immune complexes. For example, the PD-1-regulated IL-2 immune complex disclosed herein may be less prone to aggregation compared to other structures and orientations of immune complexes.

[0066] I. Definition For the purposes of this specification, “acceptor human framework” means a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived” from a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain a modification of the amino acid sequence. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is sequence-identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0067] "Administer" means a method of giving a target a dose of a compound (e.g., a PD-1 modulated IL-2 immune complex or antibody disclosed herein) or a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising a PD-1 modulated IL-2 immune complex or antibody disclosed herein). Compositions used in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarterially, intrapleurally, intratracheally, intranasally, intravitreously, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intrabladderally, intramucosa, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by local irrigation directly washing target cells, by catheter, by washing solution, in cream, or in lipid composition. The method of administration may vary depending on various factors (e.g., the compound or composition being administered, and the severity of the symptoms, disease, or disorder being treated).

[0068] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K D ). Affinity can be measured by methods common in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are also described herein.

[0069] An "affinity matured" antibody refers to an antibody having one or more changes in one or more complementarity determining regions (CDRs), and such changes result in an improvement in the affinity of the antibody for an antigen as compared to the parental antibody that does not have such changes.

[0070] The terms "anti-PD-1 antibody" and "antibody that binds to PD-1" refer to an antibody that has sufficient affinity to be able to bind to PD-1 such that the antibody is useful as a diagnostic and / or therapeutic agent in the targeting of PD-1. In one aspect, the degree of binding of an anti-PD-1 antibody to an irrelevant non-PD-1 protein is less than about 10% of the binding of the antibody to PD-1, as measured, for example, by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to PD-1 has a dissociation constant (K -8 ) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -13 [[ID=1,4]]M or less, e.g., 10 -8 M to 10 z -13 M, e.g., 10 -9 M). In certain aspects, an antibody that binds to PD-1 has a K of about 1×10 D M to about 1×10<0,000,009>M, about 1×10 -10 M to about 1×10 -12 M, or about 1×10 -11 M to about 5×10 -11 M. <ooooo14>M of KD It has. The antibody is K ≤ 1 μM. D If it has this property, it is said to "specifically bind" to PD-1.

[0071] The term “double-conjugated antibody” or “DBA” refers to an antibody that can bind to two different antigens (e.g., PD-1 and IL-2) with sufficient affinity in a mutually exclusive manner. In one embodiment, the degree of binding of the DBA to unrelated non-target proteins (e.g., other than the two target antigens) is less than about 10% of the binding of the antibody to the two target antigens (e.g., PD-1 and IL-2), as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the DBA has a viscosity of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0072] The term "antibody" is used herein in its broadest sense and encompasses, but is not limited to, a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0073] An "antibody fragment" refers to a molecule other than an intact antibody, including a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology (2005) 23:1126-1136.

[0074] A “binding domain” or “binding moiety” means a portion of a compound or molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Molecules characterized by a binding moiety include, but are not limited to, antibodies (e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof (e.g., Fab fragments, Fab'2, scFv antibodies, SMIP, domain antibodies, diabodies, minibodies, scFv-Fc, aphibodies, nanobodies, and the VH and / or VL domains of antibodies), receptors, ligands, aptamers, and other molecules having identified binding partners. In certain embodiments, molecules characterized by a binding domain include immune complexes, such as PD-1 regulated IL-2 immune complexes.

[0075] As used herein, the terms “first,” “second,” or “third” with respect to Fc subunits, etc., are used for convenience to distinguish between two or more parts of each type. The use of these terms is not intended to give any particular order or orientation of immune complexes unless expressly indicated so.

[0076] A "Fab molecule" refers to a protein that contains or consists of the VH and CH1 domains of the immunoglobulin heavy chain ("Fab heavy chain") and the VL and CL domains of the light chain ("Fab light chain").

[0077] "Fused" or "linked" means that two parts (e.g., a DBA part and the VH and VL of the IL-2 polypeptide or scFv) are linked by a covalent bond, such as a peptide bond, either directly or via one or more peptide linkers. As used herein, "linker" refers to a short polypeptide (e.g., containing 5 to 40 amino acids; e.g., containing 5 to 30 amino acids; e.g., containing 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids) used to link the other two parts (e.g., aDBA part and the VH and VL of the IL-2 polypeptide or scFv).

[0078] As used herein, the term “single-chain” refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the binding domains of an immune complex (e.g., an anti-PD-1 antibody portion) includes a single-chain Fab molecule, i.e., a Fab molecule in which a Fab light chain and a Fab heavy chain are linked by a peptide linker to form a single peptide chain. In such particular embodiments, the C-terminus of the Fab light chain in the single-chain Fab molecule is connected to the N-terminus of the Fab heavy chain.

[0079] A "single-stranded variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light (VL) chains of an antibody, linked by a linker. Specifically, the linker is a short polypeptide typically consisting of 10–25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, and can be linked from the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of a linker. For a review of scFv fragments, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269–315 (1994). See also International Publication No. 93 / 16185 and U.S. Publications No. 5,571,894 and No. 5,587,458.

[0080] A "single-stranded Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domain and the linker have one of the following sequences from the N-terminus to the C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-stranded Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-stranded Fab fragments would be further stabilized by the creation of interchain disulfide bonds through the insertion of cysteine ​​residues (for example, at position 44 of the variable heavy chain and position 100 of the variable light chain, according to Kabat numbering).

[0081] A "crossover" Fab molecule (also called a "Crossfab") refers to a Fab molecule in which the variable domains of the Fab heavy chain and the Fab light chain are exchanged (i.e., substituted for each other). In other words, a crossover Fab molecule includes a peptide chain composed of a light chain variable domain VL and a heavy chain constant domain 1 CH1 (VL-CH1, from the N-terminus to the C-terminus), and a peptide chain composed of a heavy chain variable domain VH and a light chain constant domain CL (VH-CL, from the N-terminus to the C-terminus). For clarity, in this specification, in a crossover Fab molecule in which the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain containing the heavy chain constant domain 1 CH1 is referred to as the "heavy chain" of the crossover Fab molecule.

[0082] In contrast, a “conventional” Fab molecule refers to a Fab molecule in its natural form, that is, one comprising a heavy chain (VH-CH1, N-terminus to C-terminus) composed of a heavy chain variable domain and a constant domain, and a light chain (VL-CL, N-terminus to C-terminus) composed of a light chain variable domain and a constant domain.

[0083] A diabody is an antibody fragment having two antigen-binding sites, which may be bivalent or bispecific. See, for example, European Patent No. 404,097, International Publication No. 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0084] A "single-domain antibody" is an antibody fragment that contains all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (see, for example, Domantis, Inc., Waltham, MA; U.S. Patent No. 6,248,516B1).

[0085] The terms "cancer" and "cancerous" refer to or describe physiological conditions in mammals typically characterized by uncontrolled cell proliferation. Forms of cancer include solid tumor carcinomas and non-solid tumor carcinomas. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies.More specific examples of such cancers include, but are not limited to, bladder cancer (e.g., urothelial carcinoma (UC), e.g., metastatic UC (mUC); muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC)); kidney cancer or renal cancer (e.g., renal cell carcinoma (RCC)); lung cancer, e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung; cancers of the urinary tract; breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC)); (this is Negative for strogen receptor (ER-), progesterone receptor (PR-), and HER2 (HER2-); prostate cancer, e.g., castration-resistant prostate cancer (CRPC); peritoneal cancer; hepatocellular carcinoma; gastric cancer, or stomach cancer, e.g., gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., hepatocellular carcinoma (HCC)); liver cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer Prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, e.g., superficial spreading melanoma, lentigo malignant melanoma, acral lentigo melanoma and nodular melanoma; multiple myeloma and B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleavage cell NHL; giant lesion NHL; mantle cell lymphoma This includes AIDS-associated lymphoma; and Valdenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); pilocytic cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as facomatosis-associated abnormal angiogenesis, edema (including that associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and associated metastases.

[0086] As used herein, “tumor” refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as used herein.

[0087] The terms "proliferative disorder" and "proliferative disorder" refer to disorders related to a certain degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer. In another embodiment, the proliferative disorder is a tumor.

[0088] The term "epitope" refers to a site on a proteinaceous or non-proteinaceous antigen to which a DBA moiety, anti-PD-1 antibody moiety, PD-1-regulated IL-2 immune complex, or antibody described herein binds. Epitopes may be formed from a continuous amino acid stretch site (linear epitopes) or from discontinuous amino acids (conformational epitopes), and may be formed spatially close together, for example, due to the folding of the antigen (i.e., by tertiary folding of a proteinaceous antigen). Linear epitopes are typically still bound by the DBA moiety, anti-PD-1 antibody moiety, PD-1-regulated IL-2 immune complex, or antibody described herein after exposure of a proteinaceous antigen to a denaturing agent, while conformational epitopes are typically destroyed upon treatment with a denaturing agent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 30, or at least 35, or 3-25, 3-20, 3-15, 3-10, 3-5, 30-40, 35-40, or 5-10 amino acids in its unique spatial conformation.

[0089] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) may be carried out using methods commonly used in the art, such as, but not limited to, alanine scanning, peptide blotting (see, e.g., Kobeissy et al., Meth.Mol.Biol.(2004)248:443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see, e.g., Hochleitner et al., Prot.Sci.9(2000)487-496), and cross-blocking (see, e.g., “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

[0090] Competitive binding can be used to easily determine whether an antibody or DBA binds to the same PD-1 epitope as a reference anti-PD-1 antibody or DBA that binds to PD-1, or whether it competes for binding with a reference anti-PD-1 antibody or DBA that binds to PD-1. For example, an antibody that binds to the same epitope as a reference anti-PD-1 antibody or DBA refers to an antibody or DBA that blocks the binding of the antigen to the reference anti-PD-1 antibody or DBA by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antigen to the reference antibody or DBA by 50% or more in a competitive assay. Alternatively, for example, to determine whether an antibody binds to the same epitope as a reference anti-PD-1 antibody or DBA, the reference antibody or DBA is bound to PD-1 under saturated conditions. After removing excess reference antibody, the ability of the anti-PD-1 antibody or DBA in question to bind to PD-1 is evaluated. If an anti-PD-1 antibody or DBA can bind to PD-1 after saturated binding to a reference anti-PD-1 antibody or DBA, it can be concluded that the anti-PD-1 antibody or DBA in question binds to a different epitope than the reference anti-PD-1 antibody or DBA. However, if the anti-PD-1 antibody or DBA in question cannot bind to the target epitope after saturated binding to a reference anti-PD-1 antibody or DBA, then the anti-PD-1 antibody or DBA in question may bind to the same epitope as the one bound by the reference anti-PD-1 antibody or DBA.

[0091] In some embodiments, two antibodies or DBAs are considered to bind to the same or overlapping epitopes if, when one antibody or DBA is measured in 1x, 5x, 10x, 20x, or 100x excess, it inhibits the binding of the other by at least 50%, at least 75%, at least 90%, or even 99% or more when measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50(1990) 1495-1502).

[0092] In some embodiments, two antibodies or DBAs are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody or DBA also reduce or eliminate the binding of the other antibody. Two antibodies or DBAs are considered to have an "overlapping epitope" if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody or DBA also reduces or eliminates the binding of the other antibody or DBA.

[0093] The term "chimeric" antibody refers to an antibody in which part of the heavy chain and / or light chain originates from a specific source or species, while the rest of the heavy chain and / or light chain originates from a different source or species.

[0094] The "class" of an antibody refers to the type of constant domain or constant region held by its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is the IgG1 isotype. In certain embodiments, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is the IgG2 isotype. In certain embodiments, the antibody is the IgG4 isotype and has the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0095] As used herein, the terms “human constant region” or “human constant region” refer to the constant heavy chain region and / or constant light chain kappa or lambda region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are known in the art and are described, for example, in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, e.g., Johnson, G., and Wu, TT, Nucleic Acids Res. 28(2000) 214-218; Kabat, EA, et al., Proc. Natl. Acad. Sci. USA 72(1975) 2785-2788). Unless otherwise specified herein, the numbering of amino acid residues in the constant region follows the EU numbering system (also known as the Kabat EU index), as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0096] "Effector function" refers to the biological activity resulting from the PD-1 regulatory IL-2 immune complex or the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0097] The “effective amount” of a compound, for example, the PD-1 modulated IL-2 immune complex or antibody or a composition thereof (e.g., a pharmaceutical composition) disclosed herein, is the minimum amount required to achieve a desired therapeutic or preventive outcome, such as a measurable improvement or prevention of a specific disorder (e.g., a cell proliferation disorder, e.g., cancer). The effective amount as used herein may vary depending on factors such as the patient’s disease state, age, sex, and weight, as well as the antibody’s ability to induce a desired response in the individual. The effective amount is also the amount at which the therapeutically beneficial effect outweighs any toxic or adverse effects of the treatment. In the case of prophylactic use, beneficial or desired outcomes include the elimination or reduction of the risk of disease, reduction of disease severity, or delay of disease onset, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that appear during the onset of the disease. In therapeutic use, beneficial or desired outcomes include clinical results such as a reduction in one or more symptoms caused by the disease, an improvement in the quality of life of the person affected, a reduction in the dose of other drugs required to treat the disease, an enhancement of the effect of another drug (e.g., by targeting), a delay in disease progression, and / or an extension of survival. In the case of cancer or tumors, an effective dose of the drug may have the effect of reducing the number of cancer cells, reducing tumor size, inhibiting (i.e., delaying to some extent, or preferably stopping) the invasion of cancer cells into peripheral organs, inhibiting (i.e., delaying to some extent, or preferably stopping) tumor metastasis, inhibiting tumor growth to some extent, and / or alleviating to some extent one or more of the symptoms associated with the disorder. An effective dose may be administered in one or more doses. For the purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic treatment or therapeutic action. As understood in the clinical field, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Therefore, the “effective dose” may be considered in relation to the administration of one or more therapeutic agents, and a monotherapy agent may be considered to be given in an effective dose if, when combined with one or more other agents, the desired outcome can be achieved or is achieved.

[0098] The term “Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, by expression of a particular nucleic acid molecule encoding a full-length heavy chain, antibodies produced by host cells may contain the full-length heavy chain or a cleaved variant of the full-length heavy chain. This may be the case when the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Thus, the C-terminal lysine (Lys447) or C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. In one embodiment, the heavy chain containing the Fc region specified herein, which is included in the PD-1 modulated IL-2 immunocomplex or antibody according to the present invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one embodiment, the heavy chain containing the Fc region specified herein, which is included in the PD-1 modulated IL-2 immunocomplex or antibody according to the present invention, comprises an additional C-terminal glycine residue (G446, numbering according to the EU index).

[0099] The "framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in VH (or VL) as the following sequence: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0100] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein synonymously to refer to antibodies having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region as defined herein. It should be understood that a full-length antibody includes the heavy chain variable domain and light chain variable domain as defined herein, as well as the Fc region as defined herein.

[0101] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their progeny, regardless of passage number. Progeny do not have to have exactly the same nucleic acid content as the parent cells and may contain mutations. Progeny of mutants having the same function or biological activity as those screened or selected for the original transformed cells are included in this invention.

[0102] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody, such as a human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0103] The "Human Consensus Framework" is a framework representing the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I, as described in Kabat et al. (above). In one embodiment, for VH, the subgroup is subgroup III, as described in Kabat et al. (above).

[0104] A “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody contains at least one, usually two, variable domains in all or nearly all CDRs corresponding to the variable domains of the non-human antibody, and in all or nearly all FRs corresponding to the variable domains of the human antibody. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0105] As used herein, the terms “hypervariable region” or “HVR” refer to each of the regions of an antibody variable domain whose sequence is hypervariable and which determines antigen-binding specificity, such as “complementarity-determining region” (CDR).

[0106] In certain embodiments, the antibody contains six CDRs, three of which are in the VH region (CDR-H1, CDR-H2, CDR-H3) and three in the VL region (CDR-L1, CDR-L2, CDR-L3). In certain embodiments, the antibody containing six CDRs is a full-length antibody. In certain embodiments, the antibody containing six CDRs is an antibody fragment.

[0107] Examples of CDRs used herein include: (a) Hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contact occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)) These are some examples.

[0108] Unless otherwise specified, CDRs are determined according to Kabat et al. (see above). Those skilled in the art will understand that CDR designations may also be determined according to McCallum (see above), Chothia (see above), or other scientifically recognized nomenclature.

[0109] The "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human.

[0110] An "immune complex" is an antibody (e.g., a DBA portion and / or an anti-PD-1 antibody portion) conjugated to one or more heterologous molecules (e.g., an IL-2 polypeptide), which includes but is not limited to polypeptides. In certain embodiments, the heterologous molecule is an IL-2 polypeptide, e.g., a human IL-2 polypeptide, e.g., a variant human IL-2 polypeptide as described herein.

[0111] A “PD-1-regulated IL-2 immune complex” or “PD-1-reg-IL-2v” is an immune complex containing an IL-2 polypeptide, where the IL-2 polypeptide activity (e.g., activation of IL-2R signaling) is regulated by the antigen-binding domain of the immune complex (e.g., binding to PD-1). In some embodiments, the PD-1-regulated IL-2 immune complex includes a DBA (Deep-Blocked Antibody) portion that binds to PD-1 and the IL-2 polypeptide in a substantially mutually exclusive or mutually exclusive manner. The PD-1-regulated IL-2 immune complex may further include, for example, an anti-PD-1 antibody portion that does not substantially bind to IL-2 or does not bind to IL-2.

[0112] "Isolated" immune complexes (e.g., PD-1-regulated IL-2 immune complexes) or antibodies are those separated from components of their natural environment. In some embodiments, immune complexes (e.g., PD-1-regulated IL-2 immune complexes) or antibodies are purified to a purity of over 95% or over 99% when determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing IEF, capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J.Chromatogr.B 848:79-87 (2007).

[0113] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by a base sequence, which represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone links or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of antibodies described herein in vitro and / or in vivo, for example, in a host or subject. Such DNA vectors (e.g., cDNA) or RNA vectors (e.g., mRNA) may or may not be modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject in vivo to produce an antibody. (See, for example, Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823B1).

[0114] "Isolated" nucleic acids are nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found in cells that contain nucleic acid molecules, but these nucleic acid molecules are located outside of chromosomes or in chromosomal locations different from their natural chromosomal locations.

[0115] "Isolated nucleic acid encoding an immune complex" or "isolated nucleic acid encoding an antibody" means one or more nucleic acid molecules encoding an immune complex (e.g., a PD-1 regulated IL-2 immune complex) or antibody as described herein, comprising a heavy chain and a light chain (or fragment thereof), wherein such nucleic acid molecules are contained in a single vector or separate vectors, and such nucleic acid molecules are located at one or more locations in a host cell.

[0116] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bound to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or variant antibodies that may arise during the production of a monoclonal antibody preparation, such variants generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on one antigen. Thus, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies according to this disclosure may be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0117] A "multispecific antigen-binding molecule" and a "multispecific antibody" are, respectively, monoclonal antigen-binding molecules or antibodies that have binding specificity to at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific antibody may be a "bispecific antibody" that has binding specificity to two different sites, i.e., two different epitopes on two different antigens or two different epitopes on the same antigen.

[0118] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, a native IgG antibody is a heterotetrameric glycoprotein with approximately 150,000 daltons, containing two identical light chains and two identical heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant heavy domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also called a variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0119] The term “package insert” is used to refer to the instructions typically included on the market packaging of a therapeutic product, including information relating to indications, use, dosage, administration, combination therapy, contraindications, and / or warnings for such therapeutic product.

[0120] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after introducing gaps if necessary to align the sequences for alignment purposes and achieve the maximum sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or FASTA program packages. Those skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to obtain the maximum alignment over the full length of the sequences to be compared. Alternatively, the identity percentage value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the U.S. Copyright Office (Washington DC, 20559), registered under U.S. Copyright Registration No. TXU510087, and published internationally in publication No. 2001 / 007611.

[0121] Unless otherwise specified, for the purposes of this specification, amino acid sequence identity percentage values ​​are generated using the ggsearch program and BLOSUM50 comparison matrix in FASTA package version 36.3.8c or later. The FASTA program package was written by WRPearson and DJLipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; WRPearson (1996) “Effective protein sequence comparison”, Meth.Enzymol.266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or ebi.ac.uk / Tools / sss / fasta. Alternatively, you can use the ggsearch(global protein:protein) program with default options (BLOSUM50;open:-10;ext:-2;Ktup=2) to compare sequences using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, ensuring that a global rather than local alignment is performed. The amino acid identity percentage is shown in the output alignment header.

[0122] The terms “pharmaceutical composition” and “pharmaceutical preparation” are used interchangeably herein and refer to preparations in which the biological activity of the active ingredients contained herein is effective and which do not contain additional components that are unacceptably toxic to the subject to which the pharmaceutical composition is administered.

[0123] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient that is non-toxic to the target. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0124] As used herein, the term “PD-1” refers to any native PD-1 from any vertebrate source, including mammals such as primates (e.g., humans, monkeys (cyno)) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses not only “full-length,” unprocessed PD-1, but also any form of PD-1 resulting from intracellular processing. The term also encompasses naturally occurring variants of PD-1, such as splice variants or allele variants. In certain embodiments, PD-1 is human PD-1. An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot accession number Q15116.

[0125] The terms “interleukin-2” or “IL-2,” as used herein, refer to any native IL-2 from any vertebrate source, including mammals, e.g., primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses not only “full-length,” unprocessed IL-2, but also any form of IL-2 resulting from intracellular processing. The term also encompasses naturally occurring variants of IL-2, including, for example, splice variants or allele variants. IL-2 includes, for example, human IL-2 whose polypeptide sequence is SEQ ID NO: 147 (e.g., wild-type human IL-2). As used herein, “mutant” IL-2 refers to IL-2 that includes one or more amino acid modifications compared to the wild-type IL-2 polypeptide sequence. In certain embodiments, the mutant human IL-2 polypeptide includes one or more amino acid modifications compared to the wild-type human IL-2 polypeptide of SEQ ID NO: 147. In preferred embodiments, the mutant human IL-2 polypeptide includes the amino acid sequence of SEQ ID NO: 146. In a more preferred embodiment, the mutant human IL-2 polypeptide contains the amino acid sequence of SEQ ID NO: 199.

[0126] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) refers to a clinical intervention in an attempt to alter the natural course of a disease (e.g., cancer) in the subject being treated, and may be performed for prevention (“preventive treatment” or “prophylactically treating”) or during the course of a clinicopathological condition (“therapeutic treatment” or “therapeutic treating”). Desired effects of therapeutic treatment include, but are not limited to, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of cancer metastasis, reduction of the rate of disease progression, reduction or mitigation of symptoms, and remission or improved prognosis. Desired effects of preventive treatment include, but are not limited to, prevention of the onset or recurrence of the disease. In some embodiments, the antibodies described herein are used to delay the onset of a disease or to slow the progression of a disease.

[0127] A "variable region" or "variable domain" is a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (e.g., Kindt et al. Kuby Immunology, 6) thSee ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated using the VH or VL domain of the antibody that binds to that antigen, and complementary libraries of VL or VH domains can be screened. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0128] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of the nucleic acid to which they are functionally ligated. Such vectors are referred herein to as “expression vectors.”

[0129] II. Compositions and Methods In one embodiment, the disclosure provides an immune complex (for example, an immune complex comprising at least one binding domain that binds to PD-1, conjugated directly or via a linker to an IL-2 polypeptide (for example, a binding domain comprising a double-binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, and at least one binding domain comprising an anti-PD-1 antibody moiety that binds to PD-1). In some embodiments, the immune complex comprises a first binding domain comprising an IL-2 polypeptide, a linker, and a double-binding antibody (DBA) moiety, and a second binding domain comprising an anti-PD-1 antibody moiety. The DBA moiety may comprise a heavy-chain variable region (VH) and a light-chain variable region (VL) and may bind to the PD-1 and IL-2 polypeptides in a mutually exclusive manner. In the first binding domain, the IL-2 polypeptide, linker, and DBA moiety may be linked in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide). The anti-PD-1 antibody moiety may contain VH and VL. In some examples, the anti-PD-1 antibody moiety is monospecific. In some examples, the immune complex is a PD-1 regulated IL-2 immune complex. The immune complexes and antibodies described herein are useful, for example, for the treatment of cancer.

[0130] A. Double binding antibody (DBA) portion In some embodiments, the immune complexes and antibodies described herein may include a binding domain (e.g., a first binding domain) comprising a DBA moiety. In certain embodiments, the DBA moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL) and may bind to PD-1 and IL-2 polypeptides, for example, in a mutually exclusive manner. In certain embodiments, PD-1 is human PD-1. In certain embodiments, the IL-2 polypeptide is the human IL-2 polypeptide described herein or a modified human IL-2 polypeptide. In some cases, the modified human IL-2 polypeptide contains one or more amino acids described herein (e.g., human IL-2 including the amino acid substitutions T3A, F42A, Y45A, L72G, and C125A).

[0131] In some embodiments, the DBA portion of the present invention comprises at least one CDR, at least two CDRs, at least three CDRs, at least four CDRs, at least five CDRs, or all six CDRs (e.g., one, two, three, four, five, or six CDRs) containing amino acid sequences of the SEQ ID NOs as illustrated in Table 1. In some cases, the DBA portion comprises VH and / or VL containing amino acid sequences of the SEQ ID NOs shown in Table 1. [Table 1]

[0132] In some embodiments, the present invention provides a DBA portion comprising the following six complementarity-determining regions (CDRs): CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and CDR-L3 containing the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57). In some embodiments, the DBA portion is AB002345.

[0133] In some embodiments, the DBA portion includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 69; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 70; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 71; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 72; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 60; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 61; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 62; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 63. In some embodiments, the DBA portion comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 58; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the DBA portion comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 67; (b) a VL containing the amino acid sequence of SEQ ID NO: 58; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the DBA portion is AB002345.

[0134] In some embodiments, the present invention provides a DBA moiety comprising CDR-H1 comprising the amino acid sequence of the following six CDRs:AYYIH (SEQ ID NO: 82), CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75). In some embodiments, the DBA moiety is AB003637, AB003637 deimmunized, or AB003637 deimmunized 2.

[0135] In some embodiments, the DBA portion includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 87; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 88; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 89; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 90; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 78; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 79; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 80; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 81. In some embodiments, the DBA portion comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 85; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 76; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the DBA portion comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 85; (b) a VL containing the amino acid sequence of SEQ ID NO: 76; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the DBA portion is AB003637.

[0136] In some embodiments, the DBA portion includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 87; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 88; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 89; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 90; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 78; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 79; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 80; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 155. In some embodiments, the DBA portion comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 85; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 91; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the DBA portion comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 85; (b) a VL containing the amino acid sequence of SEQ ID NO: 91; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the DBA portion is AB003637 deimmunization.

[0137] In some embodiments, the DBA portion includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 87; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 88; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 156; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 90; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 78; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 79; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 80; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 155. In some embodiments, the DBA portion comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 111; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 91; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the DBA portion comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 111; (b) a VL containing the amino acid sequence of SEQ ID NO: 91; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the DBA portion is AB003637 deimmunization 2.

[0138] In some embodiments, the DBA portion is a conventional Fab molecule. In some embodiments, the DBA portion is a conventional Fab molecule comprising a Fab heavy chain (VH-CH1, NC terminal direction) composed of a heavy chain variable domain and a constant domain, and a Fab light chain (VL-CL, NC terminal direction) composed of a light chain variable domain and a constant domain.

[0139] In some embodiments, the DBA portion is crossFab (i.e., a Fab molecule including crossover modifications), and the DBA portion comprises a Fab heavy chain containing the VH of the DBA portion and heavy chain constant domain 1 (CH1), and a Fab light chain containing the VL of the DBA portion and light chain constant domain (CL), wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other. In preferred embodiments, when the DBA portion is crossFab, the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other.

[0140] B. Anti-PD-1 antibody part In some embodiments, the immune complexes and antibodies described herein may include a binding domain (e.g., a second binding domain) containing an anti-PD-1 antibody moiety. In certain embodiments, the anti-PD-1 antibody moiety includes a heavy chain variable region (VH) and a light chain variable region (VL) and binds to PD-1. In certain embodiments, PD-1 is human PD-1. In some embodiments, the anti-PD-1 antibody moiety does not substantially bind to an IL-2 polypeptide, such as the IL-2 polypeptide described herein. In some embodiments, the PD-1 antibody moiety is monospecific.

[0141] In some embodiments, the anti-PD-1 antibody moiety of the present invention comprises at least one CDR, at least two CDRs, at least three CDRs, at least four CDRs, at least five CDRs, or all six CDRs (e.g., one, two, three, four, five, or six CDRs) containing amino acid sequences of the SEQ ID NOs shown in Table 2. In some cases, the anti-PD-1 antibody moiety comprises VH and / or VL containing amino acid sequences of the SEQ ID NOs shown in Table 2. [Table 2]

[0142] In some embodiments, the present invention provides an anti-PD-1 antibody moiety comprising the following six amino acid sequences: CDR-H1 comprising CDR:SYWMS (SEQ ID NO: 10), CDR-H2 comprising AISGSGGSRYYAESVKG (SEQ ID NO: 11), CDR-H3 comprising SPLQWIDV (SEQ ID NO: 12), CDR-L1 comprising RASQGISSWLA (SEQ ID NO: 1), CDR-L2 comprising EASSLQS (SEQ ID NO: 2), and CDR-L3 comprising QQANQFPFT (SEQ ID NO: 3). In some embodiments, the anti-PD-1 antibody moiety is AB003058.

[0143] In some embodiments, the anti-PD-1 antibody moiety comprises one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 15; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 16; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 17; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 18; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 6; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 7; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 8; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the anti-PD-1 antibody moiety comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 13; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the anti-PD-1 antibody moiety comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 13; (b) a VL containing the amino acid sequence of SEQ ID NO: 4; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the anti-PD-1 antibody moiety is AB003058.

[0144] In some embodiments, the present invention provides an anti-PD-1 antibody moiety comprising CDR-H1 comprising the amino acid sequence of the following six CDR:SYTMS (SEQ ID NO: 28), CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21). In some embodiments, the anti-PD-1 antibody moiety is 0376 deimmunized.

[0145] In some embodiments, the anti-PD-1 antibody moiety includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 33; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 34; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 35; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 36; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 24; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 25; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 26; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 27. In some embodiments, the anti-PD-1 antibody moiety comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 31; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the anti-PD-1 antibody moiety comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 31; (b) a VL containing the amino acid sequence of SEQ ID NO: 22; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the anti-PD-1 antibody moiety is 0376 deimmunized.

[0146] In some embodiments, the present invention provides an anti-PD-1 antibody moiety comprising CDR-H1 comprising the amino acid sequence of the following six CDR:SYTMS (SEQ ID NO: 166), CDR-H2 comprising the amino acid sequence of TISGGGRDIYYPDSVKG (SEQ ID NO: 167), CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 168), CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 157), CDR-L2 comprising the amino acid sequence of RSSTLES (SEQ ID NO: 158), and CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 159). In some embodiments, the anti-PD-1 antibody moiety is 0376.

[0147] In some embodiments, the anti-PD-1 antibody moiety comprises one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 171; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 172; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 173; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 174; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 162; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 163; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 164; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 165. In some embodiments, the anti-PD-1 antibody moiety comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 169; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 160; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the anti-PD-1 antibody moiety comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 169; (b) a VL containing the amino acid sequence of SEQ ID NO: 160; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the anti-PD-1 antibody moiety is 0376.

[0148] In some embodiments, the present invention provides an anti-PD-1 antibody moiety comprising the following six amino acid sequences: CDR-H1 containing CDR:SYAMS (SEQ ID NO: 46), CDR-H2 containing CDR:VITGSGGSTYYADSVKG (SEQ ID NO: 47), CDR-H3 containing CDR:GEGYAGSSYFRASDI (SEQ ID NO: 48), CDR-L1 containing CDR:RASQSISSYLN (SEQ ID NO: 37), CDR-L2 containing CDR:TASSLQS (SEQ ID NO: 38), and CDR-L3 containing CDR:QQSYSTPLT (SEQ ID NO: 39). In some embodiments, the anti-PD-1 antibody moiety is 1040 affinity matured.

[0149] In some embodiments, the anti-PD-1 antibody moiety comprises one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 51; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 52; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 53; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 54; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 42; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 43; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 44; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-PD-1 antibody moiety comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 49; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 40; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the anti-PD-1 antibody moiety comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 49; (b) a VL containing the amino acid sequence of SEQ ID NO: 40; or (c) the VH described in (a) and the VL described in (b). In some embodiments, the anti-PD-1 antibody moiety is 1040 affinity matured.

[0150] In some embodiments, the anti-PD-1 antibody portion is a conventional Fab molecule. In some embodiments, the anti-PD-1 antibody portion is a conventional Fab molecule comprising a Fab heavy chain (VH-CH1, NC terminal direction) composed of a heavy chain variable domain and a constant domain, and a Fab light chain (VL-CL, NC terminal direction) composed of a light chain variable domain and a constant domain.

[0151] In some embodiments, the anti-PD-1 antibody moiety is crossFab (i.e., a Fab molecule including crossover modification), and the anti-PD-1 antibody moiety comprises a Fab heavy chain containing the VH and heavy chain constant domain 1 (CH1) of the anti-PD-1 antibody moiety, and a Fab light chain containing the VL and light chain constant domain (CL) of the anti-PD-1 antibody moiety, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other. In preferred embodiments, when the anti-PD-1 antibody moiety is crossFab, the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other.

[0152] In some embodiments, the anti-PD-1 antibody portion is a single-stranded variable fragment (scFv). In some embodiments, the anti-PD-1 antibody is an scFv in which VH and VL are connected in the following orientation: N-VH-VL-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide). In other embodiments, the anti-PD-1 antibody is an scFv in which VH and VL are connected in the following orientation: N-VL-VH-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide). In some embodiments, the VH and VL of the scFv are connected by a linker. Any suitable linker, e.g., any linker described herein (e.g., Section D below), may be used. In some embodiments, VH, VL, and the linker are connected in the following orientation: N-VH-linker-VL-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide). In another embodiment, the anti-PD-1 antibody is scFv in which VH and VL are connected in the following orientation: N-VL-linker-VH-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0153] C.IL-2 polypeptide The immune complex of the present invention may include IL-2 polypeptides having properties advantageous for immunotherapy (e.g., human IL-2 polypeptide; e.g., mutant IL-2 polypeptide; e.g., mutant human IL-2 polypeptide). In particular, pharmacological properties of IL-2 polypeptides that contribute to toxicity but are not essential for the efficacy of the IL-2 polypeptide can be eliminated in mutant IL-2 polypeptides. Such mutant IL-2 polypeptides are described in detail in International Publication No. 2012 / 107417, which is incorporated in whole hereby for reference. Different forms of IL-2 receptors consist of different subunits and exhibit different affinities to IL-2. Intermediate affinity IL-2 receptors consist of β and γ receptor subunits, are expressed in resting effector cells, and are sufficient for IL-2 signaling. High affinity IL-2 receptors, further comprising the α-subunit of the receptor, are regulatory T(T) reg) The binding of IL-2 polypeptide to the cell is T reg It is primarily expressed on activated effector cells that can promote cell-mediated immunosuppression or activation-induced cell death (AICD). Therefore, although we do not wish to be bound by theory, reducing or eliminating the affinity of the IL-2 polypeptide to the α-subunit of the IL-2 receptor may reduce the IL-2-induced downregulation of effector cell function by regulatory T cells and the development of tumor tolerance through the AICD process. On the other hand, maintaining affinity to the intermediate affinity IL-2 receptor may maintain the induction of proliferation and activation of effector cells such as NK cells and T cells by the IL-2 polypeptide.

[0154] The mutant interleukin-2 (IL-2) polypeptides contained in the immune complex described herein may include at least one amino acid mutation that eliminates or reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, while maintaining the affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, compared to the wild-type IL-2 polypeptide.

[0155] Mutants of the human IL-2 polypeptide (hIL-2 polypeptide) with reduced affinity for CD25 can be created, for example, by amino acid substitutions at amino acid positions 35, 38, 42, 43, 45, or 72 or combinations thereof (numbering of the human IL-2 polypeptide sequence in SEQ ID NO: 147). Examples of amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. The specific mutant IL-2 polypeptides useful in immune complexes described herein include amino acid mutations, or combinations thereof, at amino acid positions corresponding to residues 42, 45, or 72 of the human IL-2 polypeptide. In one embodiment, the amino acid mutation is an amino acid substitution selected from the group F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, and more specifically, an amino acid substitution selected from the group F42A, Y45A, and L72G. These mutant IL-2 polypeptides exhibit substantially similar binding affinity to the intermediate affinity IL-2 receptor, while showing substantially reduced affinity to the α-subunit of the IL-2 receptor and the high affinity IL-2 receptor compared to the wild-type IL-2 polypeptide.

[0156] Other characteristics of useful variant IL-2 polypeptides may include the ability to induce proliferation of IL-2 receptor-containing T cells and / or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-containing T cells and / or NK cells, the ability to generate interferon (IFN)-γ as a secondary cytokine by NK cells, reduced ability to induce secondary cytokine production (particularly IL-10 and TNF-α) by peripheral blood mononuclear cells (PBMCs), reduced ability to activate regulatory T cells, reduced ability to induce apoptosis in T cells, and a reduced in vivo toxicity profile.

[0157] A specific mutant IL-2 polypeptide useful in the present invention contains three amino acid mutations that preserve the affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, but have little to no affinity for the α-subunit of the IL-2 receptor. In one embodiment, the three amino acid mutations are located at amino acid residues 42, 45, and 72 of the wild-type (WT) human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147). In one embodiment, the three amino acid mutations are amino acid substitutions. In one embodiment, the three amino acid mutations are amino acid substitutions selected from the group F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a particular embodiment, the three amino acid mutations are amino acid substitutions F42A, Y45A, and L72G (numberings for the human IL-2 polypeptide sequence of SEQ ID NO: 147).

[0158] In certain embodiments, an amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor by at least one-fifth, specifically at least one-tenth, and more specifically at least one-twenty-fifth. In certain embodiments, if there are more than one amino acid mutations that reduce the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor, the combination of these amino acid mutations may reduce the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor by at least one-thirtieth, at least one-fiftieth, or even further at least one-hundredth. In one embodiment, an amino acid mutation, or a combination of amino acid mutations, eliminates the affinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor so that binding is not detectable by surface plasmon resonance.

[0159] Substantially similar binding to the intermediate affinity receptor, i.e., conservation of the affinity of the mutant IL-2 polypeptide to the receptor, is achieved when the mutant IL-2 polypeptide exhibits more than approximately 70% of the affinity of the wild-type mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor. The mutant IL-2 polypeptides described herein may exhibit more than approximately 80%, and even more than approximately 90%, of such affinity.

[0160] Combining the removal of O-glycosylation in the IL-2 polypeptide with a reduction in the affinity of IL-2 for the α-subunit of the IL-2 receptor can yield an IL-2 protein with improved properties. For example, the absence of the O-glycosylation site results in a more homogeneous product when the mutant IL-2 polypeptide is expressed in mammalian cells such as CHO or HEK cells.

[0161] Therefore, in certain embodiments, the mutant IL-2 polypeptide contains an additional amino acid mutation that eliminates the O-glycosylation site of IL-2 at the position corresponding to residue 3 of human IL-2. In one embodiment, the additional amino acid mutation that eliminates the O-glycosylation site of IL-2 at the position corresponding to amino acid residue 3 of the human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147) is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In certain embodiments, the additional amino acid mutation is the amino acid substitution T3A.

[0162] In certain embodiments, the mutant IL-2 polypeptide is essentially a full-length IL-2 polypeptide. In certain embodiments, the mutant IL-2 polypeptide is a mutant human IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide includes the amino acid sequence of SEQ ID NO: 147 having at least one amino acid mutation that causes the mutant IL-2 polypeptide to lose or reduce its affinity for the α-subunit of the IL-2 receptor compared to an IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 147 without the mutation, but preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor. In another embodiment, the mutant IL-2 polypeptide includes the amino acid sequence of SEQ ID NO: 148 having at least one amino acid mutation that causes the mutant IL-2 polypeptide to lose or reduce its affinity for the α-subunit of the IL-2 receptor compared to an IL-2 polypeptide containing SEQ ID NO: 148 without the mutation, but preserves the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor. In another embodiment, the mutant IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 199 having at least one amino acid mutation that causes the mutant IL-2 polypeptide to lose or reduce its affinity for the α-subunit of the IL-2 receptor, but retains the affinity of the mutant IL-2 polypeptide for the intermediate affinity IL-2 receptor, compared to the IL-2 polypeptide containing SEQ ID NO: 199 without the mutation.

[0163] In specific embodiments, mutant IL-2 polypeptides can induce one or more cellular responses selected from the group consisting of activated T lymphocyte proliferation, activated T lymphocyte differentiation, cytotoxic T cell (CTL) activity, activated B cell proliferation, activated B cell differentiation, natural killer (NK) cell proliferation, NK cell differentiation, cytokine secretion by activated T cells or NK cells, and NK / lymphocyte-activated killer (LAK) antitumor cytotoxicity.

[0164] In one embodiment, the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide induces less activation-induced cell death (AICD) in T cells compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a reduced toxicity profile in vivo compared to the wild-type IL-2 polypeptide. In one embodiment, the mutant IL-2 polypeptide has a longer serum half-life compared to the wild-type IL-2 polypeptide.

[0165] The specific variant IL-2 polypeptide useful in the present invention contains four amino acid substitutions at positions corresponding to amino acid residues 3, 42, 45, and 72 of the human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147). The specific amino acid substitutions are T3A, F42A, Y45A, and L72G. As shown in International Publication No. 2012 / 107417, the quadruple variant IL-2 polypeptide does not show detectable binding to CD25 and exhibits reduced ability to induce apoptosis in T cells. reg This variant exhibits reduced ability to induce IL-2 signaling in cells and a reduced in vivo toxicity profile. However, this mutant IL-2 polypeptide retains the ability to activate IL-2 signaling in effector cells, induce effector cell proliferation, and generate IFN-γ as a secondary cytokine by NK cells.

[0166] Furthermore, the quadruple mutant IL-2 polypeptide possesses even more advantageous properties, such as reduced surface hydrophobicity, good stability, and good expression yield, as described in WO2012 / 107417. Unexpectedly, the quadruple mutant IL-2 polypeptide also offers an extended serum half-life compared to wild-type IL-2.

[0167] In the present invention, useful mutant IL-2 polypeptides may have mutations not only in the region of the IL-2 polypeptide that forms the interface between the IL-2 polypeptide and CD25 or the glycosylation site, but also in one or more mutations in the amino acid sequence outside these regions. Such additional mutations in the human IL-2 polypeptide may provide additional advantages, such as increased expression or stability. For example, as described in U.S. Patent No. 4,518,584, which is incorporated herein by reference in its entirety, the cysteine ​​at position 125 (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147) may be replaced with a neutral amino acid such as serine, alanine, threonine, or valine to obtain the C125S mutant IL-2 polypeptide, the C125A mutant IL-2 polypeptide, the C125T mutant IL-2 polypeptide, or the C125V mutant IL-2 polypeptide, respectively. As described therein, the N-terminal alanine residue of the IL-2 polypeptide may be deleted to obtain mutants such as des-A1 C125S or des-A1 C125A. Alternatively, or conjugately, mutant IL-2 polypeptides may include mutations in which the methionine normally occurring at position 104 of the wild-type human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147) is replaced by a neutral amino acid such as alanine (see U.S. Patent No. 5,206,344). The resulting mutants, e.g., des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-A1 M104A C125A IL-2, or M104A C125S IL-2 (these and other mutants are found in U.S. Patent No. 5,116,943 and Weiger et al., Eur J Biochem 180,295-300 (1989)), may be used in combination with specific IL-2 mutants of the present invention.

[0168] Therefore, in certain embodiments, the mutant IL-2 polypeptide contains an additional amino acid mutation at the position corresponding to residue 125 of the human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147). In one embodiment, the additional amino acid mutation is the amino acid substitution C125A.

[0169] IL-2 variants may also contain an amino acid substitution at the position corresponding to 126 in human IL-2 (shown in Sequence ID No. 147), specifically the amino acid substitution Q126T. The Q126T substitution reduces binding to the intermediate affinity IL-2 receptor (consisting of the β and γ subunits of the IL-2 receptor), further reducing the peripheral activity of the PD-1-reg-IL-2v molecule in vivo.

[0170] In one embodiment, the mutant IL-2 polypeptide contains 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, or 5 or fewer amino acid mutations compared to the corresponding wild-type human IL-2 polypeptide sequence, for example, the human IL-2 sequence of SEQ ID NO: 147. In a particular embodiment, the mutant IL-2 polypeptide contains 5 or fewer amino acid mutations compared to the corresponding wild-type human IL-2 polypeptide sequence, for example, the human IL-2 sequence of SEQ ID NO: 147. In another particular embodiment, the mutant IL-2 polypeptide contains 6 or fewer amino acid mutations compared to the corresponding wild-type human IL-2 polypeptide sequence, for example, the human IL-2 sequence of SEQ ID NO: 147.

[0171] In one embodiment, the mutant IL-2 polypeptide contains the sequence of SEQ ID NO: 146. In one embodiment, the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO: 146. In one embodiment, the mutant IL-2 polypeptide contains the sequence of SEQ ID NO: 199. In one embodiment, the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO: 199.

[0172] D. Linker Linkers, such as peptide linkers, contained in the immune complexes described herein may be used to bind or link two polypeptides described herein together. In some embodiments, the linkers described herein are used to link an IL-2 polypeptide (e.g., human IL-2 polypeptide, e.g., mutant IL-2 polypeptide; e.g., mutant human IL-2 polypeptide) to a DBA moiety described herein. In certain embodiments, the linker is used to link an IL-2 polypeptide to a DBA moiety, and the IL-2 polypeptide is linked to the VH of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VH]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide). In certain embodiments, a linker is used to connect an IL-2 polypeptide to a DBA portion, and the IL-2 polypeptide is connected to the VL of the DBA portion in the following orientation: N-[IL-2 polypeptide]-[linker]-[VL]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0173] In other embodiments, a linker may be used to ligate the anti-PD-1 antibody portion or Fc subunit with the IL-2 polypeptide.

[0174] In some embodiments, the linker is 5 to 30 amino acids long, preferably 20 amino acids long. In some embodiments, the linker is an amino acid sequence (G2SG2) x The linker includes, where x is an integer from 1 to 6 (sequences 149 to 154). In some embodiments, the linker includes one of the amino acid sequences from sequence numbers 149 to 154. In a preferred embodiment, the linker includes the amino acid sequence GGSGGGGSGG (sequence number 150) or GGSGGGGSGGGGSGGGGGGG (sequence number 152). In a preferred embodiment, the linker consists of the amino acid sequence GGSGGGGSGG (sequence number 150) or GGSGGGGSGGGGSGGGGGGG (sequence number 152).

[0175] In other embodiments, the linker is an amino acid sequence (G4S) xIt includes, where x is an integer from 1 to 4 (sequence numbers 192 to 195). In certain other embodiments, the linker includes the amino acid sequence of sequence number 194. In certain other embodiments, the linker consists of the amino acid sequence of sequence number 194.

[0176] E. Immune complex In some embodiments, the immune complex described herein comprises (a) a first binding domain comprising (i) an IL-2 polypeptide as described herein, (ii) a linker as described herein, and (iii) a double-binding antibody (DBA) moiety as described herein that binds to PD-1 and IL-2 polypeptides in a substantially mutually exclusive manner, wherein the first binding domain is configured such that (i) when the DBA moiety is bound to the IL-2 polypeptide, the binding of the IL-2 polypeptide to its receptor is substantially blocked, and (ii) when the DBA is bound to PD-1, the binding of the DBA moiety to the IL-2 polypeptide is substantially blocked, and the IL-2 polypeptide can bind to the IL-2 receptor; and (b) a second binding domain comprising an anti-PD-1 antibody moiety as described herein, comprising VH and VL.

[0177] For example, in some embodiments, the immune complex described herein comprises (a) a first binding domain comprising (i) an IL-2 polypeptide as described herein, (ii) a linker as described herein, and (iii) a double-binding antibody (DBA) moiety as described herein that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the IL-2 polypeptide, linker and DBA moiety are oriented as follows: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- is the N-terminus of the polypeptide). The first binding domain is connected by (where -C represents the C-terminus of the polypeptide), and the first binding domain is configured such that (i) when the DBA portion is bound to the IL-2 polypeptide, the binding of the IL-2 polypeptide to its receptor is substantially blocked, and (ii) when the DBA is bound to PD-1, the binding of the DBA portion to the IL-2 polypeptide is substantially blocked, and the IL-2 polypeptide can bind to the IL-2 receptor; and (b) a second binding domain comprising an anti-PD-1 antibody portion as described herein, including VH and VL.

[0178] In some embodiments, the immune complex further comprises an Fc domain containing a first subunit and a second subunit. In some embodiments, the DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and / or (b) the anti-PD-1 antibody portion and the second subunit are connected in the following orientation: N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0179] In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the IgG Fc domain is an IgG1Fc domain. In some embodiments, the Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain is a human IgG1Fc domain. In some embodiments, the first subunit comprises one or more CH domains selected from a first CH2 (CH21) domain and / or a first CH3 (CH31) domain, and the second subunit comprises one or more CH domains selected from a second CH2 (CH22) domain and / or a second CH3 (CH32) domain. In some embodiments, at least one of the one or more CH domains is paired with another CH domain. In some embodiments, the CH31 domain and the CH32 domain each have a projection or cavity, and the projection or cavity of the CH31 domain can be positioned in the cavity or projection of the CH32 domain, respectively. In some embodiments, the CH31 domain and the CH32 domain associate at the interface between the projection and the cavity. In some embodiments, the CH21 domain and the CH22 domain each comprise a projection or cavity, and the projection or cavity of the CH21 domain can be positioned in the cavity or projection of the CH22 domain, respectively. In some embodiments, the CH21 domain and the CH22 domain associate at the interface between the projection and the cavity. In some embodiments, (a) the first subunit comprises a tryptophan residue at position 366, and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index), or (b) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit comprises a tryptophan residue at position 366 (numbered according to the Kabat EU index).

[0180] In some embodiments, the first subunit and / or the second subunit include an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering follows the Kabat EU index).

[0181] In some embodiments, the DBA moiety and the anti-PD-1 antibody moiety bind to different epitopes of PD-1. In some embodiments, the DBA moiety and the anti-PD-1 antibody moiety bind to the same epitope of PD-1.

[0182] In some embodiments, binding of the DBA portion to PD-1 inhibits the binding of PD-1 to PD-L1, and / or binding of the anti-PD-1 antibody portion to PD-1 inhibits the binding of PD-1 to PD-L1. In some embodiments, binding of the DBA portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, or binding of the anti-PD-1 antibody portion to PD-1 does not inhibit the binding of PD-1 to PD-L1. In some embodiments, (a) binding of the DBA portion to PD-1 inhibits the binding of PD-1 to PD-L1, and binding of the anti-PD-1 antibody portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, or (b) binding of the DBA portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, and binding of the anti-PD-1 antibody portion to PD-1 inhibits the binding of PD-1 to PD-L1. In some embodiments, binding of the DBA portion to PD-1 inhibits the binding of PD-1 to PD-L1, and binding of the anti-PD-1 antibody portion to PD-1 inhibits the binding of PD-1 to PD-L1. In some embodiments, binding of the DBA portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, and binding of the anti-PD-1 antibody portion to PD-1 does not inhibit the binding of PD-1 to PD-L1.

[0183] The immune complexes described herein may comprise various combinations of the DBA moiety, anti-PD-1 antibody moiety, IL-2 polypeptide, and linker described herein.

[0184] In some embodiments, the immune complex may include any DBA portion as disclosed herein, for example, in Section A above. In some examples, the DBA portion includes at least one, at least two, at least three, at least four, at least five, or all six CDRs (e.g., including one, two, three, four, five, or six CDRs) containing the amino acid sequences of the SEQ ID NOs shown in Table 1. In some cases, the DBA portion includes VH and / or VL containing the amino acid sequences of the SEQ ID NOs shown in Table 1.

[0185] In some embodiments, the immune complex may include any anti-PD-1 antibody moiety as disclosed herein, for example, in Section B above. In some examples, the anti-PD-1 antibody moiety includes at least one CDR, at least two CDRs, at least three CDRs, at least four CDRs, at least five CDRs, or all six CDRs (e.g., including one, two, three, four, five, or six CDRs) containing amino acid sequences of the SEQ ID NOs shown in Table 2. In some cases, the anti-PD-1 antibody moiety includes VH and / or VL containing amino acid sequences of the SEQ ID NOs shown in Table 2.

[0186] In some embodiments, the immune complex may include any IL-2 portion as disclosed herein, for example, in Section C above.

[0187] In some embodiments, the immune complex may include any linker as disclosed herein, for example, in Section D above.

[0188] Specific immune complexes containing specific combinations of the DBA portion, anti-PD-1 antibody portion, IL-2 polypeptide, and linker described herein are shown in Table 3 (including immune complexes having the conventional Fab DBA portion and the crossFab anti-PD-1 antibody portion), Table 4 (including immune complexes having the crossFab DBA portion and the conventional Fab anti-PD-1 antibody portion), and Table 5 (including immune complexes having the conventional Fab DBA portion and the scFv anti-PD-1 antibody portion). [Table 3] [Table 4] [Table 5]

[0189] In some embodiments, the immune complex comprises a DBA portion and an anti-PD-1 antibody portion, (i) the DBA portion comprises CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and QQYNSFPVT (SEQ ID NO: 57). (ii) The anti-PD-1 antibody portion comprises CDR-L3, and (ii) the anti-PD-1 antibody portion comprises CDR-H1 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21). In some embodiments, (i) the DBA portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, (i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 67, and a VL containing the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 31, and a VL containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, the DBA portion is AB002345, and the anti-PD-1 antibody portion is 0376 deimmunized. In some embodiments, the immune complex has construct IDs P1AI7476, P1AI7440, P1AJ1837, P1AI7464, P1AL2287, P1AM2983, or P1AK3171.

[0190] In some embodiments, the immune complex comprises a DBA portion and an anti-PD-1 antibody portion, (i) the DBA portion comprises CDR-H1 containing the amino acid sequence AYYIH (SEQ ID NO: 82), CDR-H2 containing the amino acid sequence WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 containing the amino acid sequence GHYYGKTDY (SEQ ID NO: 84), CDR-L1 containing the amino acid sequence RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 containing the amino acid sequence GASSLQS (SEQ ID NO: 74), and the amino acid sequence QESYTSSNT (SEQ ID NO: 75). (ii) The anti-PD-1 antibody portion comprises CDR-L3 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21). In some embodiments, the DBA portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 85, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 76, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, (i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 85, and a VL containing the amino acid sequence of SEQ ID NO: 76, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 31, and a VL containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, the DBA portion is AB003637, and the anti-PD-1 antibody portion is 0376 deimmunized. In some embodiments, the immune complex has construct IDs P1AI7474, P1AI7438, P1AJ1839, P1AI7462, or P1AK3173.

[0191] In some embodiments, the immune complex comprises a DBA portion and an anti-PD-1 antibody portion, (i) the DBA portion comprises CDR-H1 containing the amino acid sequence AYYIH (SEQ ID NO: 82), CDR-H2 containing the amino acid sequence WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 containing the amino acid sequence GHYYGKTDY (SEQ ID NO: 84), CDR-L1 containing the amino acid sequence RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 containing the amino acid sequence GASSLQS (SEQ ID NO: 74), and the amino acid sequence QESYTSSNT (SEQ ID NO: 75). (ii) The anti-PD-1 antibody portion comprises CDR-L3 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21). In some embodiments, the DBA portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 103, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, (i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 103, and a VL containing the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 31, and a VL containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, the DBA portion is deimmunized with AB003637, and the anti-PD-1 antibody portion is deimmunized with 0376. In some embodiments, the immune complex has the construct ID P1AJ2534 or P1AK3185.

[0192] In some embodiments, the immune complex comprises a DBA portion and an anti-PD-1 antibody portion, (i) the DBA portion comprises CDR-H1 containing the amino acid sequence AYYIH (SEQ ID NO: 82), CDR-H2 containing the amino acid sequence WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 containing the amino acid sequence GHYYGKTDY (SEQ ID NO: 84), CDR-L1 containing the amino acid sequence RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 containing the amino acid sequence GASSLQS (SEQ ID NO: 74), and the amino acid sequence QESYTSSNT (SEQ ID NO: 75). (ii) The anti-PD-1 antibody portion comprises CDR-L3 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21). In some embodiments, the DBA portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 111, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some embodiments, (i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 111, and a VL containing the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 31, and a VL containing the amino acid sequence of SEQ ID NO: 22. In some embodiments, the DBA portion is deimmunized AB003637, and the anti-PD-1 antibody portion is deimmunized 0376. In some embodiments, the immune complex has the construct ID P1AJ2535.

[0193] In some embodiments, the immune complex comprises a DBA portion and an anti-PD-1 antibody portion, (i) the DBA portion comprises CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57). (ii) The anti-PD-1 antibody portion comprises CDR-L3 containing the sequence, and (ii) the anti-PD-1 antibody portion comprises CDR-H1 containing the amino acid sequence of SYWMS (SEQ ID NO: 10), CDR-H2 containing the amino acid sequence of AISGSGGSRYYAESVKG (SEQ ID NO: 11), CDR-H3 containing the amino acid sequence of SPLQWIDV (SEQ ID NO: 12), CDR-L1 containing the amino acid sequence of RASQGISSWLA (SEQ ID NO: 1), CDR-L2 containing the amino acid sequence of EASSLQS (SEQ ID NO: 2), and CDR-L3 containing the amino acid sequence of QQANQFPFT (SEQ ID NO: 3). In some embodiments, (i) the DBA portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 13, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, (i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 67, and a VL containing the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 13, and a VL containing the amino acid sequence of SEQ ID NO: 4. In some embodiments, the DBA portion is AB002345, and the anti-PD-1 antibody portion is AB003058. In some embodiments, the immune complex has construct IDs P1AI7473, P1AI7443, P1AI7467, or P1AK3169.

[0194] In some embodiments, the immune complex comprises a DBA portion and an anti-PD-1 antibody portion, (i) the DBA portion comprises CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and QQYNSFPVT (SEQ ID NO: 57). (ii) The anti-PD-1 antibody portion comprises CDR-H1 containing the amino acid sequence of SYAMS (SEQ ID NO: 46), CDR-H2 containing the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), CDR-H3 containing the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO: 48), CDR-L1 containing the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), CDR-L2 containing the amino acid sequence of TASSLQS (SEQ ID NO: 38), and CDR-L3 containing the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39). In some embodiments, (i) the DBA portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 49, and / or a VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 40. In some embodiments, (i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 67, and a VL containing the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 49, and a VL containing the amino acid sequence of SEQ ID NO: 40. In some embodiments, the DBA portion is AB002345, and the anti-PD-1 antibody portion is a 1040 affinity matured type. In some embodiments, the immune complex has construct IDs P1AI7441, P1AJ1838, P1AI7455, or P1AI7465.

[0195] In some embodiments, the immune complex comprises (a) a first binding domain comprising (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146, (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152, and (iii) a DBA moiety that binds to the PD-1 and IL-2 polypeptides in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, and the IL-2 polypeptide, linker, and DBA moiety are linked in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and when the DBA moiety is bound to the IL-2 polypeptide, IL (b) a first binding domain in which the binding of the IL-2 polypeptide to its receptor is substantially blocked, and when the DBA portion is bound to PD-1, the DBA portion is blocked from binding to the IL-2 polypeptide, and the IL-2 polypeptide can bind to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody portion, wherein the anti-PD-1 antibody portion is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL; (c) an Fc region comprising a first subunit and a second subunit, wherein the first subunit comprises a tryptophan residue at position 366, and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (Kabat (i) numbered according to the EU index, or (ii) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (numbered according to the Kabat EU index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).(a) The DBA portion includes an Fc region (according to the EU index), and the DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the anti-PD-1 antibody portion and the second subunit are connected according to N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and (a) the DBA portion is a conventional Fab molecule, and the anti-PD-1 antibody portion (b) A Fab molecule comprising a Fab heavy chain containing VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or (b) a Fab molecule comprising a Fab heavy chain containing VH and CH1 of the DBA portion and a Fab light chain containing VL of the DBA portion and CL of the light chain, wherein the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other, and the anti-PD-1 antibody portion is a conventional Fab molecule.

[0196] In some embodiments, the immune complex comprises (a) a first binding domain, (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146, (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152, and (iii) a double-binding antibody (DBA) moiety that binds to the PD-1 and IL-2 polypeptides in a mutually exclusive manner, and is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, wherein the IL-2 polypeptide, linker, and DBA moiety are linked by the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the DBA moiety (i) The first binding domain comprises (b) an anti-PD-1 antibody moiety, wherein the DBA moiety is blocked from binding to the IL-2 polypeptide when bound to PD-1, and the DBA moiety is blocked from binding to the IL-2 polypeptide when bound to PD-1, and the IL-2 polypeptide can bind to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is scFv comprising VH and VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein the first subunit comprises a tryptophan residue at position 366, and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (Kabat (i) numbered according to the EU index, or (ii) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (numbered according to the Kabat EU index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).The DNA comprises an Fc region (according to the EU index), the DBA portion and the first subunit connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the anti-PD-1 antibody portion and the second subunit connected according to N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0197] In some embodiments, the immune complex comprises (a) a first binding domain comprising (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 199, (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152, and (iii) a DBA moiety that binds to the PD-1 and IL-2 polypeptides in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, and the IL-2 polypeptide, linker, and DBA moiety are linked in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide (b) a first binding domain in which the peptide's binding to its receptor is substantially blocked, and when the DBA portion is bound to PD-1, the DBA portion is blocked from binding to the IL-2 polypeptide, and the IL-2 polypeptide can bind to its receptor; (c) an Fc region comprising a first subunit and a second subunit, where (i) the first subunit comprises a tryptophan residue at position 366, and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (Kabat (i) numbered according to the EU index, or (ii) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (numbered according to the Kabat EU index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).including an Fc region according to the EU index), and the DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (where N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the anti-PD-1 antibody portion and the second subunit are connected according to N-[anti-PD-1 antibody portion]-[second subunit]-C (where N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), (a) the DBA portion is a conventional Fab molecule, and the anti-PD-1 antibody portion is a Fab molecule comprising a Fab heavy chain containing the VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing the VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced with each other, or (b) the DBA portion is a Fab molecule comprising a Fab heavy chain containing the VH and CH1 of the DBA portion and a Fab light chain containing the VL and light chain CL of the DBA portion, wherein the CH1 of the Fab heavy chain and the CL of the Fab light chain are replaced with each other, and the anti-PD-1 antibody portion is a conventional Fab molecule.

[0198] In some embodiments, the immune complex comprises (a) a first binding domain, (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 199, (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152, and (iii) a double-binding antibody (DBA) moiety that binds to the PD-1 and IL-2 polypeptides in a mutually exclusive manner, and is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, wherein the IL-2 polypeptide, linker, and DBA moiety are linked in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the DBA moiety binds to the IL-2 polypeptide. (i) The first binding domain comprises (b) an anti-PD-1 antibody moiety, wherein the DBA moiety is blocked from binding to the IL-2 polypeptide when bound to PD-1, and the IL-2 polypeptide can bind to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is scFv comprising VH and VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein the first subunit comprises a tryptophan residue at position 366, and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (Kabat (i) numbered according to the EU index, or (ii) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (numbered according to the Kabat EU index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).The DNA comprises an Fc region (according to the EU index), the DBA portion and the first subunit connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the anti-PD-1 antibody portion and the second subunit connected according to N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0199] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 98. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 96, a second polypeptide having an amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 98. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 96, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 94, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 98. In some embodiments, the immune complex is P1AI7473.

[0200] In one aspect, the immune complex comprises a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one aspect, the immune complex comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 102, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108. In one aspect, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 104, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 102, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some aspects, the immune complex is P1AI7474.

[0201] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 96, a second polypeptide having an amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 96, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 94, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some embodiments, the immune complex is P1AI7476.

[0202] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 104, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 104, a second polypeptide having an amino acid sequence of SEQ ID NO: 110, a third polypeptide having an amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 104, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 110, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some embodiments, the immune complex is P1AJ2534.

[0203] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 112, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 112, a second polypeptide having an amino acid sequence of SEQ ID NO: 110, a third polypeptide having an amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 112, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 110, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some embodiments, the immune complex is P1AJ2535.

[0204] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 116, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 114, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 116, a second polypeptide having an amino acid sequence of SEQ ID NO: 114, a third polypeptide having an amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 120. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 116, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 114, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the immune complex is P1AI7438.

[0205] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence of SEQ ID NO: 122, a third polypeptide having an amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 120. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 122, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the immune complex is P1AI7440.

[0206] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 128. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence of SEQ ID NO: 122, a third polypeptide having an amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 128. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 122, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 128. In some embodiments, the immune complex is P1AI7441.

[0207] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 130, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 132. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence of SEQ ID NO: 122, a third polypeptide having an amino acid sequence of SEQ ID NO: 130, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 132. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 122, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 130, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 132. In some embodiments, the immune complex is P1AI7443.

[0208] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence of SEQ ID NO: 134, a third polypeptide having an amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 120. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 134, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the immune complex is P1AJ1837.

[0209] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 128. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 124, a second polypeptide having an amino acid sequence of SEQ ID NO: 134, a third polypeptide having an amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 128. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 134, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 128. In some embodiments, the immune complex is P1AJ1838.

[0210] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 138, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 116, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 138, a second polypeptide having an amino acid sequence of SEQ ID NO: 116, a third polypeptide having an amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 120. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 138, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 116, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the immune complex is P1AJ1839.

[0211] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 197, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 197, a second polypeptide having an amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 197, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 94, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some embodiments, the immune complex is P1AL2287.

[0212] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 198, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 198, a second polypeptide having an amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 108. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 198, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 94, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some embodiments, the immune complex is P1AM2983.

[0213] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 187, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 185, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 189, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 191. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 187, a second polypeptide having an amino acid sequence of SEQ ID NO: 185, a third polypeptide having an amino acid sequence of SEQ ID NO: 189, and a fourth polypeptide having an amino acid sequence of SEQ ID NO: 191. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 187, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 185, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 189, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 191. In some embodiments, the immune complex is P1AI7455.

[0214] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 77, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 140, and a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 141. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 77, a second polypeptide having an amino acid sequence of SEQ ID NO: 140, and a third polypeptide having an amino acid sequence of SEQ ID NO: 141. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 77, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 140, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 141. In some embodiments, the immune complex is P1AI7462.

[0215] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 141. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence of SEQ ID NO: 141. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 141. In some embodiments, the immune complex is P1AI7464.

[0216] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 142. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence of SEQ ID NO: 142. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 142. In some embodiments, the immune complex is P1AI7465.

[0217] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 143. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence of SEQ ID NO: 143. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 143. In some embodiments, the immune complex is P1AI7467.

[0218] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 144. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence of SEQ ID NO: 144. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 144. In some embodiments, the immune complex is P1AK3169.

[0219] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 145. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 59, a second polypeptide having an amino acid sequence of SEQ ID NO: 136, and a third polypeptide having an amino acid sequence of SEQ ID NO: 145. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 145. In some embodiments, the immune complex is P1AK3171.

[0220] In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 77, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 140, and a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 145. In one embodiment, the immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 77, a second polypeptide having an amino acid sequence of SEQ ID NO: 140, and a third polypeptide having an amino acid sequence of SEQ ID NO: 145. In one embodiment, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 77, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 140, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 145. In some embodiments, the immune complex is P1AK3173.

[0221] In one aspect, the immune complex comprises a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145. In one aspect, the immune complex comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 140, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 145. In one aspect, the immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 92, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 140, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 145. In some aspects, the immune complex is P1AK3185.

[0222] In some embodiments, the immune complex is a reference immune complex. In some embodiments, the reference immune complex comprises (a) a first anti-PD-1 antibody moiety and a second anti-PD-1 antibody moiety, wherein the first anti-PD-1 antibody moiety and the second anti-PD-1 antibody moiety are Fab molecules comprising a Fab heavy chain containing VH and a Fab light chain containing VL, respectively, and (b) an Fc region comprising a first subunit and a second subunit, wherein (i) the first subunit comprises a tryptophan residue at position 366, the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index), or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit comprises a tryptophan residue at position 366 (Kabat EU index). (Numbered according to the EU Index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering is Kabat (a) The first anti-PD-1 antibody moiety and the first subunit are linked by the following orientation: N-[first anti-PD-1 antibody moiety]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the second anti-PD-1 antibody moiety and the second subunit are linked by N-[second anti-PD-1 antibody moiety]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and (a) the first and second anti-PD-1 antibody moieties are conventional Fab molecules, each containing a mutant IL-2 polypeptide with the amino acid sequence of SEQ ID NO: 146, a linker with the amino acid sequence of SEQ ID NO: 194, and the first subunit are linked by N-[first subunit]-[linker]-[mutant IL-2 polypeptide]-C.

[0223] In some embodiments, each anti-PD-1 antibody portion of the reference immune complex includes CDR-H1 containing the amino acid sequence of SYTMS (SEQ ID NO: 166), CDR-H2 containing the amino acid sequence of TISGGGRDIYYPDSVKG (SEQ ID NO: 167), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 168), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 157), CDR-L2 containing the amino acid sequence of RSSTLES (SEQ ID NO: 158), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 159). In some embodiments, each anti-PD-1 antibody portion of the reference immune complex includes VH containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 169, and / or VL containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 160. In some embodiments, each anti-PD-1 antibody moiety of the reference immune complex includes VH containing the amino acid sequence of SEQ ID NO: 169 and VL containing the amino acid sequence of SEQ ID NO: 160. In some embodiments, each anti-PD-1 antibody moiety of the reference immune complex is O376.

[0224] In one embodiment, the reference immune complex comprises a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 170, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 179, and a third and a fourth polypeptide each having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 161. In one embodiment, the reference immune complex comprises a first polypeptide having an amino acid sequence of SEQ ID NO: 170, a second polypeptide having an amino acid sequence of SEQ ID NO: 179, and a third and a fourth polypeptide each having an amino acid sequence of SEQ ID NO: 161. In one embodiment, the reference immune complex comprises a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 170, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 179, and a third and a fourth polypeptide each having an amino acid sequence of SEQ ID NO: 161. In some embodiments, the reference immune complex is P1AE4422-14542 (i.e., constitutively on or constitutively on P1AE4422-14542 control).

[0225] In some embodiments, the reference immune complex is a constantially on immune complex. In some embodiments, the reference immune complex does not contain a DBA portion.

[0226] In some embodiments, the reference immune complex includes two DBA moieties. In some embodiments, the reference immune complex comprises (a) a first binding domain comprising (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 147, (ii) a linker comprising or consisting thereof the amino acid sequence of SEQ ID NO: 152, and (iii) a first DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the first DBA moiety is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, and the IL-2 polypeptide, the linker, and the first DBA moiety are linked in the following orientation: N-[IL-2 polypeptide]-[linker]-[first DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), wherein when the first DBA moiety is bound to the IL-2 polypeptide, the binding of the IL-2 polypeptide to its receptor is substantially blocked, and when the first DBA moiety is bound to PD-1, the first DBA moiety is blocked from binding to the IL-2 polypeptide (b) a first binding domain in which binding to the receptor is blocked and the IL-2 polypeptide can bind to its receptor, and (b) a second binding domain including a second DBA portion, the second DBA portion being a Fab molecule including a Fab heavy chain including VH and a Fab light chain including VL, and when the second DBA portion is bound to the IL-2 polypeptide, the binding to its receptor is substantially blocked, and when the second DBA portion is bound to PD-1, the binding to the IL-2 polypeptide is blocked and the IL-2 polypeptide can bind to its receptor, and (c) an Fc region including a first subunit and a second subunit, (i) the first subunit including a tryptophan residue at position 366, and the second subunit including a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (Kabat (i) Numbered according to the EU index, or (ii) the first subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit contains a tryptophan residue at position 366 (KabatThe first DBA portion and the first subunit each contain an Fc region (numbered according to the EU index), and each of the first and second subunits contains an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index), wherein the first DBA portion and the first subunit are connected in the following orientation: N-[first DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the second DBA portion and the second subunit are connected according to N-[second DBA portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and the first DBA portion and the second DBA portion are each conventional Fab molecules.

[0227] In one embodiment, the reference immune complex includes construct ID P1AM2158, two DBA moieties having sequence AB002345, and two DBA LCs including D1AW0211 (sequence number 59), DBA-IL-2 HC D1AN8689 (sequence number 136), and DBA HC C2184344765 (sequence number 196).

[0228] In some embodiments, the reference immune complex comprises two DBA portions, each of which comprises CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and CDR-L3 containing the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57). In some embodiments, each of the DBA portions comprises VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and / or VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58. In some embodiments, each of the DBA portions comprises VH containing the amino acid sequence of SEQ ID NO: 67 and VL containing the amino acid sequence of SEQ ID NO: 58. In some embodiments, each of the DBA portions is AB002345. In some embodiments, the reference immunocomplex has the construct ID P1AM2158 (i.e., compound C).

[0229] In one embodiment, the reference immune complex comprises a first and second polypeptide, each having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 59; a third polypeptide, each having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 136; and a fourth polypeptide, each having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 196. In one embodiment, the immune complex comprises a first and second polypeptide, each having an amino acid sequence of SEQ ID NO: 59; a third polypeptide, each having an amino acid sequence of SEQ ID NO: 136; and a fourth polypeptide, each having an amino acid sequence of SEQ ID NO: 196. In one embodiment, the immune complex comprises a first and second polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 59; a third polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 136; and a fourth polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 196. In some embodiments, the reference immune complex is P1AM2158 (compound C).

[0230] In some embodiments, the present invention provides mouse analogue versions of the immune complexes provided herein. Methods for producing mouse analogues are known in the art. The construct IDs of the mouse analogues of the immune complexes disclosed herein are shown in Table 6 below. [Table 6]

[0231] F. Additional antibodies In some embodiments, any additional antibodies described in this section may be used in association with the immune complexes described herein (e.g., in the PD-1 regulated IL-2 immune complex). In some embodiments, the immune complexes described herein (e.g., in the PD-1 regulated IL-2 immune complex) may comprise any additional antibodies described in this section, e.g., any one or more additional antibodies described in this section, any one or more linkers described herein (e.g., described in Section D), and / or any one or more IL-2 polypeptides described herein (e.g., described in Section C). In some embodiments, the IL-2 polypeptide may be linked to the N-terminus or C-terminus of either the heavy chain and / or light chain of the additional antibody, for example, via a linker, to generate the immune complex described herein. In preferred embodiments, the IL-2 polypeptide may be linked to the N-terminus of either the heavy chain and / or light chain of the additional antibody, for example, via a linker, to generate the immune complex described herein.

[0232] In some embodiments, the antibody comprises two DBA moieties, each containing the following six amino acid sequences: CDR-H1 containing CDR:RYYVH (SEQ ID NO: 64), CDR-H2 containing IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing GLFI (SEQ ID NO: 66), CDR-L1 containing RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing SASNLET (SEQ ID NO: 56), and CDR-L3 containing QQYNSFPVT (SEQ ID NO: 57). In some embodiments, each DBA moiety of the antibody is AB002345.

[0233] In some embodiments, each DBA portion of the antibody includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 69; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 70; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 71; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 72; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 60; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 61; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 62; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 63. In some embodiments, each DBA portion of the antibody comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 67; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 58; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each DBA portion of the antibody comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 67; (b) a VL containing the amino acid sequence of SEQ ID NO: 58; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each DBA portion of the antibody is AB002345.

[0234] In some embodiments, the antibody comprises two DBA moieties, each containing the following six amino acid sequences: CDR-H1 containing CDR:AYYIH (SEQ ID NO: 82), CDR-H2 containing CDR:WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 containing CDR:GHYYGKTDY (SEQ ID NO: 84), CDR-L1 containing CDR:RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 containing CDR:GASSLQS (SEQ ID NO: 74), and CDR-L3 containing CDR:QESYTSSNT (SEQ ID NO: 75). In some embodiments, each DBA moiety of the antibody is AB003637 or AB003637 deimmunized.

[0235] In some embodiments, each DBA portion of the antibody includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 87; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 88; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 89; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 90; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 78; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 79; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 80; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 81. In some embodiments, each DBA portion of the antibody comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 85; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 76; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each DBA portion of the antibody comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 85; (b) a VL containing the amino acid sequence of SEQ ID NO: 76; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each DBA portion of the antibody is AB003637.

[0236] In some embodiments, each DBA portion of the antibody includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 87; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 88; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 89; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 90; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 78; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 79; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 80; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 155. In some embodiments, the DBA portion comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 85; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 91; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each DBA portion of the antibody comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 85; (b) a VL containing the amino acid sequence of SEQ ID NO: 91; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each DBA portion of the antibody is deimmunized.

[0237] In some embodiments, the antibody comprises two anti-PD-1 antibody moieties, each containing the following six amino acid sequences: CDR-H1 containing CDR:SYWMS (SEQ ID NO: 10), CDR-H2 containing AISGSGGSRYYAESVKG (SEQ ID NO: 11), CDR-H3 containing SPLQWIDV (SEQ ID NO: 12), CDR-L1 containing RASQGISSWLA (SEQ ID NO: 1), CDR-L2 containing EASSLQS (SEQ ID NO: 2), and CDR-L3 containing QQANQFPFT (SEQ ID NO: 3). In some embodiments, each anti-PD-1 antibody moiety of the antibody is AB003058.

[0238] In some embodiments, each anti-PD-1 antibody portion of the antibody includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 15; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 16; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 17; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 18; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 6; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 7; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 8; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, each anti-PD-1 antibody portion of the antibody comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 13; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each anti-PD-1 antibody portion of the antibody comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 13; (b) a VL containing the amino acid sequence of SEQ ID NO: 4; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each anti-PD-1 antibody portion of the antibody is AB003058.

[0239] In some embodiments, the antibody comprises two anti-PD-1 antibody moieties, each containing the following six amino acid sequences: CDR-H1 (SEQ ID NO: 28), CDR-H2 (SEQ ID NO: 29), CDR-H3 (SEQ ID NO: 30), CDR-L1 (SEQ ID NO: 19), CDR-L2 (SEQ ID NO: 20), and CDR-L3 (SEQ ID NO: 21). In some embodiments, each anti-PD-1 antibody moiety of the antibody is deimmunized at 0376.

[0240] In some embodiments, each anti-PD-1 antibody portion of the antibody includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 33; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 34; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 35; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 36; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 24; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 25; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 26; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 27. In some embodiments, each anti-PD-1 antibody moiety of the antibody comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 31; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each anti-PD-1 antibody moiety of the antibody comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 31; (b) a VL containing the amino acid sequence of SEQ ID NO: 22; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each anti-PD-1 antibody moiety of the antibody is deimmunized at 0376.

[0241] In some embodiments, the antibody comprises two anti-PD-1 antibody moieties, each containing the following six amino acid sequences: CDR-H1 containing CDR:SYAMS (SEQ ID NO: 46), CDR-H2 containing CDR:VITGSGGSTYYADSVKG (SEQ ID NO: 47), CDR-H3 containing CDR:GEGYAGSSYFRASDI (SEQ ID NO: 48), CDR-L1 containing CDR:RASQSISSYLN (SEQ ID NO: 37), CDR-L2 containing CDR:TASSLQS (SEQ ID NO: 38), and CDR-L3 containing CDR:QQSYSTPLT (SEQ ID NO: 39). In some embodiments, each anti-PD-1 antibody moiety of the antibody is 1040 affinity matured.

[0242] In some embodiments, each anti-PD-1 antibody portion of the antibody includes one or more of the following eight framework regions (FRs): (i) FR-H1 containing the amino acid sequence of SEQ ID NO: 51; (ii) FR-H2 containing the amino acid sequence of SEQ ID NO: 52; (iii) FR-H3 containing the amino acid sequence of SEQ ID NO: 53; (iv) FR-H4 containing the amino acid sequence of SEQ ID NO: 54; (v) FR-L1 containing the amino acid sequence of SEQ ID NO: 42; (vi) FR-L2 containing the amino acid sequence of SEQ ID NO: 43; (vii) FR-L3 containing the amino acid sequence of SEQ ID NO: 44; and / or (viii) FR-L4 containing the amino acid sequence of SEQ ID NO: 45. In some embodiments, each anti-PD-1 antibody moiety of the antibody comprises (a) a VH containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 49; (b) a VL containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 40; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each anti-PD-1 antibody moiety of the antibody comprises (a) a VH containing the amino acid sequence of SEQ ID NO: 49; (b) a VL containing the amino acid sequence of SEQ ID NO: 40; or (c) the VH described in (a) and the VL described in (b). In some embodiments, each anti-PD-1 antibody moiety of the antibody is 1040 affinity matured.

[0243] In some embodiments, the antibody further comprises an Fc domain comprising a first subunit and a second subunit. In some embodiments, the first anti-PD-1 antibody moiety and the first subunit are connected in the following orientation: N-[first anti-PD-1 antibody moiety]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and / or (b) the second anti-PD-1 antibody moiety and the second subunit are connected in the following orientation: N-[second anti-PD-1 antibody moiety]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

[0244] In some embodiments, the Fc domain is an IgG Fc domain. In some embodiments, the IgG Fc domain is an IgG1Fc domain.

[0245] In some embodiments, the first subunit and / or the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering follows the Kabat EU index).

[0246] In some embodiments, the antibody may include any DBA portion disclosed herein, for example, in Section A above. In some examples, the DBA portion includes at least one, at least two, at least three, at least four, at least five, or all six CDRs (e.g., including one, two, three, four, five, or six CDRs) containing the amino acid sequences of the SEQ ID NOs shown in Table 1. In some cases, the DBA portion includes VH and / or VL containing the amino acid sequences of the SEQ ID NOs shown in Table 1.

[0247] In some embodiments, the antibody may comprise any anti-PD-1 antibody moiety disclosed herein, for example, in Section B above. In some examples, the anti-PD-1 antibody moiety comprises at least one CDR, at least two CDRs, at least three CDRs, at least four CDRs, at least five CDRs, or all six CDRs (e.g., one, two, three, four, five, or six CDRs) containing amino acid sequences of the SEQ ID NOs shown in Table 2. In some cases, the anti-PD-1 antibody moiety comprises VH and / or VL containing amino acid sequences of the SEQ ID NOs shown in Table 2.

[0248] The antibodies described herein may contain different DBA moieties or anti-PD-1 antibody moieties. Specific antibodies containing specific combinations of the DBA moieties or anti-PD-1 antibody moieties described herein are shown in Table 7 below. [Table 7]

[0249] In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 14; a third polypeptide, each containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 132; and a fourth polypeptide. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 14; and a third polypeptide and a fourth polypeptide, each containing the amino acid sequence of SEQ ID NO: 132. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 14; and a third polypeptide and a fourth polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 132. In some embodiments, the antibody is P1AI7512.

[0250] In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 32; a third polypeptide, each containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120; and a fourth polypeptide. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 32; and a third polypeptide and a fourth polypeptide, each containing the amino acid sequence of SEQ ID NO: 120. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 32; and a third polypeptide and a fourth polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 120. In some embodiments, the antibody is P1AH4157.

[0251] In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 50, and a third polypeptide and a fourth polypeptide, each containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 50, and a third polypeptide and a fourth polypeptide, each containing the amino acid sequence of SEQ ID NO: 128. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 50, and a third polypeptide and a fourth polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 128. In some embodiments, the antibody is P1AG3741.

[0252] In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 68, and a third polypeptide and a fourth polypeptide, each containing an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 59. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 68, and a third polypeptide and a fourth polypeptide, each containing the amino acid sequence of SEQ ID NO: 59. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 68, and a third polypeptide and a fourth polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 59. In some embodiments, the antibody is P1AI3784.

[0253] In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide, each containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 77. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide, each containing the amino acid sequence of SEQ ID NO: 77. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 77. In some embodiments, the antibody is P1AI7516.

[0254] In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide, each containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 92. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each containing the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide, each containing the amino acid sequence of SEQ ID NO: 92. In one embodiment, the antibody comprises a first polypeptide and a second polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide, each consisting of the amino acid sequence of SEQ ID NO: 92. In some embodiments, the antibody is P1AI7514.

[0255] G. Properties of immunocomplexes and / or antibodies In some examples, the immune complexes (e.g., PD-1 regulated IL-2 immune complexes) and / or antibodies (e.g., DBA and / or anti-PD-1 antibodies) disclosed herein may include any of the properties or any combination of properties shown in Sections 1 to 6 below.

[0256] 1. Antibody fragment In certain embodiments, the immune complexes described herein (e.g., PD-1 regulated IL-2 immune complexes (e.g., comprising at least one binding domain conjugated to an IL-2 polypeptide and binding to PD-1; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1) and / or antibodies (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) comprise one or more antibody fragments. In certain embodiments, the immune complexes provided herein (e.g., PD-1 regulated IL-2 immune complexes) or antibodies comprise antibody fragments.

[0257] Any suitable antibody fragment may be used. In one embodiment, the antibody fragment (e.g., the DBA portion or the anti-PD-1 antibody portion) is a Fab, Fab', Fab'-SH, or F(ab')2 fragment. In a specific example, the antibody fragment (e.g., the DBA portion or the anti-PD-1 antibody portion) is a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments (so-called "Fab" fragments), each containing the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain, in addition to the variable domains of the heavy and light chains (VH and VL, respectively). Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) of the constant domain hold a free thiol group. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of the Fab and F(ab')2 fragments that constitute salvage receptor-binding epitope residues and increase in vivo half-life, see U.S. Patent No. 5,869,046.

[0258] In another embodiment, the antibody fragment is a diabody, triabody, or tetrabody.

[0259] In a further embodiment, the antibody fragment is a single-stranded Fab fragment.

[0260] In another embodiment, the antibody fragment (e.g., the anti-PD-1 antibody portion) is a single-stranded variable fragment (scFv).

[0261] In another embodiment, the antibody fragment is a single-domain antibody.

[0262] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, the proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., Escherichia coli (E. coli)) as described herein.

[0263] 2. Chimeric and humanized immune complexes and / or antibodies In certain embodiments, the immune complexes provided herein (e.g., PD-1 regulated IL-2 immune complexes (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) and / or antibodies (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) are chimeric immune complexes or antibodies.

[0264] Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody contains a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from those of the parent antibody. A chimeric antibody contains its antigen-binding fragment.

[0265] In certain embodiments, a chimeric immune complex or antibody is a humanized immune complex or antibody. Typically, a non-human immune complex or antibody is humanized to retain the specificity and affinity of the parent non-human antibody while reducing its immunogenicity to humans. Typically, a humanized immune complex or antibody contains one or more variable domains in its CDR (or a portion thereof) derived from a non-human immune complex or antibody, and its FR (or a portion thereof) derived from a human antibody sequence. The humanized immune complex or antibody also optionally contains at least a portion of a human constant region. In some embodiments, several FR residues in the humanized immune complex or antibody are replaced with corresponding residues from a non-human immune complex or antibody (e.g., an antibody or antibody fragment(s) from which the CDR residues are derived) to restore or improve the specificity or affinity of the antibody, for example.

[0266] Humanized antibodies and methods for producing them are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents No. 5,821,337, No. 7,527,791, No. 6,982,321 and No. 7,087,409; Kashmiri et al., Methods This is described in 36:25-34 (2005) (graft junction of the specificity determination region (SDR)); Padlan, Mol.Immunol.28:489-498 (1991) ("Resurfacing" is described); Dall'Acqua et al., Methods 36:43-60 (2005) ("FR shuffling" is described); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br.J.Cancer,83:252-260 (2000) ("Guided selection" method for FR shuffling is described).

[0267] Human framework regions that may be used for humanization include: framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of the FR library (see, e.g., Baca et al. See al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996). Examples include, but are not limited to, these.

[0268] 3. Human immune complexes and antibodies In certain embodiments, the immune complexes provided herein (e.g., PD-1 regulated IL-2 immune complexes (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) and / or antibodies (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) are human immune complexes or antibodies, or comprise such a complex. Human immune complexes or antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0269] Human antibodies may be prepared by administering an immunogen to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, is located extrachromosomally, or is randomly incorporated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing the XENOMOUSE® technology; U.S. Patent No. 5,770,429 describing the HuMab® technology; U.S. Patent No. 7,041,870 describing the KM Mouse® technology; and U.S. Patent Publication No. 2007 / 0061900 describing the VelociMouse® technology. Human variable regions derived from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0270] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human xenomyeloma cell lines for producing human antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0271] Human antibodies can also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains.

[0272] 4. Multispecific immune complexes and multispecific antibodies In certain embodiments, multispecific immune complexes or multispecific antibodies are disclosed herein. A “multispecific immune complex” is a monoclonal immune complex having binding specificity to at least two distinct sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, a multispecific immune complex or multispecific antibody has three or more binding specificities. In certain embodiments, one binding specificity is for IL-2 polypeptide or PD-1 in a mutually exclusive manner, and the other specificity is for PD-1 only. Multispecific (e.g., bispecific) immune complexes or antibodies may be used to localize cytotoxic agents (e.g., IL-2 polypeptide) or cells to cells expressing PD-1. Multispecific immune complexes or antibodies may be prepared as full-length immune complexes or antibodies or antibody fragments.

[0273] Techniques for producing multispecific immune complexes or antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knob-in-hole" operations (see, for example, U.S. Patent No. 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific immune complexes or antibodies also involve manipulating the electrostatic steering effect to produce antibody Fc heterodimer molecules (see, e.g., International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992) and International Publication No. 2011 / 034605); using common light chain techniques to avoid light chain mispairing problems (see, e.g., International Publication No. 98 / 50431); and using "diabody" techniques to produce bispecific antibody fragments (see, e.g., Hollinger et al.) They may also be prepared by using single-stranded Fv(sFv) dimers (see, for example, Gruber et al., J.Immunol., 152:5368 (1994)); and by preparing the triplicate antibodies as described, for example, Tutt et al. J.Immunol. 147:60 (1991).

[0274] 5. Immune complexes and antibody variants In certain embodiments, amino acid sequence variants of immune complexes, antibodies, or IL-2 polypeptides provided herein are intended. For example, it may be desirable to alter the binding affinity and / or other biological properties of the immune complex or antibody. Amino acid sequence variants of PD-1-modulated IL-2 immune complexes or antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the immune complex or antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the immune complex, antibody, or IL-2 polypeptide. Deletions, insertions, and substitutions can be arbitrarily combined to reach the final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[0275] a) Substitution, insertion, and deletion variants In certain embodiments, an immune complex, antibody, or IL-2 polypeptide having one or more amino acid substitutions is provided. The target sites for substitutional mutagenesis include CDRs and FRs.

[0276] Conservative substitutions are shown in Table 8 under the heading "Conservative Substitutions." More substantial changes are provided in Table 8 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 8]

[0277] Amino acids can be classified as follows according to the common side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0278] Non-conservative substitution involves exchanging a member of one class with a member of another class.

[0279] One type of substitution variant involves substituting one or more hypervariable region residues of the parent antibody. Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) (e.g., increased affinity, decreased immunogenicity) in specific biological properties compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-matured antibodies and can be readily generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated, the variant antibody is displayed on a phage, and screened for specific biological activity (e.g., binding affinity).

[0280] For example, modifications (e.g., substitutions) may be made in the CDR to improve antibody affinity. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that come into contact with the antigen, and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by constructing a secondary library and then re-selecting from it is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity is the CDR-directed approach, which randomizes several CDR residues (e.g., 4-6 residues at a time). CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0281] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such alterations do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative alterations that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within a CDR. Such alterations may, for example, be outside the antigen-contact residue in the CDR. In the specific variant VH and VL sequences described above, each CDR is either unaltered or has one, two, or three or fewer amino acid substitutions.

[0282] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or target residue groups (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at the positions of amino acids that exhibit functional sensitivity to the initial substitution. Alternatively, or in addition to this, contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues may be targeted as candidate substitutions or removed. Variants may be screened to determine whether they possess the desired properties.

[0283] Amino acid insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of an antibody to an enzyme (e.g., ADEPT (for antibody-directed enzyme prodrug therapy)) or polypeptide, which increases the serum half-life of the antibody.

[0284] b) Glycosylated variants In certain embodiments, the immune complexes provided herein (e.g., PD-1 regulated IL-2 immune complexes (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) and / or antibodies (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) are modified to increase or decrease the degree to which the immune complex or antibody is glycosylated. The addition or deletion of glycosylation sites to PD-1 regulated IL-2 immune complexes or antibodies can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0285] If an immune complex or antibody contains an Fc region, the oligosaccharide bound to it may be altered. Native immune complexes or antibodies produced by mammalian cells typically contain branched or bibranched oligosaccharides, which are generally bound to Asn297 of the CH2 domain of the Fc region by N-bonding. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to the GlcNAc of the "stem" of the bibranched oligosaccharide structure. In some embodiments, the modification of oligosaccharides in antibodies described herein may be performed to produce antibody variants having specific improved properties.

[0286] In one embodiment, an antibody variant is provided having an oligosaccharide structure lacking a non-fucosylated oligosaccharide, i.e., fucose binding (direct or indirect) to the Fc region. Such a non-fucosylated oligosaccharide (also called a "defucosylated" oligosaccharide) is in particular an N-linked oligosaccharide lacking a fucose residue bound to a first GlcNAc in the stem of a branched oligosaccharide structure, and such an antibody is further referred to herein as a "defucosylated antibody." In one embodiment, an antibody variant is provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides). In certain embodiments, the defucosylation rate is approximately 65% ​​to 100%, 80% to 100%, or 80% to 95%. The rate of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides relative to the total of all oligosaccharides bound to Asn 297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2006 / 082515. Asn297 refers to the asparagine residue located at approximately 297 (EU numbering of Fc region residues) within the Fc region, although Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300 (e.g., Asn 299), due to slight sequence variations in the antibody. Antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may exhibit improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publications 2003 / 0157108 and 2004 / 0093621.

[0287] In one embodiment, the disclosure provides a defucosylated antibody variant having enhanced FcγRIIIa receptor binding. In one embodiment, the disclosure provides a defucosylated antibody variant having enhanced antibody-dependent cytotoxicity (ADCC). In one embodiment, the disclosure provides a defucosylated antibody variant having antibody-dependent phagocytic activity (ADCP).

[0288] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and International Publication No. 2004 / 056312, particularly Example 11), and knockout cell lines, e.g., alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al.) This includes cells in which GDP-fucose synthesis or transport protein is reduced or absent (see, for example, U.S. Patent Publications 2004259150, 2005031613, 2004132140, and 2004110282). See also Pereira et al., MABS (2018) 693-711.

[0289] In a further embodiment, antibody variants having bifid oligosaccharides are provided, for example, antibody variants in which a branched oligosaccharide bound to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); International Publication No. 99 / 54342, International Publication No. 2004 / 065540, and International Publication No. 2003 / 011878.

[0290] Antibody variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in International Publications 1997 / 30087; 1998 / 58964; and 1999 / 22764. c) Fc region variant

[0291] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of a PD-1-regulated IL-2 immune complex (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1) and / or an antibody (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) provided herein, thereby generating an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0292] In certain embodiments, the present invention envisions PD-1-modulated IL-2 immune complexes or antibody variants possessing some, but not all, effector functions, thereby becoming desirable candidates for applications where the half-life of the antibody in vivo is important, but specific effector functions (such as complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity) but retains FcRn binding ability. Monocytes express FcγRI, FcγRII, and FcγRIII, while NK cells, the primary cells for ADCC mediation, express only FcγRIII. The expression of FcR in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); and U.S. Patent No. 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., Acti® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, California), and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, Wisconsin)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively, or in addition to the above, the ADCC activity of the target molecule may be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and International Publication Nos. 2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J.Immunol.Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, S B et al., Int'l.Immunol. 18(12):1759-1769 (2006); International Publication No. 2013 / 120929).

[0293] Antibodies with reduced effector function include those having one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant, which has substitutions at residues 265 and 297 of alanine, as well as Fc variants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0294] Specific immune complexes or antibody variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056, International Publication No. 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001)).

[0295] In certain embodiments, an immune complex (e.g., a PD-1 regulated IL-2 immune complex (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) or antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333 and / or 334 (residue EU numbering) of the Fc region.

[0296] In certain embodiments, an immune complex (e.g., a PD-1 regulated IL-2 immune complex (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) moiety that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody moiety that binds to PD-1)) or antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., substitutions at positions 234 and 235 (residue EU numbering) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the immune complex or antibody variant further comprises D265A and / or P329G (residue EU numbering) in the Fc region derived from the human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2012 / 130831). In another embodiment, the substitutions are L234A, L235A and D265A (LALA-DA) within the Fc region derived from the human IgG1Fc region.

[0297] In certain embodiments, the Fc region includes modifications configured to facilitate association of a first Fc subunit with a second Fc subunit. A “knob-in-hole” operation of the immune complex may be used to generate a first arm containing a knob and a second arm containing a hole to which the knob of the first arm binds. In one embodiment, the knob of the multispecific antibody of the present invention may include a DBA portion. Alternatively, the knob of the immune complex or antibody of the present invention may include an anti-PD-1 antibody portion. The hole of the immune complex or antibody of the present invention may include a DBA portion. Alternatively, the hole of the immune complex or antibody of the present invention may include an anti-PD-1 antibody portion. The immune complexes and antibodies may be manipulated using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) techniques (see, e.g., International Publication No. 2009 / 080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011)). Immunocomplexes or antibodies can also be produced by manipulating the electrostatic steering effect to create antibody Fc heterodimer molecules (International Publication No. 2009 / 089004); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); or by using a leucine zipper to produce bispecific antibodies (e.g., Kostelny et al., J.Immunol., 148(5):1547-1553 (1992)).

[0298] By replacing amino acid residues in the CH3 domain of the second Fc subunit with amino acid residues having a larger side-chain volume, a projection (e.g., a knob) can be generated within the CH3 domain of the second Fc subunit that can be positioned in a cavity (e.g., a hole) within the CH3 domain of the first Fc subunit, and by replacing amino acid residues in the CH3 domain of the first Fc subunit with amino acid residues having a smaller side-chain volume, a cavity (e.g., a hole) can be generated within the CH3 domain of the first Fc subunit that can be positioned in a projection (e.g., a knob) within the CH3 domain of the second Fc subunit. In some embodiments, the CH3 domain of the second Fc subunit includes an amino acid substitution at T366, and the CH3 domain of the first Fc subunit includes amino acid substitutions at one, two, or all three of T366, L368, and / or Y407. In some embodiments, the CH3 domain of the second Fc subunit contains the amino acid substitution T366W, and the CH3 domain of the first Fc subunit contains one, two, or all three amino acid substitutions T366S, L368A, and / or Y407V.

[0299] In certain embodiments, an immune complex (e.g., a PD-1 regulated IL-2 immune complex (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) and / or an antibody (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) comprises an Fc region having one or more amino acid substitutions, e.g., substitutions at position, that improve FcγR binding (thereby improving effector function). In certain embodiments, the antibody variant includes an Fc region having at least one amino acid substitution of G236A, I332E, S298A, E333A, K334A, S239D, A330L, F243L, R292P, Y300L, V305I, P396L, L235V, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E (EU numbering of residues) (see, for example, Liu et al., Antibodies (Basel) (2020); 9(4):64).

[0300] In some embodiments, modifications are made in the Fc region that result in alterations (i.e., either improvements or reductions) to C1q binding and / or complement-dependent cell-mediated cytotoxicity (CDC), as disclosed, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0301] Antibodies responsible for the transfer of maternal IgG to the fetus, with extended half-lives and improved binding affinity to the neonatal Fc receptor (FcRn), are described in U.S. Patent Publication No. 2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Examples of such Fc variants include those with a substitution in one or more of the following Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (EU numbering of residues), for example, those with a substitution in Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0302] The Fc region residues crucial to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, for example, Dall'Acqua, WF, et al. J.Immunol 169(2002) 5171-5180). Residues i253, h310, h433, n434, and h435 (EU numbering of the residues) are involved in the interaction (Medesan, C., et al., Eur.J.Immunol.26(1996) 2533; Firan, M., et al., Int.Immunol.13(2001) 993; Kim, JK, et al., Eur.J.Immunol.24(1994) 542). Residues I253, H310, and H435 have been found to be important for the interaction between human Fc and mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are important for this interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J.Immunol.182(2009)7667-7671) have reported and investigated various mutants of residues 248-259, 301-317, 376-382, and 424-437 (EU numbering of residues).

[0303] In certain embodiments, an immune complex (e.g., a PD-1 regulated IL-2 immune complex (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) and / or an antibody (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253 and / or 310 and / or 435 (EU numbering of residues) in the Fc region. In certain embodiments, the immune complex or antibody comprises an Fc region having amino acid substitutions at positions 253, 310 and 435. In one embodiment, the substitutions are I253A, H310A and H435A within the Fc region derived from a human IgG1 Fc region. For example, see Grevys, A., et al., J.Immunol. 194(2015) 5497-5508.

[0304] In certain embodiments, an immune complex (e.g., a PD-1 regulated IL-2 immune complex (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) and / or an antibody (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 310 and / or 433 and / or 436 (residue EU numbering) of the Fc region. In certain embodiments, the immune complex or antibody comprises an Fc region having amino acid substitutions at positions 310, 433 and 436. In one embodiment, the substitutions are H310A, H433A and Y436A in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2014 / 177460).

[0305] In certain embodiments, an immune complex (e.g., a PD-1 regulated IL-2 immune complex (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) and / or an antibody (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., substitutions at positions 252 and / or 254 and / or 256 (EU numbering of residues) in the Fc region. In certain embodiments, the immune complex or antibody comprises an Fc region having amino acid substitutions at positions 252, 254 and 256. In one embodiment, the substitutions are M252Y, S254T and T256E in the Fc region derived from the human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260, 5,624,821, and International Publication No. 94 / 29351.

[0306] The C-terminus of the heavy chain of an immune complex or antibody as reported herein may be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus with one or two of the C-terminal amino acid residues removed. In one embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one embodiment of all embodiments reported herein, an antibody containing a heavy chain with the C-terminal CH3 domain as specified herein contains a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid position). In one embodiment of all embodiments presented herein, a PD-1 regulated IL-2 immune complex, or an antibody containing a heavy chain with the C-terminal CH3 domain as specified herein, contains a C-terminal glycine residue (G446, EU index numbering of amino acid position). In one of the embodiments reported herein, the antibody comprising a heavy chain containing the C-terminal CH3 domain as specified herein contains a C-terminal proline residue (P445, EU index numbering of amino acid position).

[0307] d) Cysteine-modified immune complexes and antibody variants In certain embodiments, it may be desirable to produce cysteine-modulated immune complexes or antibodies, such as THIOMAB® antibodies, in which one or more residues of the immune complex or antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues are located at accessible sites of the immune complex or antibody. By substituting these residues with cysteine, the reactive thiol group is positioned at an accessible site of the antibody and can be used to conjugate the antibody to other sites, such as a drug site or linker drug site, as further described herein, to create a PD-1 regulated IL-2 immune complex. Cysteine-modulated immune complexes or antibodies may be produced as described, for example, in U.S. Patents 7,521,541, 8,30,930, 7,855,275, 9,000,130, or International Publication 2016040856.

[0308] e) Immune complexes and antibody derivatives In certain embodiments, the immune complexes provided herein (e.g., PD-1 regulated IL-2 immune complexes (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, and an anti-PD-1 antibody moiety that binds to PD-1)) and / or antibodies (e.g., comprising two DBA moieties or two anti-PD-1 antibody moieties) may be further modified to include additional non-proteinoid moieties known and readily available in the art. Suitable moieties for derivatization of the immune complexes or antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in production due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, the polymers may be the same molecule or different molecules. In general, the number and / or types of polymers used for derivatization can be determined based on considerations such as the specific properties or functions of the antibody being improved, and whether the antibody derivative will be used for therapeutic purposes under defined conditions, but are not limited to these.

[0309] 6. Fc domain modification that promotes heterodimerization The immune complexes and antibodies of the present invention may comprise one or more DBA moieties and / or one or more anti-PD-1 antibody moieties fused to one or the other of two subunits of the Fc domain. Thus, the two subunits of the Fc domain typically comprise two non-identical polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides can result in several possible combinations of the two polypeptides. To improve the yield and purity of the immune complexes and antibodies in recombinant production, in some examples it is advantageous to introduce modifications to the Fc domain of the immune complex or antibody that facilitate the association of the desired polypeptide.

[0310] Therefore, in certain embodiments, the Fc domain of the immune complex or antibody according to the present invention may include modifications that facilitate the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is located in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located in the CH3 domain of the Fc domain.

[0311] To enhance heterodimerization, several methods exist for modifying the CH3 domain of the Fc domain, as described, for example, in International Publications 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publications 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012058768, 2013157954, and 2013096291. Typically, in such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both manipulated in a complementary manner so that each CH3 domain (or the heavy chain containing it) does not homodimerize with itself but heterodimerizes with other complementaryly manipulated CH3 domains (resulting in heterodimerization of the first and second CH3 domains, and no homodimer is formed between the two first CH3 domains or the two second CH3 domains). These different approaches to improved heavy chain heterodimerization are intended as alternatives in combination with the heavy-light chain modifications in immunocomplexes or antibodies according to the present invention (VH and VL exchange / substitution in one binding arm, as well as the introduction of oppositely charged amino acid substitutions at the CH1 / CL interface) to reduce light chain mispairing and Bence-Jones type byproducts.

[0312] In a specific embodiment, the modification that facilitates the association of the first and second subunits of the Fc domain is a so-called "knob-into-hole" modification, which includes a "knob" modification on one of the two subunits of the Fc domain and a "hole" modification on the other of the two subunits of the Fc domain.

[0313] This knob-into-hole technique is described, for example, in U.S. Patent No. 5,731,168 and 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a projection ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the projection can be positioned within the cavity to promote heterodimerization and inhibit homodimerization. The projection is constructed by replacing a smaller amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the projection is created at the interface of the second polypeptide by replacing a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0314] Accordingly, in certain embodiments, in the CH3 domain of the first subunit of the Fc domain of the immune complex or antibody, an amino acid residue is replaced with an amino acid residue having a larger side-chain volume, thereby generating a protrusion within the CH3 domain of the first subunit that can be positioned within a cavity in the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side-chain volume, thereby generating a cavity within the CH3 domain of the second subunit, in which the protrusion from the CH3 domain of the first subunit can be positioned.

[0315] Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0316] Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).

[0317] The protrusions and cavities can be created by altering the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis.

[0318] In specific embodiments, in the CH3 domain of the first subunit ("knob" subunit) of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit ("hole" subunit) of the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbering follows the Kabat EU index).

[0319] In further embodiments, in the first subunit of the Fc domain, the serine residue at position 354 is further replaced with a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is further replaced with a cysteine ​​residue (E356C), and in the second subunit of the Fc domain, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C) (numbering follows the Kabat EU index). The introduction of these two cysteine ​​residues creates a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0320] In certain embodiments, the first subunit of the Fc domain includes the amino acid substitutions S354C and T366W, and the second subunit of the Fc domain includes the amino acid substitutions Y349C, T366S, L368A and Y407V (numbering follows the Kabat EU index).

[0321] Other techniques for CH3 modification that enhance heterodimerization have been considered as alternatives to the present invention and are described, for example, in International Publication Nos. 96 / 27011, 98 / 050431, European Patent No. 1870459, International Publication Nos. 2007 / 110205, 2007 / 147901, 2009 / 089004, 2010 / 129304, 2011 / 90754, 2011 / 143545, 2012 / 058768, 2013 / 157954, and 2013 / 096291.

[0322] In one embodiment, the heterodimerization method described in European Patent Application Publication 1870459A1 is used instead. This method is based on the introduction of oppositely charged amino acids to specific amino acid positions at the CH3 / CH3 domain interface between two subunits of the Fc domain. A preferred embodiment of the immunocomplex or antibody of the present invention is an amino acid mutation R409D, K370E in one of the two CH3 domains (of the Fc domain) and an amino acid mutation D399K, E357K in the other CH3 domain of the Fc domain (numbering follows the Kabat EU index).

[0323] In another embodiment, the immune complex or antibody of the present invention comprises amino acid mutations T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally comprises amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K, E357K in the CH3 domain of the second subunit of the Fc domain (numbering follows the Kabat EU index).

[0324] In another embodiment, the immune complex or antibody of the present invention contains amino acid mutations S354C and T366W in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations Y349C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain; or the immune complex or antibody contains amino acid mutations Y349C and T366W in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations S354C, T366S, L368A, and Y407V in the CH3 domain of the second subunit of the Fc domain, and further contains amino acid mutations R409D and K370E in the CH3 domain of the first subunit of the Fc domain, and amino acid mutations D399K and E357K in the CH3 domain of the second subunit of the Fc domain (all numbering follows the Kabat EU index).

[0325] In one embodiment, the heterodimerization method described in International Publication No. 2013 / 157953 is used instead. In one embodiment, the first CH3 domain contains the amino acid mutation T366K, and the second CH3 domain contains the amino acid mutation L351D (numbering follows the Kabat EU index). In a further embodiment, the first CH3 domain contains a further amino acid mutation L351K. In a further embodiment, the second CH3 domain further contains amino acid mutations selected from Y349E, Y349D, and L368E (preferably L368E) (numbering follows the Kabat EU index).

[0326] In one embodiment, the heterodimerization method described in International Publication No. 2012 / 058768 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations L351Y, Y407A, and the second CH3 domain contains amino acid mutations T366A, K409F. In further embodiments, the second CH3 domain contains further amino acid mutations at positions T411, D399, S400, F405, N390, or K392, e.g., (a) T411N, T411R, T411Q, T411K, T411D, T411E, or T411W, (b) D399R, D399W, D399Y, or D399K (c) S400E, S400D, S400R or S400K, (d) F405I, F405M, F405T, F405S, F405V or F405W, (e) N390R, N390K or N390D, (f) K392V, K392M, K392R, K392L, K392F or K392E (numbering follows Kabat EU index). In further embodiments, the first CH3 domain includes amino acid mutations L351Y, Y407A, and the second CH3 domain includes amino acid mutations T366V, K409F. In further embodiments, the first CH3 domain includes amino acid mutation Y407A, and the second CH3 domain includes amino acid mutations T366A, K409F. In further embodiments, the second CH3 domain further includes amino acid mutations K392E, T411E, D399R, and S400R (numbering according to the Kabat EU index).

[0327] In one embodiment, the heterodimerization method described in International Publication No. 2011 / 143545 is used instead, having, for example, amino acid modifications at positions selected from the group consisting of 368 and 409 (numbering follows the Kabat EU index).

[0328] In one embodiment, the heterodimerization method described in International Publication No. 2011 / 090762 is used instead, which also employs the technique of inserting a knob into the hole described above. In one embodiment, the first CH3 domain contains the amino acid mutation T366W, and the second CH3 domain contains the amino acid mutation Y407A. In another embodiment, the first CH3 domain contains the amino acid mutation T366Y, and the second CH3 domain contains the amino acid mutation Y407T (numbering follows the Kabat EU index).

[0329] In one embodiment, the immune complex or antibody or its Fc domain is of the IgG2 subclass, and the heterodimerization method described in International Publication No. 2010 / 129304 is used as an alternative.

[0330] In alternative embodiments, modifications that facilitate the association of the first and second subunits of the Fc domain include modifications that mediate electrostatic maneuvering effects, as described, for example, in PCT Publication No. 2009 / 089004. Generally, this method involves the substitution of one or more amino acid residues with charged amino acid residues at the interface of the two Fc domain subunits such that homodimerization is electrostatically undesirable, but heterodimerization is electrostatically desirable. In one such embodiment, the first CH3 domain includes an amino acid substitution with a negatively charged amino acid of K392 or N392 (e.g., glutamic acid (E) or aspartic acid (D), preferably K392D or N392D), and the second CH3 domain includes an amino acid substitution with a positively charged amino acid of D399, E356, D356 or E357 (e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K or E357K, more preferably D399K and E356K). In a further embodiment, the first CH3 domain further includes an amino acid substitution with a negatively charged amino acid of K409 or R409 (e.g., glutamic acid (E) or aspartic acid (D), preferably K409D or R409D). In further embodiments, the first CH3 domain further comprises, or is replaced by, an amino acid substitution with negatively charged amino acids at K439 and / or K370 (e.g., glutamic acid (E) or aspartic acid (D)) (both numbered according to the Kabat EU index).

[0331] In further embodiments, the heterodimerization method described in International Publication No. 2007 / 147901 is used instead. In one embodiment, the first CH3 domain contains amino acid mutations K253E, D282K, and K322D, and the second CH3 domain contains amino acid mutations D239K, E240K, and K292D (numbering follows the Kabat EU index).

[0332] In yet another embodiment, the heterodimerization method described in International Publication No. 2007 / 110205 may be used instead.

[0333] In one embodiment, the first subunit of the Fc domain includes amino acid substitutions K392D and K409D, and the second subunit of the Fc domain includes amino acid substitutions D356K and D399K (numbering follows the Kabat EU index).

[0334] H. Recombination methods and compositions Immune complexes (e.g., PD-1 regulated IL-2 immune complexes (e.g., comprising at least one binding domain conjugated to PD-1 with an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) portion that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, and an anti-PD-1 antibody portion that binds to PD-1)) and / or antibodies (e.g., comprising two DBA portions or two anti-PD-1 antibody portions) can be produced using recombinant methods and compositions such as those described in U.S. Patent No. 4,816,567 and U.S. Patent Application Publication No. 2013 / 0078249, each of which is incorporated herein in whole by reference. In one embodiment, isolated nucleic acids (e.g., polynucleotides) or sets of isolated nucleic acids encoding the PD-1 regulated IL-2 immune complex or antibody described herein are provided. In one embodiment, isolated nucleic acids (e.g., polynucleotides) or sets of isolated nucleic acids encoding the PD-1 regulated IL-2 immune complex or antibody, or fragments thereof, are provided. Such nucleic acids or sets of nucleic acids may encode an amino acid sequence containing VL and / or an amino acid sequence containing VH of an immune complex or antibody (e.g., the light chain and / or heavy chain of either arm of the immune complex or antibody). In further embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids or sets of nucleic acids are provided.

[0335] The polynucleotides encoding the PD-1-regulated IL-2 immune complex of the present invention (e.g., comprising at least one binding domain conjugated to PD-1 on an IL-2 polypeptide; e.g., comprising a double-binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, and an anti-PD-1 antibody moiety that binds to PD-1) and / or antibody (e.g., comprising two DBA moieties or two anti-PD-1 antibody moieties) may be expressed as a single polynucleotide molecule or as a group (e.g., two or more) of polynucleotides expressed together. Polynucleotides encoded by the co-expressed polynucleotides may associate, for example, via disulfide bonds or other means to form a functional immune complex or antibody. For example, the light chain portion of the antigen-binding moiety (e.g., the DBA moiety or the anti-PD-1 antibody moiety) may be encoded by a polynucleotide, an Fc domain subunit, separate from the portion of the immune complex or antibody comprising the heavy chain portion of the antigen-binding moiety. When co-expressed, the heavy chain polypeptide binds to the light chain polypeptide to form the antigen-binding moiety. In another example, a portion of an immune complex or antibody containing one of the two Fc domain subunits may be encoded by a separate polynucleotide from the portion of the immune complex or antibody containing the other of the two Fc domain subunits. When co-expressed, the Fc domain subunits associate to form an Fc domain.

[0336] In certain embodiments, the isolated polynucleotide of the present invention encodes a fragment of a PD-1-regulated IL-2 immune complex or antibody comprising first and second antigen-binding domains and an Fc domain consisting of two subunits. In one embodiment, the isolated polynucleotide of the present invention encodes the heavy chain of the first antigen-binding moiety (e.g., the DBA moiety or the anti-PD-1 antibody moiety) and the subunit of the Fc domain. In another embodiment, the isolated polynucleotide of the present invention encodes the heavy chain of the second antigen-binding moiety and the subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide in which the Fab heavy chain (or scFv) shares a C-terminal peptide bond with the Fc domain subunit. In some embodiments, the light chains of the first and second antigen-binding moieties are co-expressed and associate with the heavy chain region to form the Fab domain.

[0337] In further embodiments, host cells comprising such nucleic acids or sets of nucleic acids are provided. In one such embodiment, the host cells comprise (e.g., transformed with): (1) a vector comprising nucleic acids encoding an amino acid sequence comprising at least one VL of an immune complex or antibody and an amino acid sequence comprising at least one VH of an immune complex or antibody; or (2) a first vector comprising nucleic acids encoding an amino acid sequence comprising the VL of an immune complex or antibody and a second vector comprising nucleic acids encoding an amino acid sequence comprising the VH of an immune complex or antibody. In one embodiment, the host cells are eukaryotic cells, e.g., Chinese hamster ovary (CHO) cells or lymphoid cells. In one embodiment, a method is provided for producing a PD-1 regulated IL-2 immune complex or antibody, comprising culturing host cells comprising nucleic acids encoding the above-mentioned immune complex or antibody under conditions suitable for the expression of the immune complex or antibody, and optionally recovering the immune complex or antibody from the host cells (or host cell culture medium).

[0338] For the recombinant production of PD-1-regulated IL-2 immune complexes or antibodies, for example, nucleic acids encoding such immune complexes or antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the immune complex or antibody), or they can be produced by recombinant methods or obtained by chemical synthesis.

[0339] Suitable host cells for cloning or expressing vectors (e.g., expression vectors) include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli (E. coli).) After expression, the immune complexes or antibodies of the present invention may be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0340] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for vectors (e.g., expression vectors), and include fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibodies with a partial or complete human glycosylation pattern. See Gerngross, TU, Nat. Biotech. 22(2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24(2006) 210-215.

[0341] Furthermore, suitable host cells for expressing (glycosylated) antibodies are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains have been identified and can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0342] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the Plantibody's® technology for antibody production in transgenic plants).

[0343] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspensions may be useful. Other examples of ...

Claims

1. It is an immunoconjugate, (a) The first binding domain, (i) IL-2 polypeptide and (ii) Linker and, (iii) A double-conjugated antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety includes a heavy chain variable region (VH) and a light chain variable region (VL), The IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), The first binding domain is (i) When the DBA portion is bound to the IL-2 polypeptide, the binding of the IL-2 polypeptide to its receptor is substantially blocked, (ii) When DBA is bound to PD-1, the DBA portion is substantially blocked from binding to the IL-2 polypeptide, and the IL-2 polypeptide can bind to the IL-2 receptor. The first binding domain is configured as follows, (b) A second binding domain comprising an anti-PD-1 antibody moiety including VH and VL, An immunoconjugate containing this substance.

2. The immunoconjugate according to claim 1, wherein the anti-PD-1 antibody portion is substantially not bound to the IL-2 polypeptide.

3. The immunoconjugate according to claim 1 or 2, wherein the DBA portion comprises a Fab molecule.

4. The immunoconjugate according to claim 3, wherein the DBA portion comprises a Fab heavy chain containing the VH and heavy chain constant domain 1 (CH1) of the DBA portion, and a Fab light chain containing the VL and light chain constant domain (CL) of the DBA portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other.

5. The immunoconjugate according to claim 3, wherein the DBA portion is a conventional Fab molecule.

6. The immunoconjugate according to any one of claims 1 to 5, wherein the anti-PD-1 antibody portion comprises a Fab molecule.

7. The immunoconjugate according to claim 6, wherein the anti-PD-1 antibody portion comprises a Fab heavy chain containing the VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing the VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other.

8. (a) The DBA portion is a conventional Fab molecule, (b) The immunoconjugate according to any one of claims 1 to 5, wherein the anti-PD-1 antibody portion is a Fab molecule comprising a Fab heavy chain containing the VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing the VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other.

9. (a) The DBA portion is a conventional Fab molecule, (b) The immunoconjugate according to any one of claims 1 to 5, wherein the anti-PD-1 antibody portion is a Fab molecule comprising a Fab heavy chain containing the VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing the VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other.

10. The immunoconjugate according to claim 6, wherein the anti-PD-1 antibody portion is a conventional Fab molecule.

11. (a) A Fab molecule in which the DBA portion comprises a Fab heavy chain containing the VH and CH1 of the DBA portion and a Fab light chain containing the VL and light chain CL of the DBA portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other, or the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other. (b) The immunoconjugate according to any one of claims 1 to 5, wherein the anti-PD-1 antibody portion is a conventional Fab molecule.

12. (a) A Fab molecule in which the DBA portion comprises a Fab heavy chain containing VH and CH1 of the DBA portion and a Fab light chain containing VL and light chain CL of the DBA portion, wherein the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other. (b) The immunoconjugate according to any one of claims 1 to 5, wherein the anti-PD-1 antibody portion is a conventional Fab molecule.

13. The immunoconjugate according to any one of claims 1 to 5, wherein the anti-PD-1 antibody portion is a single-stranded variable fragment (scFv).

14. The immunoconjugate according to any one of claims 1 to 13, further comprising an Fc domain comprising a first subunit and a second subunit.

15. The immunoconjugate according to any one of claims 1 to 14, wherein (a) the DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), and / or (b) the anti-PD-1 antibody portion and the second subunit are connected in the following orientation: N-[anti-PD-1 antibody portion]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

16. The immunoconjugate according to claim 14 or 15, wherein the Fc domain is an IgG Fc domain.

17. The aforementioned IgG Fc domain is IgG １ The immunoconjugate according to claim 16, wherein the domain is Fc.

18. The immunoconjugate according to any one of claims 14 to 17, wherein the Fc domain is a human IgG Fc domain.

19. The first subunit is the first CH2 (CH2 １ ) domain and / or first CH3 (CH3 １ The second subunit includes one or more CH domains selected from the domain, and the second CH2 (CH2 ２ ) domain and / or second CH3 (CH3 ２ The immunoconjugate according to any one of claims 14 to 18, comprising one or more CH domains selected from the domains.

20. The immunoconjugate according to claim 19, wherein at least one of the one or more CH domains is paired with another CH domain.

21. the CH3 １ domain and the CH3 ２ domain each comprise a protrusion or a cavity, and the CH3 １ protrusion or cavity within the CH3 ２ immunoconjugate according to claim 20, wherein the cavity or protrusion within the CH3 domain can be respectively disposed in the protrusion or cavity within the CH3 domain.

22. The aforementioned CH3 １ Domain and the CH3 ２ The immunoconjugate according to claim 21, wherein the domains associate at the interface between the protrusion and the cavity.

23. The aforementioned CH2 １ Domain and the CH2 ２ Each domain includes a projection or a cavity, and the CH2 １ The protrusion or cavity of the domain is the CH2 ２ The immunoconjugate according to any one of claims 20 to 22, which can be positioned in the cavities or protrusions of the domain, respectively.

24. The aforementioned CH2 １ Domain and the CH2 ２ The immunoconjugate according to claim 23, wherein the domains associate at the interface between the protrusion and the cavity.

25. (a) The first subunit contains a tryptophan residue at position 366, and the second subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index), or (b) The immunoconjugate according to any one of claims 14 to 24, wherein the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit comprises a tryptophan residue at position 366 (numbered according to the Kabat EU index).

26. The immunoconjugate according to any one of claims 14 to 25, wherein the first subunit and / or the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index).

27. The immunoconjugate according to any one of claims 1 to 26, wherein the DBA portion and the anti-PD-1 antibody portion bind to different epitopes of PD-1.

28. The immunoconjugate according to any one of claims 1 to 26, wherein the DBA portion and the anti-PD-1 antibody portion bind to the same epitope of PD-1.

29. The immunoconjugate according to any one of claims 1 to 28, wherein the binding of the DBA portion to PD-1 inhibits the binding of PD-1 to PD-L1, and / or the binding of the anti-PD-1 antibody portion to PD-1 inhibits the binding of PD-1 to PD-L1.

30. The immunoconjugate according to any one of claims 1 to 28, wherein the binding of the DBA portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, or the binding of the anti-PD-1 antibody portion to PD-1 does not inhibit the binding of PD-1 to PD-L1.

31. (a) The binding of the DBA portion to PD-1 inhibits the binding of PD-1 to PD-L1, and the binding of the anti-PD-1 antibody portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, or (b) The immunoconjugate according to any one of claims 1 to 27, 29, and 30, wherein the binding of the DBA portion to PD-1 does not inhibit the binding of PD-1 to PD-L1, and the binding of the anti-PD-1 antibody portion to PD-1 inhibits the binding of PD-1 to PD-L1.

32. The aforementioned DBA portion consists of the following six complementarity determination regions (CDRs): (a) CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and CDR-L3 containing the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57), or (b) CDR-H1 containing the amino acid sequence of AYYIH (SEQ ID NO: 82), CDR-H2 containing the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 containing the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), CDR-L1 containing the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 containing the amino acid sequence of GASSLQS (SEQ ID NO: 74), and CDR-L3 containing the amino acid sequence of QESYTSSNT (SEQ ID NO: 75) An immunoconjugate according to any one of claims 1 to 31, including the immunoconjugate described in any one of claims 1 to 31.

33. The aforementioned DBA portion, (a) VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and / or VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; (b) VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85, and / or VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 76; or (c) VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111, and / or VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 91; or (d) VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85, and / or VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

91. The immunoconjugate according to claim 32, including the immunoconjugate described in claim 32.

34. The aforementioned DBA portion, (a) VH containing the amino acid sequence of SEQ ID NO: 67, and VL containing the amino acid sequence of SEQ ID NO: 58; (b) VH containing the amino acid sequence of SEQ ID NO: 85, and VL containing the amino acid sequence of SEQ ID NO: 76; or (c) VH containing the amino acid sequence of SEQ ID NO: 111, and VL containing the amino acid sequence of SEQ ID NO: 91; or (d) VH containing the amino acid sequence of SEQ ID NO: 85, and VL containing the amino acid sequence of SEQ ID NO: 91 The immunoconjugate according to claim 33, including the immunoconjugate described in claim 33.

35. The anti-PD-1 antibody portion consists of the following six CDRs: (a) CDR-H1 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21); (b) CDR-H1 containing the amino acid sequence of SYAMS (SEQ ID NO: 46), CDR-H2 containing the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), CDR-H3 containing the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO: 48), CDR-L1 containing the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), CDR-L2 containing the amino acid sequence of TASSLQS (SEQ ID NO: 38), and CDR-L3 containing the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39); or (c) CDR-H1 containing the amino acid sequence of SYWMS (SEQ ID NO: 10), CDR-H2 containing the amino acid sequence of AISGSGGGSRYYAESVKG (SEQ ID NO: 11), CDR-H3 containing the amino acid sequence of SPLQWIDV (SEQ ID NO: 12), CDR-L1 containing the amino acid sequence of RASQGISSWLA (SEQ ID NO: 1), CDR-L2 containing the amino acid sequence of EASSLQS (SEQ ID NO: 2), and CDR-L3 containing the amino acid sequence of QQANQFPFT (SEQ ID NO: 3) An immunoconjugate according to any one of claims 1 to 34, including the immunoconjugate described in any one of claims 1 to 34.

36. The anti-PD-1 antibody portion is (a) VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and / or VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (b) VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49, and / or VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40; or (c) VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13, and / or VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

4. The immunoconjugate according to claim 35, including the immunoconjugate described in claim 35.

37. The anti-PD-1 antibody portion is (a) VH containing the amino acid sequence of SEQ ID NO: 31, and / or VL containing the amino acid sequence of SEQ ID NO: 22; (b) VH containing the amino acid sequence of SEQ ID NO: 49, and / or VL containing the amino acid sequence of SEQ ID NO: 40; or (c) VH containing the amino acid sequence of SEQ ID NO: 13, and / or VL containing the amino acid sequence of SEQ ID NO: 4 The immunoconjugate according to claim 36, including the immunoconjugate described in claim 36.

38. (a)(i) The DBA portion comprises CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and CDR-L3 containing the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57), and (ii) The anti-PD-1 antibody portion comprises CDR-H1 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21); (b) (i) The DBA portion comprises CDR-H1 containing the amino acid sequence AYYIH (SEQ ID NO: 82), CDR-H2 containing the amino acid sequence WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), CDR-H3 containing the amino acid sequence GHYYGKTDY (SEQ ID NO: 84), CDR-L1 containing the amino acid sequence RASQGIRNDLG (SEQ ID NO: 73), CDR-L2 containing the amino acid sequence GASSLQS (SEQ ID NO: 74), and CDR-L3 containing the amino acid sequence QESYTSSNT (SEQ ID NO: 75), and (ii ) The anti-PD-1 antibody portion includes CDR-H1 containing the amino acid sequence of SYTMS (SEQ ID NO: 28), CDR-H2 containing the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), CDR-H3 containing the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), CDR-L1 containing the amino acid sequence of KASESVDTSDNSFH (SEQ ID NO: 19), CDR-L2 containing the amino acid sequence of RASTLES (SEQ ID NO: 20), and CDR-L3 containing the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21); or (c) (i) The DBA portion comprises CDR-H1 containing the amino acid sequence of RYYVH (SEQ ID NO: 64), CDR-H2 containing the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), CDR-H3 containing the amino acid sequence of GLFI (SEQ ID NO: 66), CDR-L1 containing the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), CDR-L2 containing the amino acid sequence of SASNLET (SEQ ID NO: 56), and CDR-L3 containing the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57), and (ii ) The anti-PD-1 antibody portion includes CDR-H1 containing the amino acid sequence of SYAMS (SEQ ID NO: 46), CDR-H2 containing the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), CDR-H3 containing the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO: 48), CDR-L1 containing the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), CDR-L2 containing the amino acid sequence of TASSLQS (SEQ ID NO: 38), and CDR-L3 containing the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39). An immunoconjugate according to any one of claims 1 to 31, including the immunoconjugate described in any one of claims 1 to 31.

39. (a)(i) the DBA portion comprises a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (b) (i) the DBA portion comprises a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103, and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (c) (i) the DBA portion comprises a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111, and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31, and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; or (d) (i) The DBA portion comprises a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67, and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58, and (ii) The anti-PD-1 antibody portion comprises a VH containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49, and / or a VL containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40, The immunoconjugate according to claim 38.

40. (a)(i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 67 and a VL containing the amino acid sequence of SEQ ID NO: 58, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 31 and a VL containing the amino acid sequence of SEQ ID NO: 22; (b) (i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 103 and a VL containing the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 31 and a VL containing the amino acid sequence of SEQ ID NO: 22; (c) (i) the DBA portion comprises a VH containing the amino acid sequence of SEQ ID NO: 111 and a VL containing the amino acid sequence of SEQ ID NO: 109, and (ii) the anti-PD-1 antibody portion comprises a VH containing the amino acid sequence of SEQ ID NO: 31 and a VL containing the amino acid sequence of SEQ ID NO: 22; or (d) (i) The DBA portion comprises VH containing the amino acid sequence of SEQ ID NO: 67 and VL containing the amino acid sequence of SEQ ID NO: 58, and (ii) The anti-PD-1 antibody portion comprises VH containing the amino acid sequence of SEQ ID NO: 49 and VL containing the amino acid sequence of SEQ ID NO: 40, The immunoconjugate according to claim 39.

41. The immunoconjugate according to any one of claims 1 to 40, wherein the linker has a length of 5 to 30 amino acids.

42. The immunoconjugate according to claim 41, wherein the linker has a length of 20 amino acids.

43. The linker has an amino acid sequence (G ２ SG ２ ) ｘ An immunoconjugate according to any one of claims 1 to 41, comprising, in the formula, where x is an integer from 1 to 6 (Sequence IDs 149 to 154).

44. The immunoconjugate according to claim 43, wherein x is 2 (SEQ ID NO: 150) or 4 (SEQ ID NO: 152).

45. The immunoconjugate according to any one of claims 1 to 44, wherein the IL-2 polypeptide is connected to the VH of the DBA portion in the following orientation: N-[IL-2 polypeptide]-[linker]-[VH]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

46. The immunoconjugate according to any one of claims 1 to 44, wherein the IL-2 polypeptide is connected to the VL of the DBA portion in the following orientation: N-[IL-2 polypeptide]-[linker]-[VL]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

47. The immunoconjugate according to any one of claims 1 to 46, wherein the IL-2 polypeptide is a mutant human IL-2 polypeptide comprising an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72 and / or an alanine residue at position 125 (numbered relative to the human IL-2 sequence of SEQ ID NO: 147).

48. The immunoconjugate according to claim 47, wherein the mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:

146.

49. The immunoconjugate according to any one of claims 1 to 46, wherein the IL-2 polypeptide is a mutant human IL-2 polypeptide comprising an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, an alanine residue at position 125 and / or a threonine residue at position 126 (numbered relative to the human IL-2 sequence of SEQ ID NO: 147).

50. The immunoconjugate according to claim 49, wherein the mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO:

199.

51. The immunoconjugate according to any one of claims 1 to 50, wherein the PD-1 is human PD-1.

52. It is an immunoconjugate, (a) The first binding domain, (i) IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 146, (ii) A linker comprising or consisting of the amino acid sequence of Sequence ID No. 150 or 152, (iii) A DBA moiety that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, comprising a DBA moiety that is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, The IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), When the DBA portion is bound to the IL-2 polypeptide, the binding of the IL-2 polypeptide to its receptor is substantially blocked; when the DBA portion is bound to PD-1, the binding of the DBA portion to the IL-2 polypeptide is blocked; and the IL-2 polypeptide can bind to its receptor, comprising a first binding domain, (b) A second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, (c) An Fc region comprising a first subunit and a second subunit, (i) The first subunit contains a tryptophan residue at position 366, and the second subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index), or (ii) The first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit comprises a tryptophan residue at position 366 (numbered according to the Kabat EU index), Each of the first subunit and the second subunit includes an Fc region comprising an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index), The DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), The anti-PD-1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), (a) a Fab molecule in which the DBA portion is a conventional Fab molecule, and the anti-PD-1 antibody portion comprises a Fab heavy chain containing the VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing the VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other; or (b) An immunoconjugate in which the DBA portion is a Fab molecule comprising a Fab heavy chain containing the VH and CH1 of the DBA portion and a Fab light chain containing the VL and light chain CL of the DBA portion, wherein the CH1 of the Fab heavy chain and the CL of the Fab light chain are substituted for each other, and the anti-PD-1 antibody portion is a conventional Fab molecule.

53. It is an immunoconjugate, (a) The first binding domain, (i) IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 199, (ii) A linker containing or consisting of the amino acid sequence of Sequence ID No. 152, (iii) A DBA moiety that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, comprising a DBA moiety that is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, The IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), When the DBA portion is bound to the IL-2 polypeptide, the binding of the IL-2 polypeptide to its receptor is substantially blocked; when the DBA portion is bound to PD-1, the binding of the DBA portion to the IL-2 polypeptide is blocked; and the IL-2 polypeptide can bind to its receptor, comprising a first binding domain, (b) A second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain containing VH and a Fab light chain containing VL, (c) An Fc region comprising a first subunit and a second subunit, (i) The first subunit contains a tryptophan residue at position 366, and the second subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index), or (ii) The first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit comprises a tryptophan residue at position 366 (numbered according to the Kabat EU index), Each of the first subunit and the second subunit includes an Fc region comprising an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index), The DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), The anti-PD-1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), An immunoconjugate in which the DBA portion is a conventional Fab molecule, and the anti-PD-1 antibody portion is a Fab molecule comprising a Fab heavy chain containing the VH and CH1 of the anti-PD-1 antibody portion and a Fab light chain containing the VL and CL of the anti-PD-1 antibody portion, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are substituted for each other.

54. It is an immunoconjugate, (a) The first binding domain, (i) IL-2 polypeptide containing the amino acid sequence of SEQ ID NO: 146, (ii) A linker comprising or consisting of the amino acid sequence of Sequence ID No. 150 or 152, (iii) A double-binding antibody (DBA) moiety that binds to PD-1 and IL-2 polypeptides in a mutually exclusive manner, comprising a DBA molecule which includes a Fab heavy chain containing VH and a Fab light chain containing VL, The IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), When the DBA portion is bound to the IL-2 polypeptide, the binding of the IL-2 polypeptide to its receptor is substantially blocked; when the DBA portion is bound to PD-1, the binding of the DBA portion to the IL-2 polypeptide is blocked; and the IL-2 polypeptide can bind to its receptor, comprising a first binding domain, (b) A second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is an scFv comprising VH and VL, (c) An Fc region comprising a first subunit and a second subunit, (i) The first subunit contains a tryptophan residue at position 366, and the second subunit contains a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to the Kabat EU index), or (ii) The first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the second subunit comprises a tryptophan residue at position 366 (numbered according to the Kabat EU index), Each of the first subunit and the second subunit includes an Fc region comprising an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to the Kabat EU index), The DBA portion and the first subunit are connected in the following orientation: N-[DBA portion]-[first subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide), An immunoconjugate in which the anti-PD-1 antibody moiety and the second subunit are linked according to N-[anti-PD-1 antibody moiety]-[second subunit]-C (wherein N- represents the N-terminus of the polypeptide and -C represents the C-terminus of the polypeptide).

55. The immunoconjugate, (a) a first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (b) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 104; a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 110; a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (c) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 112; a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 110; a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106; and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (d) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 124; a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134; a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 128; (e) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 124; a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134; a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120; (f) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 122; a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134; a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 118; and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 120; (g) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 122; a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 134; a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 126; and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 128; (h) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 197, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 108; or (i) A first polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 198, a second polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

108. An immunoconjugate according to any one of claims 1 to 51, including the immunoconjugate described in any one of claims 1 to 51.

56. The immunoconjugate, (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (b) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (c) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (d) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128; (e) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120; (f) A first polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120; (g) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128; (h) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; or (i) A first polypeptide containing the amino acid sequence of SEQ ID NO: 198, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO:

108. The immunoconjugate according to claim 55, including the immunoconjugate described in claim 55.

57. An immunoconjugate comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 96, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO:

108.

58. An immunoconjugate comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 104, a second polypeptide containing the amino acid sequence of SEQ ID NO: 110, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO:

108.

59. An immunoconjugate comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 124, a second polypeptide containing the amino acid sequence of SEQ ID NO: 134, a third polypeptide containing the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO:

128.

60. An immunoconjugate comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 124, a second polypeptide containing the amino acid sequence of SEQ ID NO: 134, a third polypeptide containing the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO:

120.

61. An immunoconjugate comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 197, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO:

108.

62. An immunoconjugate comprising a first polypeptide containing the amino acid sequence of SEQ ID NO: 198, a second polypeptide containing the amino acid sequence of SEQ ID NO: 94, a third polypeptide containing the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide containing the amino acid sequence of SEQ ID NO:

108.

63. An isolated polynucleotide or set of isolated polynucleotides encoding an immunoconjugate according to any one of claims 1 to 62.

64. A vector or set of vectors comprising an isolated polynucleotide or a set of isolated polynucleotides as described in claim 63.

65. (i) a host cell or a set of host cells comprising an isolated polynucleotide or a set of isolated polynucleotides as described in claim 63, or a vector or a set of vectors as described in claim 64.

66. (a) A method for producing an immunoconjugate, comprising the step of culturing the host cells or set of host cells described in claim 65 under conditions suitable for the expression of the immunoconjugate.

67. The method according to claim 66, further comprising recovering the immunoconjugate.

68. The method according to claim 66, wherein the host cell expresses the first binding domain and the second binding domain.

69. The method according to claim 66, wherein a first host cell expresses the first binding domain and a second host cell expresses the second binding domain.

70. The method according to claim 68 or 69, further comprising recovering the first binding domain and the second binding domain.

71. The method according to claim 70, further comprising contacting the recovered first binding domain with the recovered second binding domain.

72. An immunoconjugate produced by the method described in any one of claims 66 to 71.

73. A pharmaceutical composition comprising an immunoconjugate according to any one of claims 1 to 62 and 72, and a pharmaceutically acceptable carrier.

74. An immunoconjugate according to any one of claims 1 to 62 and 72, or a pharmaceutical composition according to claim 73, for use as a pharmaceutical.

75. Use of an immunoconjugate according to any one of claims 1 to 62 and 72 or a pharmaceutical composition according to claim 73 in the manufacture of a pharmaceutical product.

76. An immunoconjugate according to any one of claims 1 to 62 and 72 or a pharmaceutical composition according to claim 73, for use in the treatment of cancer in subjects requiring cancer treatment.

77. Use of an immunoconjugate according to any one of claims 1 to 62 and 72 or a pharmaceutical composition according to claim 73 in the manufacture of a pharmaceutical for the treatment of cancer in a subject requiring treatment for cancer.

78. Use of an immunoconjugate according to any one of claims 1 to 62 and 72 or a pharmaceutical composition according to claim 73 for treating cancer in a subject requiring treatment for cancer.

79. A method for treating cancer in a subject, comprising administering to the subject an effective amount of an immunoconjugate according to any one of claims 1 to 62 and 72 or a pharmaceutical composition according to claim 73.

80. The immunoconjugate for use, pharmaceutical composition for use, use, or method according to any one of claims 76 to 79, wherein the cancer is a PD-1 positive cancer.

81. An immunoconjugate for use, a pharmaceutical composition for use, a use, or a method according to any one of claims 76 to 79, further comprising administering an additional therapeutic agent to the subject.

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