PD-L1-targeted chimeric protein and its use

Abstract

The present invention provides improved immunotherapeutic agents that can deliver targeted therapy against cancer while minimizing the side effects they cause, as well as methods for treating or preventing cancer. [Solution] The present invention relates in part to agents that bind to PD-L1, and their use as diagnostic and therapeutic agents. The present invention further relates to pharmaceutical compositions comprising a PD-L1 targeting moiety, and their use in the treatment of various diseases. In various embodiments, the present invention relates to conjugates having at least one targeting moiety that specifically binds to PD-1 or PD-L1. In various embodiments, these conjugates bind to PD-1 or PD-L1 and functionally modulate it (e.g., partially or completely neutralize it).

Description

[Technical Field]

[0001] The present invention relates, in part, to a targeting moiety that recognizes and binds to PD-L1, and to its use as a diagnostic and therapeutic agent. The present invention further relates to a pharmaceutical composition comprising a chimeric protein having a PD-L1 targeting moiety, and to its use in the treatment of various diseases, including cancer.

[0002] Cross-reference of related applications This application claims the interests of U.S. Provisional Patent Application No. 62 / 906,447, filed on 26 September 2019, and the entire disclosure of that patent is incorporated herein by reference.

[0003] Sequence List This application includes an array listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created on 23 September 2020, is named "ORN-068PC_ST25" and has a size of 182,668 bytes. [Background technology]

[0004] Immunotherapy has been developed to direct the body's immune system towards cancer. Immunotherapy offers the advantage of cell specificity that other therapies, such as chemotherapy and radiation, lack. Therefore, methods to enhance the effectiveness of immunotherapy can be clinically beneficial. For example, immune checkpoint molecules that provide co-stimulatory or co-inhibitory signals play a central role in regulating the immune response against tumor cells.

[0005] However, despite impressive patient responses to checkpoint molecule-targeted drugs, including the success of YERVOY, KEYTRUDA, and OPDIVO, immunotherapies such as checkpoint inhibitors still fail in the vast majority of patients. Furthermore, many immunotherapies are complicated by side effects that significantly narrow the patient's time window for treatment and make patients more susceptible to other diseases.

[0006] Therefore, there remains a need for improved immunotherapies that can provide targeted therapy against cancer while minimizing the side effects they cause. [Overview of the Initiative]

[0007] In various embodiments, the present invention relates to a conjugate having at least one targeting moiety that specifically binds to PD-1 or PD-L1. In various embodiments, these conjugates bind to PD-1 or PD-L1 and functionally modulate it (e.g., partially or completely neutralize it). In various embodiments, these conjugates bind to PD-1 or PD-L1 but do not functionally modulate it (e.g., do not partially or completely neutralize it). Thus, in various embodiments, the conjugates of the present invention are used, for example, to recruit PD-1-expressing cells or PD-L1-expressing cells directly or indirectly to a site of interest, while still allowing the cells to signal via either PD-1 or PD-L1 (i.e., binding of the PD-1 or PD-L1 conjugate does not reduce or eliminate PD-1 or PD-L1 signaling at the site of interest). In some embodiments, the targeting moiety is a single-domain antibody (VHH).

[0008] In some embodiments, the present invention provides a PD-L1 targeting moiety comprising a recognition domain, the recognition domain comprising (i) three complementarity-determining regions (CDR1, CDR2, and CDR3), wherein (a) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 2 or 5, (b) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (c) CDR3 comprises an amino acid sequence selected from either SEQ ID NO: 4 or 7, or (ii) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and (i) or (ii) further comprises one or more mutations at positions D54 and G55 numbered relative to SEQ ID NO: 1.

[0009] In this embodiment, the PD-L1 targeting portion, which includes the recognition domain, further comprises one or more mutations at positions Q1, Q5, A14, A63, T74, K76, S79, K86, and Q110.

[0010] In the embodiments, the mutation is a substitution, which is optionally selected from arginine (R) and lysine (K), an aromatic, polar, and positively charged hydrophilic residue including histidine (H), a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0011] In embodiments, the mutation is selected from one or more of the following: a hydrophobic, aliphatic amino acid at position D54, optionally D54G, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); a polar, positively charged hydrophilic residue at position D54, optionally D54K, selected from arginine (R) and lysine (K); a polar, neutrally charged hydrophilic residue at position G54T, optionally D54T, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); and a polar, positively charged hydrophilic residue at position G55, optionally G55R, selected from arginine (R) and lysine (K).

[0012] In the embodiment, the mutation is optionally Q1D, a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E) at position Q1; optionally Q5V, a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q5; optionally A14P, a polar and neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C) at position A14; optionally A63V, a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position A63; optionally T74S, asparagine (N) at position T74, One or more of the following are selected: a polar and neutrally charged hydrophilic residue selected from glutamine (Q), serine (S), proline (P), and cysteine ​​(C); a polar and neutrally charged hydrophilic residue at position K76, optionally K76N, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); a hydrophobic, aromatic amino acid selected at position S79, optionally S79Y, selected from phenylalanine (F), tryptophan (W), and tyrosine (Y) at position S79; a hydrophobic, aliphatic amino acid selected at position Q110, optionally Q110L, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0013] In the embodiment, the mutation is one or more of Q1D, Q5V, A14P, A63V, T74S, S79Y, K86R, and Q110L, or optionally selected from all of Q1D, Q5V, A14P, D54G, T74S, K76N, S79Y, K86R, and Q110L.

[0014] In some embodiments, the present invention relates to a PD-L1 targeting moiety including a recognition domain, wherein the recognition domain includes (i) three complementarity-determining regions (CDR1, CDR2, and CDR3), where (a) CDR1 includes an amino acid sequence selected from either SEQ ID NO: 2 or 5, (b) CDR2 includes an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (c) CDR3 includes an amino acid sequence selected from either SEQ ID NO: 4 or 7, or (ii) includes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and (i) or (ii) further includes one or more mutations at positions D54, G55, K76, and S79 numbered relative to SEQ ID NO: 1. In some embodiments, the PD-L1 targeting moiety includes one or more mutations at positions T74, K86, and Q110.

[0015] In some embodiments, the present invention relates to a PD-L1 targeting moiety including a recognition domain, wherein the recognition domain comprises (i) three complementarity-determining regions (CDR1, CDR2, and CDR3), where (a) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 27 or 30, (b) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 28 or 31, and (c) CDR3 comprises an amino acid sequence selected from either SEQ ID NO: 29 or 32, or (ii) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, and (i) or (ii) further comprises one or more mutations at positions N32, D33, and M97 numbered relative to SEQ ID NO: 26.

[0016] In the embodiment, the PD-L1 targeting moiety including the recognition domain includes one or more of the following mutations (relative to SEQ ID NO: 26): Q1D, Q5V, A14P, A62S, A74S, M77T, M78V, S79Y, K86R, and Q109L, and optionally further includes all of Q1D, Q5V, A14P, D33H, A62S, A74S, M77T, M78V, K86R, and M97V.

[0017] In another embodiment, the present invention relates to a chimeric protein or chimeric protein complex having at least one targeting moiety that specifically binds to PD-L1. In various embodiments, the chimeric protein or chimeric protein complex further comprises signaling molecules, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be modified to reduce their activity.

[0018] In some embodiments, the present invention relates to an Fc-based chimeric protein complex, which comprises (A) a targeting moiety, (a) three complementarity-determining regions (CDR1, CDR2, and CDR3), where (i) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 2 or 5, (ii) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (iii) CDR3 comprises an amino acid sequence selected from either SEQ ID NO: 4 or 7, or (b) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and (a) or (b) is The invention comprises: a targeting moiety further comprising one or more mutations at positions D54 and G55 numbered relative to Sequence ID No. 1; (B) a signaling molecule which is a) a wild-type signaling molecule, or b) a modified signaling molecule which has one or more mutations that confer improved safety to the wild-type signaling molecule; and (C) an Fc domain which optionally has one or more mutations that reduce or eliminate the effector function of one or more Fc domains, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain.

[0019] In this embodiment, the PD-L1 targeting portion, which includes the recognition domain, further comprises one or more mutations at positions Q1, Q5, A14, A63, T74, K76, S79, K86, and Q110.

[0020] In the embodiments, the mutation is a substitution, which is optionally selected from arginine (R) and lysine (K), an aromatic, polar, and positively charged hydrophilic residue including histidine (H), a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0021] In embodiments, the mutation is selected from one or more of the following: a hydrophobic, aliphatic amino acid at position D54, optionally D54G, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); a polar, positively charged hydrophilic residue at position D54, optionally D54K, selected from arginine (R) and lysine (K); a polar, neutrally charged hydrophilic residue at position G54T, optionally D54T, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); and a polar, positively charged hydrophilic residue at position G55, optionally G55R, selected from arginine (R) and lysine (K).

[0022] In the embodiment, the mutation is optionally Q1D, a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E) at position Q1; optionally Q5V, a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q5; optionally A14P, a polar and neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C) at position A14; optionally A63V, a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position A63; optionally T74S, asparagine (N) at position T74, One or more of the following are selected: a polar and neutrally charged hydrophilic residue selected from glutamine (Q), serine (S), proline (P), and cysteine ​​(C); a polar and neutrally charged hydrophilic residue at position K76, optionally K76N, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); a hydrophobic, aromatic amino acid selected at position S79, optionally S79Y, selected from phenylalanine (F), tryptophan (W), and tyrosine (Y) at position S79; a hydrophobic, aliphatic amino acid selected at position Q110, optionally Q110L, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V). In the embodiment, the mutation is one or more of Q1D, Q5V, A14P, A63V, T74S, S79Y, K86R, and Q110L, or optionally selected from all of Q1D, Q5V, A14P, D54G, T74S, K76N, S79Y, K86R, and Q110L.

[0023] In some embodiments, the present invention relates to an Fc-based chimeric protein complex, which includes (A) a targeting portion comprising (a) three complementarity-determining regions (CDR1, CDR2, and CDR3), wherein (i) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 2 or 5, (ii) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (iii) CDR3 comprises an amino acid sequence selected from either SEQ ID NO: 4 or 7, or (b) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, wherein (a) or (b) is SEQ ID NO: The invention comprises: (B) a targeting moiety further comprising one or more mutations at positions D54, G55, K76, and S79 numbered relative to 1; (B) a signaling molecule which is a) a wild-type signaling molecule, or b) a modified signaling molecule which has one or more mutations that confer improved safety to the wild-type signaling molecule; and (C) an Fc domain which optionally has one or more mutations that reduce or eliminate the effector function of one or more Fc domains, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain.

[0024] In some embodiments, the present invention also relates to an Fc-based chimeric protein complex, which (A) a targeting portion comprising (a) three complementarity-determining regions (CDR1, CDR2, and CDR3), wherein (i) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 27 or 30, (ii) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 28 or 31, and (iii) CDR3 comprises an amino acid sequence selected from either SEQ ID NO: 29 or 32, or (b) an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, and (a) or (b) The present invention further comprises: (B) a targeting moiety comprising one or more mutations at positions N32, D33, and M97 numbered relative to SEQ ID NO: 26; (A) a signaling molecule which is a) a wild-type signaling molecule, or b) a modified signaling molecule which has one or more mutations that confer improved safety to the wild-type signaling molecule; and (C) an Fc domain which optionally has one or more mutations that reduce or eliminate the effector function of one or more Fc domains, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain. In some embodiments, the present invention also comprises recombinant nucleic acids encoding the PD-L1 targeting moiety or chimeric protein or chimeric protein complex of the present invention. In other embodiments, the present invention comprises a host cell comprising recombinant nucleic acids encoding the PD-L1 targeting moiety or chimeric protein or chimeric protein complex of the present invention. In the embodiment, the PD-L1 targeting moiety including the recognition domain includes one or more of the following mutations (relative to SEQ ID NO: 26): Q1D, Q5V, A14P, A62S, A74S, M77T, M78V, S79Y, K86R, and Q109L, and optionally further includes all of Q1D, Q5V, A14P, D33H, A62S, A74S, M77T, M78V, K86R, and M97V.

[0025] In various embodiments, the chimeric protein or chimeric protein complex includes an additional targeting moiety that binds to another target of interest (e.g., an antigen, a receptor). In some embodiments, the other target of interest (e.g., an antigen, a receptor) is located on tumor cells. In other embodiments, the other target of interest (e.g., an antigen, a receptor) is located on immune cells. In some embodiments, the chimeric protein or chimeric protein complex of the present invention can recruit immune cells directly or indirectly to a site of action (a non-limiting example being the tumor microenvironment). In some embodiments, the chimeric protein or chimeric protein complex of the present invention promotes phagocytosis of target cells (e.g., tumor cells).

[0026] In various embodiments, the chimeric proteins or chimeric protein complexes of the present invention are used to treat various diseases or disorders such as cancer, infectious diseases, immune disorders, and other diseases and disorders, and the present invention encompasses various therapeutic methods.

[0027] In some embodiments, the present invention relates to a chimeric protein complex comprising one or more signaling molecules, one or more targeting molecules, and one or more fragment crystallizable domains (Fc domains). These Fc-based chimeric protein complexes of the present invention are highly target-selective, enable conditional and / or controlled modulation of receptor signaling, are highly active and / or long-acting, and / or long-acting, while simultaneously inducing minimal side effects. [Brief explanation of the drawing]

[0028] [Figure 1] The wild-type sequence of 2LIG99 VHH is shown. The highlighted portion of the sequence represents the ABM format CDR, and the underlined portion represents the Kabat format CDR. [Figure 2] The wild-type sequence of 2LIG189 VHH is shown. The highlighted portion of the sequence represents the ABM format CDR, and the underlined portion represents the Kabat format CDR. [Figure 3] This table shows the affinity of 2LIG99 humanized and isomerized variants. For SEQ ID NO: 14, dissociation could not be measured during the 5-minute time interval of the assay. [Figure 4] This table shows the affinity of the second wave of 2LIG99 humanized and isomerized variants. [Figure 5] This shows the neutralization of the PD-L1 / PD-1 interaction by the 2LIG99 variant on HL116 cells. [Figure 6] This table shows the affinity of humanized, deamidated, and oxidized variants of 2LIG189. [Figure 7] This table shows the affinity of the humanized, deamidated, and oxidized variants of 2LIG189 in the second wave. [Figure 8] This shows neutralization of the PD-L1 / PD-1 interaction on HL116 cells by the 2LIG189 variant. [Figure 9-27] Figures 9A-F, 10A-H, 11A-H, 12A-D, 13A-F, 14A-J, 15A-D, 16A-F, 17A-J, 18A-F, 19A-L, 20A-L, 21A-F, 22A-L, 23A-L, 24A-J, 25A-J, 26A-F, and 27A-F show various non-limiting schematic diagrams of the Fc-based chimeric protein complex of the present invention. In some embodiments, each schematic diagram represents a composition of the present invention. Where applicable in the drawings, "TM" refers to the "Targeting Portion" as described herein, "SA" refers to the "Signaling Substance" as described herein, and [ka] " is an optional "linker" as described herein, where two long parallel rectangles are, for example, human Fc domains derived from IgG1, IgG2, or IgG4 as described herein, and optionally having effector knockout and / or stabilizing mutations as described herein, and two long parallel rectangles having one protrusion and the other recessed are, for example, human Fc domains derived from IgG1, IgG2, or IgG4 as described herein, and optionally having knob-in-hole and / or ion pair (also known as charge pair, ionic bond, or charge residue pair) mutations as described herein, and optionally having effector knockout and / or stabilizing mutations as described herein. [Figures 9A-9F] Examples of homodimeric double-chain complexes are shown. These figures illustrate the structures of typical homodimeric double-chain complexes. [Figure 10A] Examples of homodimer double-chain complexes having two targeting moieties (TM) (in some embodiments, more targeting moieties may be present as described herein) are shown. In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 10G and 10H) have a signaling molecule (SA) between TM1 and TM2, or between TM1 and Fc. [Figure 10B] Examples of homodimer double-chain complexes having two targeting moieties (TM) (in some embodiments, more targeting moieties may be present as described herein) are shown. In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 10G and 10H) have a signaling molecule (SA) between TM1 and TM2, or between TM1 and Fc. [Figure 10C]Examples of homodimer double-chain complexes having two targeting moieties (TM) (in some embodiments, more targeting moieties may be present as described herein) are shown. In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 10G and 10H) have a signaling molecule (SA) between TM1 and TM2, or between TM1 and Fc. [Figure 10D] Examples of homodimer double-chain complexes having two targeting moieties (TM) (in some embodiments, more targeting moieties may be present as described herein) are shown. In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 10G and 10H) have a signaling molecule (SA) between TM1 and TM2, or between TM1 and Fc. [Figure 10E] Examples of homodimer double-chain complexes having two targeting moieties (TM) (in some embodiments, more targeting moieties may be present as described herein) are shown. In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 10G and 10H) have a signaling molecule (SA) between TM1 and TM2, or between TM1 and Fc. [Figure 10F] Examples of homodimer double-chain complexes having two targeting moieties (TM) (in some embodiments, more targeting moieties may be present as described herein) are shown. In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 10G and 10H) have a signaling molecule (SA) between TM1 and TM2, or between TM1 and Fc. [Figure 10G]Examples of homodimer double-chain complexes having two targeting moieties (TM) (in some embodiments, more targeting moieties may be present as described herein) are shown. In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 10G and 10H) have a signaling molecule (SA) between TM1 and TM2, or between TM1 and Fc. [Figure 10H] Examples of homodimer double-chain complexes having two targeting moieties (TM) (in some embodiments, more targeting moieties may be present as described herein) are shown. In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 10G and 10H) have a signaling molecule (SA) between TM1 and TM2, or between TM1 and Fc. [Figure 11A] Examples of homodimer double-chain complexes having two signaling molecules (in some embodiments, more signaling molecules may be present, as described herein) are shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 11G and 11H) have TM between SA1 and SA2, or TM at the N-terminus or C-terminus). [Figure 11B] Examples of homodimer double-chain complexes having two signaling molecules (in some embodiments, more signaling molecules may be present, as described herein) are shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 11G and 11H) have TM between SA1 and SA2, or TM at the N-terminus or C-terminus). [Figure 11C]Examples of homodimer double-chain complexes having two signaling molecules (in some embodiments, more signaling molecules may be present, as described herein) are shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 11G and 11H) have TM between SA1 and SA2, or TM at the N-terminus or C-terminus). [Figure 11D] Examples of homodimer double-chain complexes having two signaling molecules (in some embodiments, more signaling molecules may be present, as described herein) are shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 11G and 11H) have TM between SA1 and SA2, or TM at the N-terminus or C-terminus). [Figure 11E] Examples of homodimer double-chain complexes having two signaling molecules (in some embodiments, more signaling molecules may be present, as described herein) are shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 11G and 11H) have TM between SA1 and SA2, or TM at the N-terminus or C-terminus). [Figure 11F] Examples of homodimer double-chain complexes having two signaling molecules (in some embodiments, more signaling molecules may be present, as described herein) are shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 11G and 11H) have TM between SA1 and SA2, or TM at the N-terminus or C-terminus). [Figure 11G]Examples of homodimer double-chain complexes having two signaling molecules (in some embodiments, more signaling molecules may be present, as described herein) are shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 11G and 11H) have TM between SA1 and SA2, or TM at the N-terminus or C-terminus). [Figure 11H] Examples of homodimer double-chain complexes having two signaling molecules (in some embodiments, more signaling molecules may be present, as described herein) are shown. In some embodiments, the positions of SA1 and SA2 are interchangeable. In some embodiments, the constructs shown in the box (i.e., Figures 11G and 11H) have TM between SA1 and SA2, or TM at the N-terminus or C-terminus). [Figures 12A-12D] An example of a heterodimer two-chain complex with separated TM and SA chains, i.e., TM on the Fc knob chain and SA on the Fc hole chain, is shown. [Figures 13A-13F] An example of a heterodimer two-chain complex is shown, having separated TM and SA chains, i.e., both TMs on the knob chain of Fc and SA on the hole chain of Fc, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 14A] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14B]Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14C] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14D] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14E] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14F]Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14G] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14H] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14I] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 14J]Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., TM on the knob chain of Fc and SA on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figures 15A-15D] An example of a heterodimer two-chain complex with separated TM and SA chains, i.e., SA on the Fc knob chain and TM on the Fc hole chain, is shown. [Figures 16A-16F] An example of a heterodimer two-chain complex is shown, having separated TM and SA chains, i.e., both TMs on the Fc knob chain and on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 17A] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17B] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17C] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17D] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17E] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17F] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17G]Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17H] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17I] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 17J] Examples of heterodimer two-chain complexes are shown, having separated TM and SA chains, i.e., SA on the knob chain of Fc and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In these orientations and / or structures, one type of SA is on the knob chain and one type of SA is on the hole chain. In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figures 18A-18F] An example of a heterodimer two-chain complex is shown, in which TM and SA are on the same chain, i.e., both SA and TM are on the Fc knob chain. [Figure 19A] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19B] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19C] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19D] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19E]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19F] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19G] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19H] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19I]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19J] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19K] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 19L] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two targeting moieties (in some embodiments, as described herein, there may be more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 20A]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20B] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20C] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20D] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20E] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20F]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20G] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20H] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20I] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20J] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20K]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 20L] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc knob chain, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figures 21A-21F] An example of a heterodimer two-chain complex is shown, in which TM and SA are on the same chain, i.e., both SA and TM are on the Fc hole chain. [Figure 22A] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22B] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22C]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22D] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22E] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22F] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22G]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22H] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22I] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22J] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22K]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 22L] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the Fc hole chain, and having two targeting moieties (in some embodiments, as described herein, there are more targeting moieties). In some embodiments, the positions of TM1 and TM2 are interchangeable. In some embodiments, TM1 and TM2 may be identical. [Figure 23A] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23B] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23C] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23D]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23E] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23F] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23G] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23H] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23I]An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23J] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23K] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 23L] An example of a heterodimer two-chain complex is shown, having both TM and SA chains on the same chain, i.e., both SA and TM on the hole chain of Fc, and having two signaling molecules (in some embodiments, as described herein, more signaling molecules may be present). In some embodiments, the positions of SA1 and SA2 are interchangeable. [Figure 24A] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24B]An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24C] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24D] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24E] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24F] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24G] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24H]An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24I] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 24J] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the knob Fc and TM on each chain, is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25A] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25B] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25C] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25D]An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25E] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25F] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25G] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25H] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25I] An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figure 25J]An example of a heterodimer two-chain complex having two targeting regions (in some embodiments, there may be more targeting regions as described herein), with SA on the hole Fc and TM on each chain is shown. In some embodiments, TM1 and TM2 may be identical. [Figures 26A-26F] An example of a heterodimer two-chain complex having two signaling molecules (as described herein, there may be more signaling molecules in some embodiments) and separated SA and TM chains: SA on the knob and TM on the hole Fc is shown. [Figures 27A-27F] An example of a heterodimer two-chain complex having two signaling molecules (as described herein, there may be more signaling molecules in some embodiments) and separated SA and TM chains: TM on the knob and SA on the hole Fc is shown. [Figure 28] The biological activity of PD-L1-targeted IFNa2_R149A (top), IFNa1 (middle), and IFNa2_A145G (bottom) AFNs is shown as mean luciferase activity (±STDEV) in HL116 cells stimulated for 6 hours with serially diluted wild-type IFNa2 or IFNa1 AFN. [Figure 29] This study demonstrates the inhibition of PD-1 / PD-L1 interaction by PD-L1-targeted AFN in an AlpaLisa setting. Here, PD-L1 acceptor beads were pre-incubated with serially diluted PD-L1 AFN or atezolzimab before adding donor beads. The mean AlphaLisa counts from repeated measurements were plotted ±STDEV. [Figure 30] This study demonstrates the inhibition of CD80 / PD-L1 interaction by PD-L1-targeted AFN in a plate-binding assay. Here, PD-L1-coated plates were pre-incubated with serially diluted PD-L1 AFN before the addition of biotinylated CD80. Binding was measured using HRP-coupled streptavidin and a colorimetric peroxidase substrate. [Figure 31A]This graph shows the affinity of PD-L1 VHH AFN for human PD-L1 using biolayer interferometry (BLI). In each set of graphs, at 300 seconds, the points on the graph from top to bottom represent the doses shown from left to right. [Figure 31B] This shows the affinity of PD-L1 VHH AFN for PD-L1 in cynomolgus monkeys using biolayer interferometry (BLI). In each set of graphs, at 300 seconds, the points on the graph from top to bottom are equal to the doses shown from left to right. [Figure 31C] This is a table showing the dynamic parameters of the affinity. [Figures 32A-32B] This shows 2LIG99 and 2LIG189 VHH epitope binning in biolayer interferometry (BLI). [Figure 33A] This study demonstrates the stability of the PD-L1 AFN variant after freeze-thaw cycles. Here, the sample was analyzed by analytical sizing (SEC). 2LIG99-IFNa2_R149A. [Figure 33B] This study demonstrates the stability of the PD-L1 AFN variant after freeze-thaw cycles. Here, the sample was analyzed by analytical sizing (SEC). 2LIG189-IFNa2_R149A. [Figure 33C] This study demonstrates the stability of the PD-L1 AFN variant after freeze-thaw cycles. Here, the sample was analyzed by analytical sizing (SEC). (2LIG99)2-IFNa2_R149A. [Figure 33D] This demonstrates the stability of the PD-L1 AFN variant after freeze-thaw cycles. Here, the sample was analyzed by analytical sizing (SEC). (2LIG189)2-IFNa2_R149A. [Figure 33E] This study demonstrates the stability of the PD-L1 AFN variant after freeze-thaw cycles. Here, the sample was analyzed by analytical sizing (SEC). 2LIG99-IFNa1. [Figure 33F] This study demonstrates the stability of the PD-L1 AFN variant after freeze-thaw cycles. Here, the sample was analyzed by analytical sizing (SEC). 2LIG189-IFNa1. [Figure 33G]This demonstrates the stability of the PD-L1 AFN variant after freeze-thaw cycles. Here, the sample was analyzed by analytical sizing (SEC). (2LIG99)2-IFNa1. [Figure 33H] This demonstrates the stability of the PD-L1 AFN variant after freeze-thaw cycles. Here, the sample was analyzed by analytical sizing (SEC). (2LIG189)2-IFNa1. [Figure 34] This shows tumor growth in humanized mice treated with PD-L1 VHH AFN, plotting the median (mm3) of 5-6 animals at each time point. [Modes for carrying out the invention]

[0029] The present invention is partly based on the discovery of a conjugate (e.g., an antibody such as VHH, to an extent not limited thereto) that recognizes and binds to PD-L1. In some embodiments, the conjugate of the present invention is part of a chimeric or fusion protein having one or more targeting moieties and / or one or more signaling molecules. In various embodiments, these conjugates bind to PD-L1 and functionally modulate it (e.g., partially or completely neutralize it). In some embodiments, these conjugates bind to PD-L1 but do not functionally modulate it. Surprisingly, the inventors have discovered that various mutations of parental VHH against PD-L1 can have beneficial properties as demonstrated herein.

[0030] The present invention further provides pharmaceutical compositions comprising binding substances, and their use in the treatment of various diseases, including cancer, autoimmune diseases, and / or neurodegenerative diseases.

[0031] PD-L1 binding substance / targeting portion In various embodiments, the present invention relates to PD-L1-binding substances, which are protein-based substances capable of specifically binding to PD-L1. In various embodiments, the PD-L1-binding substance is a protein-based substance capable of specifically binding to PD-L1 without functional modification of PD-L1 (e.g., partial or complete neutralization).

[0032] In various embodiments, the present invention provides PD-L1-binding substances. Programmed death ligand 1 (PD-L1), also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1), is a type 1 transmembrane protein that is presumed to play a crucial role in suppressing the immune system. PD-L1 is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and on T cells and B cells during TCR and B cell receptor signaling.

[0033] In various embodiments, the PD-L1 binding material of the present invention includes a targeting moiety having an antigen-recognition domain that recognizes an epitope present on PD-L1. In one embodiment, the antigen-recognition domain recognizes one or more linear epitopes present on PD-L1. As used herein, a linear epitope refers to any continuous sequence of amino acids present on PD-L1. In another embodiment, the antigen-recognition domain recognizes one or more structural epitopes present on PD-L1. As used herein, a structural epitope refers to a portion (which may be discontinuous) of one or more amino acids that form a three-dimensional surface having features and / or shape and / or tertiary structure that can be recognized by the antigen-recognition domain.

[0034] In various embodiments, the present invention relates to mutations in a parent PD-L1 targeting moiety, including a recognition domain, to yield remarkably beneficial properties. For example, in various embodiments, the PD-L1 targeting moiety of the present invention has improved affinity to the parent PD-L1 targeting moiety. In some embodiments, the PD-L1 targeting moiety has an affinity increased by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times compared to the parent PD-L1 targeting moiety. In some embodiments, the PD-L1 targeting moiety of the present invention has a dissociation rate reduced by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times compared to the parent PD-L1 targeting moiety.

[0035] In some embodiments, the present invention provides a PD-L1 targeting moiety comprising a recognition domain, the recognition domain comprising (i) three complementarity-determining regions (CDR1, CDR2, and CDR3), wherein (a) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 2 or 5, (b) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (c) CDR3 comprises an amino acid sequence selected from either SEQ ID NO: 4 or 7, or (ii) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and (i) or (ii) further comprises one or more mutations at positions D54 and G55 numbered relative to SEQ ID NO: 1.

[0036] In this embodiment, the PD-L1 targeting portion, which includes the recognition domain, further comprises one or more mutations at positions Q1, Q5, A14, A63, T74, K76, S79, K86, and Q110.

[0037] In the embodiments, the mutation is a substitution, which is optionally selected from arginine (R) and lysine (K), an aromatic, polar, and positively charged hydrophilic residue including histidine (H), a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0038] In embodiments, the mutation is selected from one or more of the following: a hydrophobic, aliphatic amino acid at position D54, optionally D54G, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); a polar, positively charged hydrophilic residue at position D54, optionally D54K, selected from arginine (R) and lysine (K); a polar, neutrally charged hydrophilic residue at position G54T, optionally D54T, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); and a polar, positively charged hydrophilic residue at position G55, optionally G55R, selected from arginine (R) and lysine (K).

[0039] In the embodiment, the mutation is optionally Q1D, a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E) at position Q1; optionally Q5V, a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q5; optionally A14P, a polar and neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C) at position A14; optionally A63V, a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position A63; optionally T74S, asparagine (N) at position T74, One or more of the following are selected: a polar and neutrally charged hydrophilic residue selected from glutamine (Q), serine (S), proline (P), and cysteine ​​(C); a polar and neutrally charged hydrophilic residue at position K76, optionally K76N, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); a hydrophobic, aromatic amino acid selected at position S79, optionally S79Y, selected from phenylalanine (F), tryptophan (W), and tyrosine (Y) at position S79; a hydrophobic, aliphatic amino acid selected at position Q110, optionally Q110L, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0040] In the embodiment, the mutation is one or more of Q1D, Q5V, A14P, A63V, T74S, S79Y, K86R, and Q110L, or optionally selected from all of Q1D, Q5V, A14P, D54G, T74S, K76N, S79Y, K86R, and Q110L.

[0041] In some embodiments, the present invention relates to a PD-L1 targeting portion including a recognition domain, wherein the recognition domain is (i) Three complementarity determination regions (CDR1, CDR2, and CDR3), (a) CDR1 contains an amino acid sequence selected from either SEQ ID NO: 2 or 5, (b) CDR2 contains an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (c) CDR3 contains three complementarity-determining regions, each containing an amino acid sequence selected from either SEQ ID NO: 4 or 7, or (ii) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, wherein (i) or (ii) further comprises one or more mutations at positions D54, G55, K76, and S79 numbered relative to SEQ ID NO: 1.

[0042] In some embodiments, the PD-L1 targeting portion further includes one or more mutations at positions T74, K86, and Q110 with respect to SEQ ID NO: 1. In some embodiments, the PD-L1 targeting moiety has a mutation which is a substitution, and optionally the substitution is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), an aromatic, polar, positively charged hydrophilic residue including histidine (H), a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0043] In some embodiments, the mutation is selected from one or more of the following: • Optionally, D54G is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position D54, or optionally, D54K is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), or optionally, D54T is a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). • A polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K) at position G55, which is optionally G55R. • A polar and neutrally charged hydrophilic residue at position T74, selected from asparagine (N), glutamine (Q), serine (S), proline (P), and cysteine ​​(C), which is optionally T74S. • A polar and neutrally charged hydrophilic residue at position K76, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), which is optionally K76N. • A hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y) at position S79, optionally being S79Y. · Arginine (R) at position K86, which is K86R, and • A hydrophobic, aliphatic amino acid at position Q110, optionally Q110L, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0044] In various embodiments, the aforementioned mutant PD-L1 targeting moiety (i.e., the one disclosed for SEQ ID NO: 1) has improved affinity to the parent PD-L1 targeting moiety of SEQ ID NO: 1.

[0045] In some embodiments, the PD-L1 targeting portion of the present invention comprises (i) three complementarity determination regions (CDR1, CDR2, and CDR3), (a) CDR1 contains an amino acid sequence selected from either SEQ ID NO: 27 or 30, (b) CDR2 contains an amino acid sequence selected from either SEQ ID NO: 28 or 31, and (c) CDR3 contains three complementarity-determining regions, each containing an amino acid sequence selected from either SEQ ID NO: 29 or 32, or (ii) comprising a recognition domain comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, wherein (i) or (ii) further comprises one or more mutations at positions N32, D33, and M97 numbered relative to SEQ ID NO: 26. In some embodiments, the PD-L1 targeting moiety has a mutation that is a substitution for SEQ ID NO: 26. In some embodiments, the substitution is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), or an aromatic, polar, positively charged hydrophilic residue comprising histidine (H). In some embodiments, the substitution is a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). In some embodiments, the substitution is a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E). In some embodiments, the substitution is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0046] In some embodiments, the PD-L1 targeting moiety is a positively hydrophilic residue and has a substitution at position N32 selected from arginine (R) and lysine (K). In some embodiments, the substitution at position N32 is a polar and neutral hydrophilic residue selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). In some embodiments, the substitution at position N32 is N32Q or N32R for SEQ ID NO: 26.

[0047] In some embodiments, the PD-L1 targeting moiety has a substitution at position D33, which is D33H relative to SEQ ID NO: 26. In some embodiments, the PD-L1 targeting moiety is an aliphatic hydrophobic residue, which has a substitution at position M97, which is selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) relative to SEQ ID NO: 26. In some embodiments, the PD-L1 targeting moiety has a substitution at position M97 relative to SEQ ID NO: 26, which is M97I, M97L, or M97V.

[0048] In the embodiment, the PD-L1 targeting moiety including the recognition domain includes one or more of the following mutations (relative to SEQ ID NO: 26): Q1D, Q5V, A14P, A62S, A74S, M77T, M78V, S79Y, K86R, and Q109L, and optionally further includes all of Q1D, Q5V, A14P, D33H, A62S, A74S, M77T, M78V, K86R, and M97V.

[0049] In various embodiments, the aforementioned mutant PD-L1 targeting moiety (i.e., the one disclosed for SEQ ID NO: 26) has improved affinity to the parent PD-L1 targeting moiety of SEQ ID NO: 26.

[0050] In some embodiments, the PD-L1 targeting portion of the present invention includes an amino acid sequence having at least 90% sequence identity with any one of the amino acid sequences selected from SEQ ID NOs: 1, 8-26, and 33-74.

[0051] In some embodiments, the PD-L1 targeting portion of the present invention includes one or more additional recognition domains. In some embodiments, these additional recognition domains bind to CD8, CD13, CD20, NKp46, Clec9A, Clec4c, PD-1, PD-L1, PD-L2, SIRP1α, FAP, XCR1, tenascin CA1, Flt3, or ECM proteins.

[0052] In various embodiments, the PD-L1 conjugates of the present invention may bind to the full-length and / or mature forms, and / or isoforms, and / or splice variants, and / or fragments of human PD-L1, and / or any other natural or synthetic analogs, variants, or variants. In various embodiments, the PD-L1 conjugates of the present invention may bind to any type of human PD-L1. In some embodiments, the PD-L1 conjugates bind to phosphorylated PD-L1. In some embodiments, the PD-L1 conjugates bind to acetylated PD-L1.

[0053] In some embodiments, the PD-L1 targeting moiety recognizes tumor antigens and optionally modulates them functionally. In various embodiments, the PD-L1 targeting moiety recognizes antigens on immune cells and optionally modulates them functionally. The immune cells are selected from T cells, B cells, dendritic cells, macrophages, neutrophils, NK cells, and NKT cells. In some embodiments, the PD-L1 targeting moiety of the present invention recruits cytotoxic T cells to tumor cells or the tumor environment.

[0054] In one embodiment, the PD-L1 binding substance of the present invention includes a targeting moiety having an antigen recognition domain that recognizes one or more epitopes present on human PD-L1. In one embodiment, human PD-L1 includes the following amino acid sequence (signal peptide is underlined): Isoform 1: [ka] Isoform 2: [ka] Isoform 3: [ka]

[0055] In various embodiments, the PD-L1 conjugate of the present invention includes a targeting moiety capable of specific binding. In various embodiments, the PD-L1 conjugate includes a targeting moiety having an antigen-recognition domain such as an antibody or a derivative thereof. In one embodiment, the PD-L1 conjugate includes a targeting moiety that is an antibody. In various embodiments, the antibody is a full-length multimer protein comprising two heavy chains and two light chains. Each heavy chain has one variable region (e.g., V H ) and at least three steady regions (e.g., CH1, CH2, and CH3), and each light chain has one variable region (V L ) and one steady-state region (C L The variable region determines the specificity of the antibody. Each variable region contains three high-frequency variable regions, also known as complementarity-determining regions (CDRs), which are flanked by four relatively conserved framework regions (FRs). The three CDRs are called CDR1, CDR2, and CDR3 and contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0056] In some embodiments, the PD-L1 conjugate comprises a targeting moiety that is an antibody derivative or format. In some embodiments, the PD-L1 conjugate of the present invention is recognized by U.S. Patent No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Patent No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Patent No. 7,838,629, U.S. Patent No. 7,186,524, U.S. U.S. Patent No. 6,004,746, U.S. Patent No. 5,475,096, U.S. Patent Application Publication No. 2004 / 146938, U.S. Patent Application Publication No. 2004 / 157209, U.S. Patent No. 6,994,982, U.S. Patent No. 6,794,144, U.S. Patent Application Publication No. 2010 / 239633, U.S. Patent No. 7,803,907, U.S. Patent Application Publication No. 2010 / 119446, and / or U.S. Patent No. 7,166,697 (this As described in (the entire contents of which are incorporated herein by reference), the targeting moieties include single-domain antibodies, recombinant heavy-chain-only antibodies (VHH), single-chain antibodies (scFv), shark heavy-chain-only antibodies (VNAR), microproteins (cysteine ​​knot protein, Notchin), DARPin; tetranectin; afibody; transbody; anticarin; adnectin; affin; afimer; microbody; aptamer; alterase; plastic antibody; phylomer; stradobody; maxibody; shrimpbody; finomer; armadillo repeat protein; Knitz domain, avimer, atrimer, probody, immunobody, triomab, trobody, pepbody, vacibody, unibody; duobody, Fv, Fab, Fab', F(ab')2, peptide mimetic molecules, or synthetic molecules. See also Storz MAbs.2011 May-Jun;3(3):310-317.

[0057] In some embodiments, the PD-L1 conjugate includes a targeting moiety that is a single-domain antibody such as VHH. VHH may be derived from organisms that produce VHH antibodies, such as camels or sharks, or VHH may be a designed VHH. VHH is an antibody-derived therapeutic protein that incorporates the unique structural and functional properties of naturally occurring heavy-chain antibodies. VHH technology is based on fully functional antibodies derived from camels that lack a light chain. These heavy-chain antibodies have a single variable domain (V H It contains H) and two constant domains (CH2 and CH3).

[0058] In some embodiments, the PD-L1 binding substance includes VHH. In some embodiments, VHH is humanized VHH or camelized VHH.

[0059] In some embodiments, VHH is a complete human V H Domains, for example, human bodies (Crescendo Biologics, Cambridge, UK). In some embodiments, fully human V H The domain, for example, the human body, is monovalent, divalent, or trivalent. In some embodiments, a complete human V H Domains, such as human bodies, are single-specific, double-specific, or triple-specific. (e.g., fully human V) H Examples of domains, such as human bodies, are described, for example, in WO2016 / 113555 and WO2016 / 113557, the full disclosures of which are incorporated herein by reference.

[0060] In some embodiments, the PD-L1 binding substance comprises a targeting moiety which is a VHH containing a single amino acid chain having four “framework regions” or FRs and three “complementarity-determining regions” or CDRs. As used herein, “framework regions” or “FRs” refers to regions within a variable domain located between CDRs. As used herein, “complementarity-determining regions” or “CDRs” refers to variable regions in a VHH containing an amino acid sequence that can specifically bind to an antigen target.

[0061] In various embodiments, the PD-L1 binding material comprises a VHH having a variable domain containing at least one CDR1, CDR2, and / or CDR3 sequence. In various embodiments, the PD-L1 binding material comprises a VHH having a variable region containing at least one FR1, FR2, FR3, and FR4 sequence.

[0062] In some embodiments, the CDR1 sequence of the PD-L1 binding material is selected from GTIFSINRMD (SEQ ID NO: 2), GTIFS (SEQ ID NO: 5), GKIFSGNDMG (SEQ ID NO: 27), or GKIFS (SEQ ID NO: 30).

[0063] In some embodiments, the CDR2 sequence of the PD-L1 binding agent is selected from LITSDGTPA (SEQ ID NO: 3), LITSDGTPAYADSAKG (SEQ ID NO: 6), IITSGGITD (SEQ ID NO: 28), or IITSGGITDADAVKG (SEQ ID NO: 31).

[0064] In some embodiments, the CDR3 sequence of the PD-L1 binding agent is selected from SSGVYNY (SEQ ID NO: 4), SSGVYNY (SEQ ID NO: 7), RDRTIW (SEQ ID NO: 29), or RDRTIW (SEQ ID NO: 32).

[0065] In various exemplary embodiments, the PD-L1 binding substance comprises an amino acid sequence selected from the following sequences: • Sequence ID 8-P-1659:2LIG99_OPT1 (Q1D_Q5V_A14P_T74S_K86R_Q110L) DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNSKKTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS • Sequence ID 9-P-1660:2LIG99_OPT2 (Q1D_Q5V_A14P_T23A_T74S_K86R_Q110L) DVQLVESGGGLVQPGGSLRLSCAASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNSKKTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS • Sequence ID 10-P-1661:2LIG99_OPT3 (Q1D_Q5V_A14P_A63V_T74S_K86R_Q110L) DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSVKGRFTISRDNSKKTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS • Sequence ID 11-P-1662:2LIG99_OPT4 (Q1D_Q5V_A14P_T74S_K76N_K86R_Q110L) DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNSKNTVSLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS • Sequence ID 12-P-1663:2LIG99_OPT5 (Q1D_Q5V_A14P_T74S_S79Y_K86R_Q110L) DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNSKKTVYLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS • Sequence ID 13-P-1664:2LIG99_OPT6 (Q1D_Q5V_A14P_T23A_A63V_T74S_K76N_S79Y_K86R_Q110L) DVQLVESGGGLVQPGGSLRLSCTASGTIFSINRMDWFRQAPGKQRELVALITSDGTPAYADSAKGRFTISRDNSKKTVYLQMNSLRPEDTAVYYCHVSSGVYNYWGQGTLVTVSS • Sequence ID 14-P-1665: 2LIG99_D54G [ka] • Sequence ID 15-P-1666:2LIG99_D54K [ka] • Sequence ID 16-P-1667:2LIG99_D54T [ka] • Sequence ID 17-P-1668:2LIG99_G55R [ka] • Sequence ID 18-P-2049: 2LIG99_OPT_D54G (Q1D_Q5V_A14P_D54G_T74S_K86R_Q110L) [ka] • Sequence ID 19-P-2050:2LIG99_OPT_D54G_A63V (Q1D_Q5V_A14P_D54G_A63V_T74S_K86R_Q110L) [ka] • Sequence ID 20-P-2051:2LIG99_OPT_D54G_K76N (Q1D_Q5V_A14P_D54G_T74S_K76N_K86R_Q110L) [ka] • Sequence ID 21-P-2052: 2LIG99_OPT_D54G_S79Y (Q1D_Q5V_A14P_D54G_T74S_S79Y_K86R_Q110L) [ka] • Sequence ID 22-P-2053:2LIG99_OPT_D54G_A63V_K76N (Q1D_Q5V_A14P_D54G_A63V_T74S_K76N_K86R_Q110L) [ka] • Sequence ID 23-P-2054:2LIG99_OPT_D54G_A63V_S79Y (Q1D_Q5V_A14P_D54G_A63V_T74S_K86R_S97Y_Q110L) [ka] • Sequence ID 24-P-2055: 2LIG99_OPT_D54G_K76N_S79Y (Q1D_Q5V_A14P_D54G_T74S_K76N_K86R_S79Y_Q110L) [ka] • Sequence ID 25-P-2056:2LIG99_OPT_D54G_A63V_K76N_S79Y (Q1D_Q5V_A14P_D54G_A63V_T74S_K76N_K86R_S79Y_Q110L) [ka]

[0066] In various exemplary embodiments, the PD-L1 binding material comprises an amino acid sequence having or not having a terminal histidine tag sequence (i.e., HHHHHH; SEQ ID NO: 78).

[0067] In some embodiments, the PD-L1 binding material comprises an amino acid sequence with or without an HA tag (i.e., YPYDVPDYGS; SEQ ID NO: 79).

[0068] In some embodiments, the PD-L1 binding material comprises an amino acid sequence that may or may not have an AAA linker.

[0069] In some embodiments, the PD-L1 binding material comprises an amino acid sequence having or not having an AAA linker, an HA tag, and a terminal histidine tag sequence (i.e., AAAYPYDVPDYGSHHHHHH; SEQ ID NO: 80).

[0070] In various embodiments, the present invention intends to utilize any natural or synthetic analogs, variants, alleles, homologs, and orthologs (collectively referred to herein as “Analogs”) of the PD-L1 conjugates of the present invention described herein. In various embodiments, the amino acid sequence of the PD-L1 conjugate further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0071] In various embodiments, the PD-L1 binding material includes a targeting moiety comprising a sequence that is at least 60% identical to any one of the sequences disclosed herein. For example, the PD-L1 binding material is at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least Approximately 97%, at least approximately 98%, at least approximately 99%, or 100% identical (for example, approximately 60%, or approximately 61%, or approximately 62%, or approximately 63%, or approximately 64%, or approximately 65%, or approximately 66%, or approximately 67%, or approximately 68%, or approximately 69%, or approximately 70%, or approximately 71%, or approximately 72%, or approximately 73%, or approximately 74%, or approximately 75%, or approximately 76%, or approximately 77%, or approximately 7 The targeting portion may include sequences that have 8%, or approximately 79%, or approximately 80%, or approximately 81%, or approximately 82%, or approximately 83%, or approximately 84%, or approximately 85%, or approximately 86%, or approximately 87%, or approximately 88%, or approximately 89%, or approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99%, or approximately 100% sequence identity.

[0072] In various embodiments, the PD-L1 conjugate includes a targeting moiety comprising an amino acid sequence having one or more amino acid mutations with respect to any one of the sequences disclosed herein. In various embodiments, the PD-L1 conjugate includes a targeting moiety comprising an amino acid sequence having 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 15, or 20 amino acid mutations with respect to any one of the sequences disclosed herein. In some embodiments, one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0073] In some embodiments, amino acid mutations are amino acid substitutions, which may include conservative and / or non-conservative substitutions.

[0074] "Conservative substitutions" can be made, for example, based on the similarity of the amino acid residues involved in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties. The 20 native amino acids can be classified into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0075] As used herein, “conservative substitution” is defined as the exchange of one amino acid with another amino acid listed within the same group of the six standard amino acid groups described above. For example, the exchange of Asp with Glu retains one negative charge in the thus modified polypeptide. Furthermore, glycine and proline may be substituted for each other based on their ability to disrupt their α-helices.

[0076] As used herein, “non-conservative substitution” is defined as the replacement of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) described above.

[0077] In various embodiments, substitutions may also include non-classical amino acids. Examples of non-classical amino acids include, but are not limited to, selenocysteine, pyrrollysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, C-α-methylamino acids, N-α-methylamino acids, and common amino acid analogs.

[0078] In various embodiments, the amino acid mutation may be within the CDR of the targeting region (e.g., CDR1, CDR2, or CDR3 region). In another embodiment, the amino acid change may be within the framework region (FR) of the targeting region (e.g., FR1, FR2, FR3, or FR4 region).

[0079] Modification of amino acid sequences can be achieved using any known technique in the art, such as site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0080] In various embodiments, the mutation does not substantially reduce the ability of the PD-L1 binding substance of the present invention to specifically bind to PD-L1. In various embodiments, the mutation does not substantially reduce the ability of the PD-L1 binding substance of the present invention to specifically bind to PD-L1 without functionally regulating (e.g., partially or completely neutralizing) PD-L1.

[0081] In various embodiments, the binding affinity of the PD-L1 binding substance of the present invention for the full-length and / or mature form, and / or isoform, and / or splice variant, and / or fragment, and / or monomer and / or dimer form, and / or any other natural or synthetic analog, variant, or mutant (including monomer and / or dimer form) of human PD-L1 can be described by the equilibrium dissociation constant (K D ). In various embodiments, the PD-L1 binding substance has a K D of about 1 uM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, or about 5 nM, or less than about 1 nM, and includes a targeting moiety that binds to the full-length and / or mature form, and / or isoform, and / or splice variant, and / or fragment, and / or any other natural or synthetic analog, variant, or mutant (including monomer and / or dimer form) of human PD-L1.

[0082] In various embodiments, the PD-L1 conjugate includes a targeting moiety that binds to the target antigen, i.e., PD-L1, but does not functionally modulate it (e.g., does not partially or completely neutralize it). For example, in various embodiments, the targeting moiety of the PD-L1 conjugate merely targets the antigen and does not substantially functionally modulate (e.g., does not partially or completely inhibit, reduce, or neutralize) the biological effect of the antigen. In various embodiments, the targeting moiety of the PD-L1 conjugate binds to an epitope that is physically distant from the antigenic site important to its biological activity (e.g., the active site of the antigen).

[0083] In various embodiments, these binding materials bind to PD-L1 and functionally modulate it (e.g., partially or completely neutralize it).

[0084] Therapeutic drugs containing a PD-L1 targeting portion In various embodiments, the PD-L1 targeting moiety of the present invention is part of a chimeric or fusion protein with one or more targeting substances or signaling molecules. Accordingly, the present invention provides a chimeric or fusion protein comprising, for example, a targeting moiety for PD-L1 and one or more signaling molecules. In some embodiments, the present invention provides one or more targeting moieties in which at least one targeting moiety is for PD-L1 and one or more signaling molecules.

[0085] In various embodiments, a signaling molecule is modified to have reduced affinity or activity to one or more of its receptors, thereby enabling attenuation of the activity (including agonism or antagonistism) of a chimeric or fusion protein and / or preventing nonspecific signaling or undesirable sequestration of the chimeric or fusion protein. In various embodiments, a signaling molecule is an antagonist in its wild-type form and has one or more mutations that weaken its antagonist activity. In various embodiments, a signaling molecule is an antagonist due to one or more mutations; for example, an agonist signaling molecule is converted to an antagonist signaling molecule, and such converted signaling molecule optionally also has one or more mutations that weaken its antagonist activity (e.g., as described in WO 2015 / 007520, the entirety of which is incorporated herein by reference).

[0086] Therefore, in various embodiments, the signaling molecule is a modified (e.g., mutant) signaling molecule having one or more mutations. In various embodiments, the modification (e.g., mutation) allows the modified signaling molecule to have one or more weakened activities, such as reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, compared to the unmodified or unmutated, i.e., wild-type, signaling molecule (e.g., comparing the wild-type and modified or mutant forms of the same signaling molecule). In some embodiments, a mutation that weakens or reduces binding or affinity includes a mutation that substantially reduces or eliminates binding or activity. In some embodiments, a mutation that weakens or reduces binding or affinity is different from a mutation that substantially reduces or eliminates binding or activity. As a result, in various embodiments, the mutation allows the signaling molecule to have improved safety, such as reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the unmutated, i.e., wild-type, signaling molecule (e.g., comparing the wild-type and modified or mutant forms of the same signaling molecule).

[0087] In some embodiments, the targeting portion of the present invention restores the affinity or activity of a modified signaling molecule to the receptor of the signaling molecule.

[0088] As described herein, a drug may have improved safety due to one or more modifications, e.g., mutations. In various embodiments, improved safety means that the chimeric protein or chimeric protein complex of the present invention results in lower toxicity (e.g., systemic toxicity and / or tissue / organ-related toxicity), and / or reduced or substantially eliminated side effects, and / or enhanced tolerability, reduced or substantially eliminated adverse events, and / or reduced or substantially eliminated off-target effects, and / or an extended therapeutic time range.

[0089] In various embodiments, the signaling molecule is modified to have one or more mutations that reduce its binding affinity or activity to one or more of its receptors. In some embodiments, the signaling molecule is modified to have one or more mutations that substantially reduce or eliminate its binding affinity or activity to the receptor. In some embodiments, the activity provided by the wild-type signaling molecule is agonism to the receptor (e.g., activation of a cellular effect at the site of treatment). For example, the wild-type signaling molecule may activate its receptor. In such embodiments, the mutation results in a signaling molecule modified to reduce or eliminate its activating effect on the receptor. For example, the mutation may result in a signaling molecule modified to send a reduced activation signal to the target cell, or the activation signal may be eliminated. In some embodiments, the action provided by the wild-type signaling molecule is antagonism to the receptor (e.g., blockage or suppression of a cellular effect at the site of treatment). For example, the wild-type signaling molecule may antagonize or inhibit the receptor. In these embodiments, the mutation results in a signaling molecule modified to reduce or eliminate its antagonistic activity to the receptor. For example, a mutation may result in a signaling molecule that is modified to send a reduced inhibitory signal to a target cell, or the inhibitory signal may be eliminated. In various embodiments, the signaling molecule is an antagonist due to one or more mutations, for example, an agonist signaling molecule being converted into an antagonist signaling molecule (for example, as described in International Publication No. 2015 / 007520, the entirety of which is incorporated herein by reference), and such converted signaling molecule also has one or more mutations that optionally reduce its binding affinity or activity to one or more of its receptors, or reduce or eliminate its binding affinity or activity to one or more of its receptors.

[0090] In some embodiments, the reduced affinity or activity to the receptor can be restored by binding to one or more of the targeting moieties described herein (e.g., the targeting moiety for PD-L1 or any other targeting moieties described herein). In other embodiments, the reduced affinity or activity to the receptor is not substantially restored by the action of one or more of the targeting moieties.

[0091] In various embodiments, the chimeric protein or chimeric protein complex of the present invention reduces off-target effects because its signaling molecule has mutations that weaken or eliminate its binding affinity or activity to a receptor. In various embodiments, this reduction in side effects is observed, for example, compared to the wild-type signaling molecule. In various embodiments, the signaling molecule is active against target cells because the targeting moiety(s) compensates for any missing / insufficient binding (e.g., but not limited to and / or binding affinity) required for substantial activation. In various embodiments, the modified signaling molecule is substantially inactive on its way to the site of therapeutic action and substantially exerts its effect on the cell type specifically targeted, thereby significantly reducing undesirable side effects.

[0092] In some embodiments, the signaling molecule may include one or more mutations that weaken or reduce binding or affinity to one receptor (i.e., a therapeutic receptor), and one or more mutations that substantially reduce or eliminate binding or activity to a second receptor. In such embodiments, these mutations may be in the same or different positions (i.e., the same or multiple mutations). In some embodiments, one or more mutations that reduce binding and / or activity to one receptor are different from one or more mutations that substantially reduce or eliminate activity to another receptor. In some embodiments, one or more mutations that reduce binding and / or activity to one receptor are the same as one or more mutations that substantially reduce or eliminate activity to another receptor. In some embodiments, the chimeric protein or chimeric protein complex of the present invention has a modified signaling molecule having both mutations that weaken binding and / or activity to a therapeutic receptor (e.g., compared to a wild-type signaling molecule), thus enabling a more controlled on-target therapeutic effect, and mutations that substantially reduce or eliminate binding and / or activity to another receptor (e.g., compared to a wild-type signaling molecule), thus reducing side effects.

[0093] In some embodiments, a substantial reduction or elimination of binding or activity is not substantially recoverable by the targeting moiety (e.g., the targeting moiety for PD-L1 or any other targeting moiety described herein). In some embodiments, a substantial reduction or elimination of binding or activity is recoverable by the targeting moiety. In various embodiments, a substantial reduction or elimination of binding or activity to a second receptor may also prevent adverse effects mediated by the other receptor. Alternatively, or in addition, a substantial reduction or elimination of binding or activity to other receptors improves the therapeutic effect by reducing or eliminating the sequestering of the therapeutic chimeric protein or chimeric protein complex away from the therapeutic site of action. For example, in some embodiments, this eliminates the need for high doses of the chimeric protein or chimeric protein complex of the present invention to compensate for the loss at the other receptor. The ability to reduce such dosages further reduces the potential for side effects.

[0094] In various embodiments, the modified signaling molecule has reduced, substantially reduced, or removed affinity to one or more of its receptors, e.g., binding (e.g., KD), and / or activation (e.g., if the modified signaling molecule is an agonist of its receptor, e.g., KD). A and / or EC 50 (Measurable as), and / or inhibition (for example, if the modified signaling molecule is an antagonist of its receptor, e.g., K I and / or IC 50The modified signaling molecule comprises one or more mutations that cause it to have (measurable as) an affinity for the receptor. In various embodiments, the reduced affinity of the signaling molecule to the receptor allows for attenuation of activity (including agonism or antagonism). In such embodiments, the modified signaling molecule has an affinity for the receptor of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, or about 80%-100% compared to the wild-type signaling molecule. In some embodiments, the binding affinity is at least about 2 times lower, about 3 times lower, about 4 times lower, about 5 times lower, about 6 times lower, about 7 times lower, about 8 times lower, about 9 times lower, at least about 10 times lower, at least about 15 times lower, at least about 20 times lower, at least about 25 times lower, at least about 30 times lower, at least about 35 times lower, at least about 40 times lower, at least about 45 times lower, at least about 50 times lower, at least about 100 times lower, at least about 150 times lower, or about 10 to 50 times lower, about 50 to 100 times lower, about 100 to 150 times lower, about 150 to 200 times lower, or more than 200 times lower than the wild-type signaling molecule.

[0095] In some embodiments, a chimeric protein or chimeric protein complex comprises a modified signaling molecule having mutations that reduce binding to one receptor and substantially reduce or eliminate binding to a second receptor, wherein the attenuation or reduction of the binding affinity of the modified signaling molecule to one receptor is less than the substantially reduction or elimination of affinity to the other receptor. In some embodiments, the attenuation or reduction of the binding affinity of the modified signaling molecule to one receptor is about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% less than the substantially reduction or elimination of affinity to the other receptor. In various embodiments, substantial reduction or elimination refers to a reduction in binding affinity and / or activity that is greater than attenuation or reduction.

[0096] In various embodiments, the modified signaling molecule includes, for example, one or more mutations that reduce the endogenous activity of the signaling molecule to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, compared to the wild-type signaling molecule.

[0097] In some embodiments, the modified signaling molecule comprises one or more mutations that cause the signaling molecule to have a reduced affinity to its receptor(s), which is lower than the binding affinity of the targeting moiety(s)

[0098] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be evaluated by scatchard plot analysis and computer fitting of binding data (e.g., Scatchard, 1949), or by reflection interference spectroscopy under flow-through conditions, as described by Brecht et al. (1993) (all of these are incorporated herein by reference).

[0099] In various embodiments, the signaling molecule is one or more immunomodulatory agents, such as interleukins, interferons, and tumor necrosis factors, any of which are optionally modified or mutated. In some embodiments, the modified signaling molecule is selected from human: IFNα2, IFNα1, IFNβ, IFNγ, consensus interferon, TNF, TNFR, TGF-α, TGF-β, VEGF, EGF, PDGF, FGF, TRAIL, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-33, IGF-1, or EPO.

[0100] In some licenses, the signaling molecule is an interleukin or modified interleukin, including, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, or a fragment, variant, analog, or family member thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulating the proliferation of immune cells (e.g., helper T cells, B cells, eosinophils, and lymphocytes), migrating neutrophils and T lymphocytes, and / or inhibiting interferons. Interleukin activity can be measured using assays known in the art: Matthews et al., in Lymphokines and Interferons: A Practical Approach, Clemens et al., eds, IRL Press, Washington, DC 1987, pp. 221-225, and Orencole & Dinarello (1989) Cytokine 1, 14-20.

[0101] In some embodiments, the signaling molecule is an interferon, or a modified interferon such as interferon types I, II, and III. Examples of interferons include, for example, interferon-α-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon-β and interferon-γ, interferon-κ, interferon-ε, interferon-τ, and interferon-ω.

[0102] In some embodiments, the signaling molecule is tumor necrosis factor (TNF), or a modified form of tumor necrosis factor (TNF), or a protein of the TNF family, and includes, but is not limited to, TNF-α, TNF-β, LT-β, ​​CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

[0103] The amino acid sequences of the wild-type signaling molecules described herein are well known in the art. Therefore, in various embodiments, the modified signaling substance is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, and This means that the sequence identity is at least approximately 91%, or at least approximately 92%, or at least approximately 93%, or at least approximately 94%, or at least approximately 95%, or at least approximately 96%, or at least approximately 97%, or at least approximately 98%, or at least approximately 99% (for example, about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 7 It contains an amino acid sequence having sequence identity of 3%, or approximately 74%, or approximately 75%, or approximately 76%, or approximately 77%, or approximately 78%, or approximately 79%, or approximately 80%, or approximately 81%, or approximately 82%, or approximately 83%, or approximately 84%, or approximately 85%, or approximately 86%, or approximately 87%, or approximately 88%, or approximately 89%, or approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99%.

[0104] In various embodiments, the modified signaling substance has at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least Also, approximately 91%, or at least approximately 92%, or at least approximately 93%, or at least approximately 94%, or at least approximately 95%, or at least approximately 96%, or at least approximately 97%, or at least approximately 98%, or at least approximately 99% sequence identity (e.g., approximately 60%, or approximately 61%, or approximately 62%, or approximately 63%, or approximately 64%, or approximately 65%, or approximately 66%, or approximately 67%, or approximately 68%, or approximately 69%, or approximately 70%, or approximately 71%, or approximately 72%, or approximately 73%). It contains an amino acid sequence having sequence identity of approximately 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0105] In various embodiments, the modified signaling molecule comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions and may include conserved substitutions and / or non-conserved substitutions, as described elsewhere in this specification.

[0106] In various embodiments, substitutions may also include non-classical amino acids, as described elsewhere in this specification.

[0107] As described herein, modified signaling molecules have mutations that affect affinity and / or activity to one or more receptors. In various embodiments, there is reduced affinity and / or activity to therapeutic receptors, e.g., receptors through which the desired therapeutic effect is mediated (e.g., agonism or antagonistism). In various embodiments, the modified signaling molecules have mutations that substantially reduce or eliminate affinity and / or activity to receptors, e.g., receptors through which the desired therapeutic effect is not mediated (e.g., as a result of a disrupted binding state). The receptors of any signaling molecules described herein are known in the art.

[0108] Examples of mutations resulting in reduced affinity and / or activity (e.g., agonist activity) to receptors are found in WO 2013 / 107791 and PCT / EP2017 / 061544 (e.g., with respect to interferon), WO 2015 / 007542 (e.g., with respect to interleukin), and WO 2015 / 007903 (e.g., with respect to TNF), the full contents of each of these are incorporated herein by reference. Examples of mutations that reduce affinity and / or activity (e.g., antagonist activity) to therapeutic receptors are found in International Publication 2015 / 007520, the full contents of which are incorporated herein by reference.

[0109] In some embodiments, the modified signaling molecule comprises one or more mutations that cause the signaling molecule to have reduced affinity and / or activity for a receptor of the type I cytokine receptor, type II cytokine receptor, chemokine receptor, tumor necrosis factor receptor (TNFR) superfamily receptor, TGF-beta receptor, immunoglobulin (Ig) superfamily receptor, and / or tyrosine kinase superfamily receptor.

[0110] In various embodiments, the receptor for the signaling molecule is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2 (beta subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and receptors for thrombopoietin (TPO), prolactin, and growth hormone. Exemplary type I cytokine receptors include, but are not limited to, the GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and type I IL receptor.

[0111] In various embodiments, the receptor for the signaling molecule is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits and are primarily receptors for interferons. This receptor family includes, but is not limited to, receptors for interferon alpha, interferon beta, and interferon gamma, IL10, IL22, and tissue factor. Examples of type II cytokine receptors include, but are not limited to, the IFN-alpha receptor (e.g., IFNAR1 and IFNAR2), IFN-beta receptor, IFN-gamma receptor (e.g., IFNGR1 and IFNGR2), and type II IL receptor.

[0112] In various embodiments, the receptor for a signaling molecule is a G protein-coupled receptor. A chemokine receptor is a G protein-coupled receptor having a seven-transmembrane structure that binds to a G protein for signal transduction. Examples of chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors (XCR1). Examples of chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

[0113] In various embodiments, the receptor for the signaling substance is a TNFR family member. Tumor necrosis factor receptor (TNFR) family members share a cysteine-rich domain (CRD) formed from three disulfide bonds surrounding a CXXCXXC core motif that creates an elongated molecule. Examples of the tumor necrosis factor receptor family include the following: CD120a (TNFRSF1A), CD120b (TNFRSF1B), lymphotoxin beta receptor (LTBR), TNFRSF3, CD134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSF10A (TNFRSF10A), TNFRSF10B (TNFRSF10B), TNFRSF10C (TNFRSF10C), TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11B), TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C), TNFRSF14 (TNFRSF14), nerve growth factor receptor (NGFR, TNFRSF16), TNFRSF17 (TNFRSF17), TNFRSF18 (TNFRSF18), TNFRSF19 (TNFRSF19), TNFRSF21 (TNFRSF21), and TNFRSF25 (TNFRSF25). In one embodiment, the TNFR family member is CD120a (TNFRSF1A) or TNF-R1. In another embodiment, the TNFR family member is CD120b (TNFRSF1B) or TNF-R2.

[0114] In various embodiments, the receptor for the signaling substance is a TGF-beta receptor. The TGF-beta receptor is a single-pass transmembrane serine / threonine kinase receptor. TGF-beta receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.

[0115] In various embodiments, the receptors for signaling molecules are Ig superfamily receptors. Receptors of the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Examples of Ig superfamily receptors, but not limited to, include interleukin-1 receptors, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

[0116] In various embodiments, the receptor for the signaling molecule is a tyrosine kinase superfamily receptor. Receptors of the tyrosine kinase superfamily are well known in the art. There are approximately 58 receptor tyrosine kinases (RTKs) classified into 20 subfamilies. Examples of tyrosine kinase superfamily receptors include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0117] In some embodiments, the modified signaling molecule is interferon-alpha. In such embodiments, the modified IFNα substance has reduced affinity and / or activity toward the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNα substance has substantially reduced or eliminated affinity and / or activity toward the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0118] Mutant interferon-α is known to those skilled in the art. In one exemplary embodiment, the modified signaling molecule is allelic IFNα2a having the amino acid sequence of SEQ ID NO: 81.

[0119] In one exemplary embodiment, the modified signaling molecule is an allele-type IFNα2b having the amino acid sequence of SEQ ID NO: 82 (which differs from IFNα2a at amino acid position 23):

[0120] In some embodiments, the IFN-α2 variant (IFN-α2a or IFN-α2b) is mutated at one or more amino acids at positions 144–154, e.g., amino acid positions 145, 148, 149, and / or 153. In some embodiments, the IFN-α2 variant includes one or more mutations selected from L153A, R149A, M148A, and A145G. Variants are described, for example, in WO2013 / 107791 and Piehler et al., (2000) J. Biol. Chem, 275:40425-33, all of which are incorporated herein by reference.

[0121] In some embodiments, the IFNα2 variant has reduced affinity and / or activity for IFNAR1. In some embodiments, as described in International Publication No. 2010 / 030671, the IFNα2 variant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A. The entire contents of this patent are incorporated herein by reference.

[0122] In some embodiments, as described in International Publication No. 2008 / 124086, the IFNα2 variant comprises one or more mutations selected from K133A, R144A, R149A, and L153A. The entire contents of this patent are incorporated herein by reference.

[0123] In some embodiments, as described in International Publication No. 2015 / 007520 and International Publication No. 2010 / 030671, the IFNα2 variant comprises one or more mutations selected from R120E and R120E / K121E. The entire contents of these patents are incorporated herein by reference. In such embodiments, the IFNα2 variant antagonistizes wild-type IFNα activity 2. In such embodiments, the mutant IFNα2 has reduced affinity and / or activity for IFNAR1, but retains affinity and / or activity for IFNR2.

[0124] In some embodiments, the human IFNα2 variant comprises (1) one or more mutations selected from R120E and R120E / K121E (these produce antagonist effects, although we do not wish to be bound by theory), and (2) one or more mutations selected from K133A, R144A, R149A, and L153A (these enable, for example, attenuation effects on IFNAR2, although we do not wish to be bound by theory). In one embodiment, the human IFNα2 variant comprises R120E and L153A.

[0125] In some embodiments, the human IFNα2 variants include one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156A, as disclosed in International Publication No. 2013 / 059885, the entirety of which is incorporated herein by reference. In some embodiments, as disclosed in International Publication No. 2013 / 059885, the human IFNα2 variant includes mutations H57Y, E58N, Q61S, and / or L30A. In some embodiments, as disclosed in International Publication No. 2013 / 059885, the human IFNα2 variant includes mutations H57Y, E58N, Q61S, and / or R33A. In some embodiments, as disclosed in International Publication No. 2013 / 059885, the human IFNα2 variant includes mutations H57Y, E58N, Q61S, and / or M148A. In some embodiments, as disclosed in International Publication No. 2013 / 059885, the human IFNα2 variant includes mutations H57Y, E58N, Q61S, and / or L153A. In some embodiments, as disclosed in International Publication No. 2013 / 059885, the human IFNα2 variants include mutations N65A, L80A, Y85A, and / or Y89A. In some embodiments, as disclosed in International Publication No. 2013 / 059885, the human IFNα2 variants include mutations N65A, L80A, Y85A, Y89A, and / or D114A. In some embodiments, the human IFN-α2 variants include one or more mutations selected from R144X1, A145X2, and R33A, where X1 is selected from A, S, T, Y, L, and I, and X2 is selected from G, H, Y, K, and D. In some embodiments, the signaling molecule is optionally a modified IFNα2 having the R149A mutation with respect to the amino acid sequence of SEQ ID NO: 81 or 82.

[0126] In some embodiments, the human IFN-α2 variant contains a mutation at T106. In some embodiments, T106 is replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y.

[0127] In some embodiments, the modified signaling molecule is interferon-α1. In some embodiments, IFN-α1 comprises the amino acid sequence of SEQ ID NO: 83 or a variant thereof. In some embodiments, IFN-α1 is modified, i.e., a variant, and comprises one or more mutations. In some embodiments, one or more mutations reduce the biological activity of IFN-α1. For example, one or more mutations may reduce the affinity of IFN-α1 interferon to a therapeutic receptor. In some embodiments, the therapeutic receptor is an interferon-α / β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits. In some embodiments, modified IFN-α1 comprises one or more mutations that reduce its affinity for IFNAR1. In other embodiments, modified IFN-α1 comprises one or more mutations that reduce its affinity for IFNAR2. In some embodiments, modified IFN-α1 comprises one or more mutations that reduce its affinity for IFNAR1 and one or more mutations that reduce its affinity for IFNAR2. In some embodiments, the chimeric protein or Fc-based chimeric protein complex comprises one or more additional signaling molecules, which may be modified, such as, but are not limited to, interferon, interleukin, and tumor necrosis factor. In various embodiments, the chimeric protein or Fc-based chimeric protein complex of the present invention provides an improved safety and / or therapeutic activity and / or pharmacokinetic profile (e.g., an extended serum half-life) compared to untargeted IFN-α1 or unmodified wild-type IFN-α, such as IFN-α1.

[0128] In various embodiments, wild-type IFN-α1 includes the following amino acid sequence: CDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (Sequence ID 83).

[0129] In various embodiments, the chimeric protein or Fc-based chimeric protein complex of the present invention includes a modified IFN-α1, i.e., an IFN-α1 variant, including an IFN-α1 mutant, as a signaling molecule. In various embodiments, the IFN-α1 variant includes a variant, functional derivative, analog, precursor, isoform, splice variant, or fragment of interferon.

[0130] Additional IFN-α1 variant sequences are known in the art. In various embodiments, the modified IFN-α1 is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or less At least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity (for example, about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%). It contains an amino acid sequence having sequence identity of %, or approximately 74%, or approximately 75%, or approximately 76%, or approximately 77%, or approximately 78%, or approximately 79%, or approximately 80%, or approximately 81%, or approximately 82%, or approximately 83%, or approximately 84%, or approximately 85%, or approximately 86%, or approximately 87%, or approximately 88%, or approximately 89%, or approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99%.

[0131] In some embodiments, IFN-α1 interferon is modified to have mutations in one or more amino acids at positions L15, A19, R23, S25, L30, D32, R33, H34, Q40, C86, D115, L118, K121, R126, E133, K134, K135, R145, A146, M149, R150, S153, L154, and N157 with respect to SEQ ID NO: 83. The mutations may be optionally hydrophobic and may be selected from, for example, alanine, valine, leucine, and isoleucine. In some embodiments, IFN-α1 interferon is L15A, A19W, R23A, S25A, L30A, L30V, D32A, R33K, R33A, R33Q, H34A, Q40A, C86S, C86A, C86Y, D115R, L118A, K121A, K121E, R126A, R126E, E133A, K134A, K135A, R145A, R145D, R145E, R145G, R145H, R145I, R The molecule is modified to have one or more mutations selected from 145K, R145L, R145N, R145Q, R145S, R145T, R145V, R145Y, A146D, A146E, A146G, A146H, A146I, A146K, A146L, A146M, A146N, A146Q, A146R, A146S, A146T, A146V, A146Y, M149A, M149V, R150A, S153A, L154A, and N157A. In some embodiments, the IFN-α1 variant includes one or more mutations selected from L30A / H58Y / E59N_Q62S, R33A / H58Y / E59N / Q62S, M149A / H58Y / E59N / Q62S, L154A / H58Y / E59N / Q62S, R145A / H58Y / E59N / Q62S, D115A / R121A, L118A / R121A, L118A / R121A / K122A, R121A / K122A, and R121E / K122E with respect to SEQ ID NO: 83.

[0132] In certain embodiments, the IFN-α1 interferon is modified to have a mutation at amino acid position C86 with respect to SEQ ID NO: 83. The mutation at position C86 can be, for example, C86S or C86A. These C86 mutants of IFN-α1 are referred to as aggregation mutants by reducing cysteine.

[0133] In some embodiments, the modified signaling substance is interferon β. In such embodiments, the modified interferon β substance has a reduced affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified interferon β substance has a substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0134] In certain embodiments, the modified signaling substance is interferon β. In such embodiments, the modified interferon β substance has a reduced affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified IFNβ substance has a substantially reduced or eliminated affinity and / or activity for the IFNα / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0135] In one exemplary embodiment, the modified signaling molecule is IFN-β. In various embodiments, IFNβ encompasses functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFNβ. In various embodiments, IFNβ encompasses IFNβ from any species. In one embodiment, a chimeric protein or chimeric protein complex contains modified mouse IFN-β. In another embodiment, a chimeric protein or chimeric protein complex contains modified human IFN-β. Human IFNβ is a polypeptide with a molecular weight of approximately 22 kDa, containing 166 amino acid residues. The amino acid sequence of human IFNβ is SEQ ID NO: 84.

[0136] In some embodiments, human IFNβ is IFNβ1a, which is a glycosylated form of human IFNβ. In some embodiments, IFNβ is IFNβ1b, which is a non-glycosylated form of human IFNβ having a Met-1 deletion and a mutation to Ser at Cys-17.

[0137] In various embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity to the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFNβ has reduced affinity and / or activity to IFNAR1. In various embodiments, the modified IFNβ is human IFNβ having one or more mutations at positions F67, R71, L88, Y92, I95, N96, K123, and R124. In some embodiments, one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In one embodiment, the modified IFNβ includes the F67G mutation. In one embodiment, the modified IFNβ includes the K123G mutation. In one embodiment, the modified IFNβ includes the F67G and R71A mutations. In some embodiments, the modified IFNβ includes L88G and Y92G mutations. In some embodiments, the modified IFNβ includes Y92G, I95A, and N96G mutations. In some embodiments, the modified IFNβ includes K123G and R124G mutations. In some embodiments, the modified IFNβ includes F67G, L88G, and Y92G mutations. In some embodiments, the modified IFNβ includes F67S, L88S, and Y92S mutations.

[0138] In some embodiments, the modified IFNβ has one or more mutations that reduce its binding or affinity to the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFNβ has reduced affinity and / or activity to IFNAR2. In various embodiments, the modified IFNβ is human IFNβ having one or more mutations at positions W22, R27, L32, R35, V148, L151, R152, and Y155. In some embodiments, one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In some embodiments, the modified IFNβ includes the W22G mutation. In some embodiments, the modified IFNβ includes the L32A mutation. In some embodiments, the modified IFNβ includes the L32G mutation. In some embodiments, the modified IFNβ includes the R35A mutation. In some embodiments, the modified IFNβ includes the R35G mutation. In some embodiments, the modified IFNβ includes the V148G mutation. In some embodiments, the modified IFNβ includes the R152A mutation. In some embodiments, the modified IFNβ includes the R152G mutation. In some embodiments, the modified IFNβ includes the Y155G mutation. In some embodiments, the modified IFNβ includes the W22G and R27G mutations. In some embodiments, the modified IFNβ includes the L32A and R35A mutations. In some embodiments, the modified IFNβ includes the L151G and R152A mutations. In some embodiments, the modified IFNβ includes the V148G and R152A mutations.

[0139] In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H. In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with C17S or C17A.

[0140] In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H, in combination with any of the other IFN-β mutations described herein.

[0141] The crystal structure of human IFN-β is known and described in Karpusas et al., (1998) PNAS, 94(22):11813-11818. In particular, the structure of human IFNβ has been shown to include five α-helices (i.e., A, B, C, D, and E) and four loop regions (i.e., AB, BC, CD, and DE loops) linking these helices. In various embodiments, modified IFNβ has one or more mutations in the A, B, C, D, E helices and / or the AB, BC, CD, and DE loops that reduce its binding affinity or activity to therapeutic receptors such as IFNARs. Examples of mutations are described in WO2000 / 023114 and US2015 / 0011732, the full contents of which are incorporated herein by reference. In exemplary embodiments, the modified IFN-β is human IFN-β containing alanine substitutions at amino acid positions 15, 16, 18, 19, 22, and / or 23. In exemplary embodiments, the modified IFN-β is human IFN-β containing alanine substitutions at amino acid positions 28-30, 32, and 33. In exemplary embodiments, the modified IFN-β is human IFN-β containing alanine substitutions at amino acid positions 36, 37, 39, and 42. In exemplary embodiments, the modified IFN-β is human IFN-β containing alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In exemplary embodiments, the modified IFN-β is human IFN-β containing alanine substitutions at amino acid positions 71-73. In exemplary embodiments, the modified IFN-β is human IFN-β containing alanine substitutions at amino acid positions 92, 96, 99, and 100. In exemplary embodiments, the modified IFN-β is human IFN-β containing alanine substitutions at amino acid positions 128, 130, 131, and 134. In exemplary embodiments, the modified IFN-β is human IFN-β containing alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and includes a mutation in W22, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0142] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and includes a mutation at R27, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0143] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at W22, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further includes a mutation at R27, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0144] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and includes a mutation at L32, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0145] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and includes a mutation at R35, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0146] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at L32, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further includes a mutation at R35, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0147] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and includes a mutation at F67, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0148] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and includes a mutation in R71, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0149] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at F67, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further includes a mutation at R71, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0150] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and contains a mutation at L88, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0151] In some embodiments, the mutant IFNβ includes sequence number 84 and includes a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0152] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, with a mutation at F67, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); and a mutation at L88, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0153] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at L88, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further includes a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0154] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at I95, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and further includes a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0155] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at N96, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further includes a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0156] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), includes a mutation at I95, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and includes a mutation at N96, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0157] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and includes a mutation at K123, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0158] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and contains a mutation at R124, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0159] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at K123, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further includes a mutation at R124, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0160] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and contains a mutation at L151, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0161] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and contains a mutation at R152, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0162] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, and includes a mutation at L151, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and further includes a mutation at R152, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0163] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and contains a mutation in V148, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0164] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84, includes a mutation at V148, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and further includes a mutation at R152, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0165] In some embodiments, the mutant IFNβ includes SEQ ID NO: 84 and includes a mutation at Y155, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0166] In some embodiments, the present invention relates to a chimeric protein or chimeric protein complex, which comprises (a) a modified IFN-β having the amino acid sequence of SEQ ID NO: 84 and having a mutation at position W22, wherein the mutation is an aliphatic hydrophobic residue, and (b) one or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest (e.g., Clec9A), wherein the modified IFN-β and one or more targeting moieties are optionally linked by one or more linkers. In various embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M, and V. In various embodiments, the mutation at position W22 is G.

[0167] Examples of additional IFNβ variants are provided in PCT / EP2017 / 061544, the entire disclosure of which is incorporated herein by reference.

[0168] In some embodiments, the modified signaling molecule is interferon-γ. In such embodiments, the modified interferon-γ substance has reduced affinity and / or activity to the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon-γ substance has substantially reduced or eliminated affinity and / or activity to the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and / or IFNGR2 chains.

[0169] IFN-γ is the only member of the type II class of interferons. IFN-γ is produced primarily by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response. IFN-γ is also produced by CD4 Th1 and CD8 cytotoxic T lymphocytes (CTLs), effector T cells, macrophages, dendritic cells, and B cells. Activated IFN-γ forms a dimer that acts via a heterodimer receptor (i.e., IFN-γ receptor or IFN-γR) composed of IFN-γ receptor 1 and IFN-γ receptor 2 subunits. IFN-γ receptor 1 is the primary ligand-binding subunit, while IFN-γ receptor 2 is required for signaling and enhances the affinity of IFN-γ receptor 1 for its ligand. Binding of the IFN-γ dimer to the receptor activates the JAK-STAT signaling pathway, inducing various biological effects.

[0170] In various embodiments, the modified signaling molecule comprises a modified IFN-γ as the signaling molecule. In various embodiments, IFN-γ comprises a functional derivative, analog, precursor, isoform, splice variant, or fragment of IFN-γ. In various embodiments, IFN-γ comprises IFN-γ from any species. In one embodiment, the modified signaling molecule comprises a modified mouse IFN-γ. In another embodiment, the modified signaling molecule comprises a modified human IFN-γ.

[0171] Human IFN-γ is a polypeptide containing 166 amino acid residues. In one embodiment, human IFN-γ has the amino acid sequence of SEQ ID NO: 85, and the signal peptide contains the first 23 amino acids.

[0172] [ka]

[0173] As used herein, human IFN-γ may also refer to mature human IFN-γ that does not have an N-terminal signal peptide. In this embodiment, mature human IFN-γ comprises 143 amino acids and has the following amino acid sequence: QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ (Sequence ID 86).

[0174] In some embodiments, human IFN-γ is glycosylated human IFN-γ. In some embodiments, human IFN-γ is unglycosylated human IFN-γ.

[0175] The sequence of IFN-γ is known in the art. In various embodiments, the modified IFN-γ is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91% of the known wild-type amino acid sequence of IFN-γ. , or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or It contains an amino acid sequence having approximately 74%, or approximately 75%, or approximately 76%, or approximately 77%, or approximately 78%, or approximately 79%, or approximately 80%, or approximately 81%, or approximately 82%, or approximately 83%, or approximately 84%, or approximately 85%, or approximately 86%, or approximately 87%, or approximately 88%, or approximately 89%, or approximately 90%, or approximately 91%, or approximately 92%, or approximately 93%, or approximately 94%, or approximately 95%, or approximately 96%, or approximately 97%, or approximately 98%, or approximately 99% sequence identity.

[0176] In some embodiments, the modified IFN-γ is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least Sequence identity of approximately 91%, or at least approximately 92%, or at least approximately 93%, or at least approximately 94%, or at least approximately 95%, or at least approximately 96%, or at least approximately 97%, or at least approximately 98%, or at least approximately 99% (e.g., approximately 60%, or approximately 61%, or approximately 62%, or approximately 63%, or approximately 64%, or approximately 65%, or approximately 66%, or approximately 67%, or approximately 68%, or approximately 69%, or approximately 70%, or approximately 71%, or approximately 72%, or approximately 73%). Or contains an amino acid sequence having sequence identity of approximately 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0177] In some embodiments, the modified IFN-γ is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least Sequence identity of approximately 91%, or at least approximately 92%, or at least approximately 93%, or at least approximately 94%, or at least approximately 95%, or at least approximately 96%, or at least approximately 97%, or at least approximately 98%, or at least approximately 99% (e.g., approximately 60%, or approximately 61%, or approximately 62%, or approximately 63%, or approximately 64%, or approximately 65%, or approximately 66%, or approximately 67%, or approximately 68%, or approximately 69%, or approximately 70%, or approximately 71%, or approximately 72%, or approximately 73%). Or contains an amino acid sequence having sequence identity of approximately 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0178] In various embodiments, the modified IFN-γ comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0179] In some embodiments, amino acid mutations are amino acid substitutions, which may include conservative and / or non-conservative substitutions.

[0180] "Conservative substitutions" can be made, for example, based on the similarity of the amino acid residues involved in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties. The 20 native amino acids can be classified into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0181] As used herein, “conservative substitution” is defined as the exchange of one amino acid with another amino acid listed within the same group of the six standard amino acid groups described above. For example, the exchange of Asp with Glu retains one negative charge in the thus modified polypeptide. Furthermore, glycine and proline may be substituted for each other based on their ability to disrupt their α-helices.

[0182] As used herein, “non-conservative substitution” is defined as the replacement of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) described above.

[0183] In various embodiments, substitutions may also include non-classical amino acids (e.g., designer amino acids such as selenocysteine, pyrrolelysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methylamino acids, C α-methylamino acids, N α-methylamino acids, and common amino acid analogs).

[0184] In various embodiments, IFN-γ is modified to have one or more mutations. In some embodiments, the mutations allow the modified IFN-γ to have one or more attenuated activities compared to the non-mutant form, e.g., wild-type IFN-γ, such as reduced binding affinity, reduced endogenous activity, and reduced specific biological activity. For example, one or more of the attenuated activities compared to the non-mutant form, e.g., wild-type IFN-γ, such as reduced binding affinity, reduced endogenous activity, and reduced specific biological activity may be directed towards therapeutic receptors, such as the IFN-γ receptor. As a result, in various embodiments, the mutations allow the modified soluble substance to have reduced systemic toxicity, reduced side effects, and reduced off-target effects compared to the non-mutant form, e.g., wild-type IFN-γ.

[0185] In various embodiments, IFN-γ is modified to have mutations that reduce its binding affinity and / or activity to therapeutic receptors, such as IFN-γ receptors containing IFN-γ receptor 1 and IFN-γ receptor 2 subunits. In some embodiments, the activity conferred by wild-type IFN-γ is agonism to the therapeutic receptor (e.g., activation of cellular effects at the site of treatment). For example, wild-type IFN-γ can activate the therapeutic receptor. In such embodiments, the mutation results in a modified IFN-γ with reduced activating activity towards the therapeutic receptor.

[0186] In some embodiments, reduced affinity and / or activity to a therapeutic receptor (e.g., IFN-γ receptor) is recoverable by binding to a targeting moiety. In other embodiments, reduced affinity and / or activity to a therapeutic receptor is not substantially recoverable by binding to a targeting moiety. In various embodiments, the therapeutic chimeric protein or chimeric protein complex of the present invention reduces off-target effects because IFN-γ has mutations that weaken its binding affinity and / or activity to the therapeutic receptor. In various embodiments, this reduces side effects observed with, for example, wild-type IFN-γ. In various embodiments, the modified IFN-γ is substantially inactive on its way to the site of therapeutic action and substantially exerts its effect on the specifically targeted cell type, which greatly reduces undesirable side effects.

[0187] In various embodiments, the modified IFN-γ has one or more mutations that cause IFN-γ to have weakened or reduced affinity and / or activity, e.g., binding (e.g., KD) and / or activation (e.g., measurable as KA and / or EC50) to one or more therapeutic receptors (e.g., IFN-γ receptors). In various embodiments, the reduced affinity and / or activity to therapeutic receptors allows for attenuation of activity and / or signaling from the therapeutic receptors.

[0188] In various embodiments, modified IFN-γ has one or more mutations that reduce its binding or affinity and / or biological activity to the IFN-γ receptor 1 subunit. In one embodiment, modified IFN-γ has reduced affinity and / or activity to the IFN-γ receptor 1 subunit. In various embodiments, modified IFN-γ is human IFN-γ having one or more mutations in amino acid residues involved in binding to the IFN-γ receptor 1 subunit. In some embodiments, modified IFN-γ is human IFN-γ having one or more mutations in amino acids located at the interface with the IFN-γ receptor 1 subunit. In various embodiments, one or more mutations are selected from, but are not limited to, the amino acids Q1, V5, E9, K12, H19, S20, V22, A23, D24, N25, G26, T27, L30, K108, H111, E112, I114, Q115, A118, E119, and K125 (each being wild-type human IFN-γ lacking its N-terminal signal sequence, with respect to SEQ ID NO: 86). In some embodiments, one or more mutations are substitutions selected from V5E, S20E, V22A, A23G, A23F, D24G, G26Q, H111A, H111D, I114A, Q115A, and A118G (each being SEQ ID NO: 86). In the embodiment, one or more mutations are substitutions selected from V22A, A23G, D24G, H111A, H111D, I114A, Q115A, and A118G.

[0189] In one embodiment, the modified IFN-γ includes mutations A23G and D24G. In another embodiment, the modified IFN-γ includes mutations I114A and A118G. In yet another embodiment, the modified IFN-γ includes mutations V5E, S20E, A23F, and G26Q.

[0190] In various embodiments, the modified IFN-γ has one or more of the following mutations: deletion of residue A23, deletion of residue D24, S20I substitution, A23V substitution, D21K substitution, and D24A substitution.

[0191] In some embodiments, the modified IFN-γ has one or more mutations that reduce its binding or affinity to the IFN-γ receptor 2 subunit and / or its biological activity.

[0192] In some embodiments, the modified IFN-γ has one or more mutations that reduce its binding or affinity and / or biological activity to both IFN-γ receptor 1 and IFN-γ receptor 2 subunits.

[0193] In some embodiments, the modified IFN-γ has one or more mutations that reduce its binding to or affinity for and / or biological activity to IFN-γ receptor 1, and one or more mutations that substantially reduce or eliminate its binding to or affinity for and / or biological activity to IFN-γ receptor 2. In some embodiments, a chimeric protein or chimeric protein complex having such a modified IFN-γ can provide target-selective IFN-γ receptor 1 activity (e.g., IFN-γ receptor 1 activity is recoverable via targeting through a targeting moiety).

[0194] In some embodiments, the modified IFN-γ has one or more mutations that reduce its binding or affinity and / or biological activity to IFN-γ receptor 1, and one or more mutations that reduce its binding or affinity and / or biological activity to IFN-γ receptor 1. In some embodiments, a chimeric protein or chimeric protein complex having such a modified IFN-γ can provide target-selective IFN-γ receptor 1 and / or IFN-γ receptor 1 activity (e.g., IFN-γ receptor 1 and IFN-γ receptor 2 activity are recoverable via targeting through the targeting moiety).

[0195] In various embodiments, the modified IFN-γ is truncated at the C-terminus. In some embodiments, the modified IFN-γ is a mature IFN-γ containing the amino acid sequence of SEQ ID NO: 86 having a C-terminal deletion. In such embodiments, the mature IFN-γ may contain C-terminal truncation of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acid residues. In one embodiment, the modified IFN-γ is a mature IFN-γ containing the amino acid sequence of SEQ ID NO: 86 having a C-terminal deletion of 5 amino acids. In another embodiment, the modified IFN-γ is a mature IFN-γ containing the amino acid sequence of SEQ ID NO: 86 having a C-terminal deletion of 7 amino acids. In one embodiment, the modified IFN-γ is a mature IFN-γ containing the amino acid sequence of SEQ ID NO: 86 having a C-terminal deletion of 14 amino acids. In another embodiment, the modified IFN-γ is a mature IFN-γ containing the amino acid sequence of SEQ ID NO: 86 having a C-terminal deletion of 15 amino acids. In yet another embodiment, the modified IFN-γ is a mature IFN-γ containing the amino acid sequence of SEQ ID NO: 86 having a C-terminal deletion of 16 amino acids. Additional modified IFN-γs having C-terminal truncation that can be used in the present invention are described in Haelewyn et al., Biochem. J. (1997), 324:591-595 and Lundell et al., Protein Eng. (1991) 4:335-341, the full contents of which are incorporated herein by reference.

[0196] In various embodiments, the modified IFN-γ is a single-chain IFN-γ as described, for example, in Randal et al. (2001) Structure 9:155-163 and Randal et al. (1998) Protein Sci. 7:1057-1060, the full contents of which are incorporated herein by reference. In some embodiments, the single-chain IFN-γ comprises a first IFN-γ chain linked at its C-terminus to the N-terminus of a second IFN-γ chain. In various embodiments, the first and second IFN-γ chains are linked by a linker, as described elsewhere herein.

[0197] In some embodiments, the first IFN-γ chain includes a C-terminal truncation of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acid residues. In some embodiments, the first IFN-γ chain includes a C-terminal truncation of about 24 amino acid residues. In some embodiments, the second IFN-γ chain includes an N-terminal truncation of at least about 1, about 2, about 3, about 4, or about 5 amino acid residues. In some embodiments, the second IFN-γ chain includes an N-terminal truncation of about 3 amino acid residues. In some embodiments, the second IFN-γ chain includes a C-terminal truncation of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acid residues. In various embodiments, the first and / or second IFN-γ chain includes one or more amino acid mutations in Q1, V5, E9, K12, H19, S20, V22, A23, D24, N25, G26, T27, L30, K108, H111, E112, I114, Q115, A118, E119, and K125, as described elsewhere in this specification. In various embodiments, the first and / or second IFN-γ chain includes one or more substitutions selected from V5E, S20E, V22A, A23G, A23F, D24G, G26Q, H111A, H111D, I114A, Q115A, and A118G. In various embodiments, the first and / or second IFN-γ chain includes one or more substitutions selected from V22A, A23G, D24G, H111A, H111D, I114A, Q115A, and A118G. In various embodiments, the first and / or second IFN-γ chain includes A23G and D24G substitutions. In various embodiments, the first and / or second IFN-γ chain includes I114A and A118G substitutions. In another embodiment, the mutations are V5E, S20E, A23F, and G26Q.

[0198] In various embodiments, the first and / or second IFN-γ chain comprises one or more substitutions disclosed herein, and the first and / or second IFN-γ chain comprises a C-terminal truncation disclosed herein.

[0199] In various embodiments, the first and / or second IFN-γ chains include one or more substitutions and C-terminal truncation as disclosed herein.

[0200] The crystal structure of human IFN-γ is known and is described, for example, Ealick et al., (1991) Science, 252:698-702. In particular, the structure of human IFN-γ has been shown to contain a core of six α-helices and an extended unfolded sequence in the C-terminal region. In various embodiments, modified IFN-γ has one or more mutations in one or more helices that reduce its binding affinity and / or bioactivity to a therapeutic receptor (e.g., the IFN-γ receptor).

[0201] In various embodiments, modified IFN-γ has affinity and / or bioactivity to a therapeutic receptor (e.g., the IFN-γ receptor or one of its IFN-γ receptor 1 and IFN-γ receptor 2 subunits) of about 1%, or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 10% to 20%, about 20% to 40%, about 50%, about 40% to 60%, about 60% to 80%, or about 80% to 100% compared to wild-type IFN-γ. In some embodiments, the binding affinity and / or bioactivity is at least about 2 times lower, about 3 times lower, about 4 times lower, about 5 times lower, about 6 times lower, about 7 times lower, about 8 times lower, about 9 times lower, at least about 10 times lower, at least about 15 times lower, at least about 20 times lower, at least about 25 times lower, at least about 30 times lower, at least about 35 times lower, at least about 40 times lower, at least about 45 times lower, at least about 50 times lower, at least about 100 times lower, at least about 150 times lower, or about 10 to 50 times lower, about 50 to 100 times lower, about 100 to 150 times lower, about 150 to 200 times lower, or more than 200 times lower than wild-type IFN-γ.

[0202] In various embodiments, the modified IFN-γ includes, for example, one or more mutations that reduce the endogenous activity of IFN-γ to about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, compared to wild-type IFN-γ.

[0203] In some embodiments, the modified IFN-γ comprises one or more mutations that cause the modified IFN-γ to have reduced affinity and / or bioactivity to the receptor. In some embodiments, the binding affinity and / or bioactivity of the modified IFN-γ to the receptor is lower than the binding affinity and / or bioactivity of the targeting moiety to that receptor. In some embodiments, this difference in binding affinity and / or bioactivity lies between the modified IFN-γ / receptor and the targeting moiety / receptor on the same cell. In some embodiments, this difference in binding affinity and / or bioactivity allows the modified IFN-γ to have a localized on-target effect and minimize the off-target effects underlying the side effects observed with wild-type IFN-γ. In some embodiments, this binding affinity and / or bioactivity is at least about 2 times, or at least about 5 times, or at least about 10 times, or at least about 15 times lower, or at least about 25 times, or at least about 50 times lower, or at least about 100 times, or at least about 150 times lower.

[0204] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or binding activity can be evaluated by scatchard plot analysis and computer fitting of binding data (e.g., Scatchard, 1949), or by reflection interference spectroscopy under flow-through conditions, as described by Brecht et al. (1993) (all of these are incorporated herein by reference).

[0205] In some embodiments, the modified signaling molecule is a consensus interferon. The consensus interferon is generated by scanning the sequences of several human non-allelic IFNα subtypes and assigning the most frequently observed amino acids at the corresponding positions. The consensus interferon differs from IFNα2b in 20 of its 166 amino acids (88% homology), and in comparison with IFNβ, it shows identity at more than 30% of the amino acid positions. In various embodiments, the consensus interferon includes the amino acid sequence of SEQ ID NO: 87.

[0206] In some embodiments, the consensus interferon contains the amino acid sequence of SEQ ID NO: 88, which differs from the amino acid sequence of SEQ ID NO: 87 by only one amino acid, i.e., SEQ ID NO: 88 lacks the first methionine residue of SEQ ID NO: 87.

[0207] In various embodiments, the consensus interferon includes a modified consensus interferon, i.e., a consensus interferon variant, as a signaling molecule. In various embodiments, the consensus interferon variant includes a functional derivative, analog, precursor, isoform, splice variant, or fragment of the consensus interferon.

[0208] In some embodiments, the consensus interferon variant is selected from the consensus interferon variants disclosed in U.S. Patents 4,695,623, 4,897,471, 5,541,293, and 8,496,921. The entire contents of these documents are incorporated herein by reference. For example, the consensus interferon variant may include an amino acid sequence of IFN-CON2 or IFN-CON3, as disclosed in U.S. Patents 4,695,623, 4,897,471, and 5,541,293. In some embodiments, the consensus interferon variant includes the amino acid sequence of IFN-CON2 (SEQ ID NO: 89).

[0209] In one embodiment, the consensus interferon variant includes the amino acid sequence of IFN-CON3 (SEQ ID NO: 90).

[0210] In one embodiment, the consensus interferon variant comprises one amino acid sequence of any of the variants disclosed in U.S. Patent No. 8,496,921. For example, the consensus variant may comprise the amino acid sequence of SEQ ID NO: 91.

[0211] In another embodiment, the consensus interferon variant may include the amino acid sequence of SEQ ID NO: 92.

[0212] In some embodiments, the consensus interferon variant may be pegylated, i.e., include a PEG portion. In some embodiments, the consensus interferon variant may include a PEG portion ligated at position S156C of SEQ ID NO: 92.

[0213] In some embodiments, the modified interferon is a variant of human IFNα2a, and the insertion of Asp near position 41 of the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQ ID NO: 93) yields Glu-Glu-Phe-Asp-Gly-Asn-Gln (SEQ ID NO: 94) (this results in a sequence renumbering for the IFNα2a sequence), and has the following mutations: Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, and Lys136Thr. All embodiments of this specification describing the consensus interferon also apply to this genetically modified interferon.

[0214] In various embodiments, the consensus interferon comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations.

[0215] In some embodiments, amino acid mutations are amino acid substitutions, which may include conservative and / or non-conservative substitutions.

[0216] In various embodiments, substitutions may also include non-classical amino acids (e.g., designer amino acids such as selenocysteine, pyrrolelysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, β-methylamino acids, C-α-methylamino acids, N-α-methylamino acids, and generally amino acid analogs).

[0217] In various embodiments, consensus interferon is modified to have one or more mutations. In some embodiments, the mutations allow the consensus interferon variant to have one or more attenuated activities compared to the non-mutant, e.g., wild-type form of consensus interferon (e.g., consensus interferon having the amino acid sequence of SEQ ID NO: 87 or 88), such as reduced binding affinity, reduced endogenous activity, and reduced specific biological activity. For example, one or more of the attenuated activities compared to the non-mutant, e.g., wild-type form of consensus interferon, such as reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, may be to therapeutic receptors such as IFNARs. As a result, in various embodiments, the mutations allow the consensus interferon variant to have reduced systemic toxicity, reduced side effects, and reduced off-target effects compared to the non-mutant, e.g., wild-type form of consensus interferon.

[0218] In various embodiments, consensus interferons are modified to have mutations that reduce their binding affinity or activity to therapeutic receptors such as IFNARs. In some embodiments, the activity provided by consensus interferons is agonism to therapeutic receptors (e.g., activation of cellular effects at the site of treatment). For example, consensus interferons can activate therapeutic receptors. In such embodiments, the mutations result in consensus interferon variants that have reduced activating activity to therapeutic receptors.

[0219] In some embodiments, reduced affinity or activity to the therapeutic receptor is recoverable by binding to a targeting moiety (e.g., PD-L1). In other embodiments, reduced affinity or activity to the therapeutic receptor is not substantially recoverable by binding to a targeting moiety. In various embodiments, the therapeutic chimeric protein or chimeric protein complex of the present invention reduces off-target effects because the consensus interferon variant has mutations that weaken its binding affinity or activity to the therapeutic receptor. In various embodiments, this reduces side effects observed with, for example, wild-type consensus interferon. In various embodiments, the consensus interferon variant is substantially inactive on its way to the site of therapeutic action and substantially exerts its effect on the specifically targeted cell type, which greatly reduces undesirable side effects.

[0220] In various embodiments, the consensus interferon variant is characterized by having a weakened or reduced affinity for one or more therapeutic receptors, for example, binding (e.g., K D ) and / or activation (e.g., K A and / or EC 50 It has one or more mutations that cause it to have (measurable as) a therapeutic receptor. In various embodiments, the reduced affinity to the therapeutic receptor allows for attenuation of activity and / or signaling from the therapeutic receptor.

[0221] In various embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to the IFNAR1 subunit of IFNAR. In one embodiment, the consensus interferon variant has reduced affinity and / or activity to IFNAR1. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to the IFNAR2 subunit of IFNAR. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to both the IFNAR1 and IFNAR2 subunits.

[0222] In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding to or affinity for IFNAR1, and one or more mutations that substantially reduce or eliminate its binding to or affinity for IFNAR2. In some embodiments, a chimeric protein or chimeric protein complex having such a consensus interferon variant can provide target-selective IFNAR1 activity (e.g., IFNAR1 activity is recoverable via targeting by a targeting moiety, e.g., PD-L1).

[0223] In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding to or affinity for IFNAR2, and one or more mutations that substantially reduce or eliminate its binding to or affinity for IFNAR1. In some embodiments, a chimeric protein or chimeric protein complex having such a consensus interferon variant can provide target-selective IFNAR2 activity (e.g., IFNAR2 activity is recoverable via targeting by a targeting moiety, e.g., PD-L1).

[0224] In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding to or affinity for IFNAR1, and one or more mutations that reduce its binding to or affinity for IFNAR2. In some embodiments, a chimeric protein or chimeric protein complex having such a consensus interferon variant can provide target-selective IFNAR1 and / or IFNAR2 activity (for example, IFNAR1 and / or IFNAR2 activity is recoverable via targeting by a targeting moiety, e.g., PD-L1).

[0225] In some embodiments, the consensus interferon is modified to have a mutation in one or more amino acids at positions 145-155, e.g., amino acid positions 149, 150, and / or 154, with respect to SEQ ID NO: 88. The substitution is optionally hydrophobic and selected from alanine, valine, leucine, and isoleucine. In some embodiments, the consensus interferon variant is selected from M149A, R150A, and L154A and contains one or more mutations with respect to SEQ ID NO: 88.

[0226] In one embodiment, the consensus interferon is modified to have a mutation at amino acid position 121 (i.e., K121) with respect to SEQ ID NO: 88. In another embodiment, the consensus interferon contains the K121E mutation with respect to SEQ ID NO: 88.

[0227] In various embodiments, the modified signaling molecules are selected from modified forms of cytokines, growth factors, and hormones. Examples of such cytokines, growth factors, and hormones include conventional polypeptide hormones such as lymphokines, monokines, human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and tumor necrosis factor-β; Müllerian duct inhibitors; mouse gonadotropin-related peptides; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-α; platelet growth factor; and transforming growth factors such as TGF-α and TGF-β. Factors (TGF); insulin-like growth factors-I and II; bone induction factors; interferons such as interferon-α, interferon-β, and interferon-γ (and interferon types I, II, and III); colony-stimulating factors (CSF), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, and IL-18; tumor necrosis factors such as TNF-α or TNF-β; and other polypeptide factors, such as LIF and kit ligand (KL). As used herein, cytokines, growth factors, and hormones include proteins of natural origin or proteins produced from recombinant bacteria, eukaryotes, or mammalian cell culture systems, and biologically active equivalents of naturally occurring sequence cytokines.

[0228] In some embodiments, the modified signaling molecules are modified versions of growth factors selected from, but are not limited to, transforming growth factors (TGF) such as TGF-α and TGF-β (and its subtypes including various subtypes of TGFβ, including TGFβ1, TGFβ2, and TGFβ3), epidermal growth factor (EGF), insulin-like growth factors such as insulin-like growth factor-I and II, fibroblast growth factor (FGF), heregulin, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF).

[0229] In some embodiments, the growth factor is a modified fibroblast growth factor (FGF). Examples of FGFs include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, mouse FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0230] In some embodiments, the modified signaling molecule is vascular endothelial growth factor (VEGF). VEGF is a potent growth factor that plays a crucial role in both physiological and pathological angiogenesis, regulating vascular permeability and acting as a growth factor on cells expressing VEGF receptors. Further functions include, in particular, stimulation of cell migration of macrophage lineages and endothelial cells. In addition to at least three receptors (VEGFR1, VEGFR2, and VEGFR3), several members of the VEGF growth factor family exist. Members of the VEGF family can bind to and activate two or more VEGFR types. For example, VEGF-A can bind to VEGFR1 and VEGFR2, while VEGF-C can bind to VEGFR2 and VEGFR3. Activation of VEGFR-1 and VEGFR-2 regulates angiogenesis, while VEGFR-3 activation is involved in lymphangiogenesis. The majority of pro-angiogenic signals are generated from the activation of VEGFR2. VEGFR1 activation has been reported to be potentially associated with a negative role in angiogenesis. VEGFR1 signaling has also been reported to be important for in vivo tumor progression via bone marrow-derived VEGFR1-positive cells (contributing to the formation of the pre-metastatic microenvironment in bone). Several VEGF-A-based therapies directed at or neutralizing therapeutic antibodies have been developed, primarily for use in the treatment of various human tumors that depend on angiogenesis. However, these are not without side effects. This is not surprising considering that they act as general non-cell / tissue-specific VEGF / VEGFR interaction inhibitors. Therefore, it would be desirable to limit VEGF (e.g., VEGF-A) / VEGFR2 inhibition against specific target cells (e.g., tumor vascular endothelial cells).

[0231] In some embodiments, VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E and VEGF 121 , VEGF 121 b, VEGF 145 , VEGF 165 , VEGF 165 b, VEGF 189, and VEGF 206 These isoforms include various VEGF-A isoforms such as [list of isoforms]. In some embodiments, the modified signaling molecule has reduced affinity and / or activity to VEGFR-1(Flt-1) and / or VEGFR-2(KDR / Flk-1). In some embodiments, the modified signaling molecule has reduced or removed affinity and / or activity to VEGFR-1(Flt-1) and / or VEGFR-2(KDR / Flk-1). In some embodiments, the modified signaling molecule has reduced affinity and / or activity to VEGFR-2(KDR / Flk-1) and / or reduced or removed affinity and / or activity to VEGFR-1(Flt-1). Such embodiments are used, for example, in wound healing methods or in the treatment of ischemia-related diseases (not intended to be theoretically bound, but mediated by the effects of VEGFR-2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1(Flt-1), which is associated with oncological and pro-inflammatory activity, is avoided. In various embodiments, VEGFR-1 (Flt-1) functions as a decoy receptor, thereby substantially reducing or eliminating affinity for this receptor and avoiding the isolation of therapeutic agents. In some embodiments, the modified signaling molecule has reduced or eliminated affinity and / or activity for VEGFR-1 (Flt-1) and / or reduced or eliminated affinity and / or activity for VEGFR-2 (KDR / Flk-1). In some embodiments, VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signaling molecule has reduced affinity and / or activity for VEGFR-3. Alternatively, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for VEGFR3.

[0232] Pro-angiogenic therapies are also important in various diseases (e.g., ischemic heart disease, bleeding, etc.) and include VEGF-based therapies. Activation of VEGFR2 is pro-angiogenic (acting on endothelial cells). VEGFR1 can stimulate the migration of inflammatory cells (e.g., macrophages) and lead to inflammation associated with vascular hyperpermeability. Activation of VEGFR1 can also activate bone marrow, which is involved in tumor microenvironment formation. Therefore, VEGF-based therapies that are selective for VEGFR2 activation would be desirable in this case. Furthermore, cells that specifically target endothelial cells, for example, would be desirable.

[0233] In some embodiments, the modified signaling molecule has reduced affinity and / or activity (e.g., antagonist activity) for VEGFR-2 and / or substantially reduced or removed affinity and / or activity for VEGFR-1. When targeting tumor vascular endothelial cells via a targeting moiety that binds to tumor endothelial cell markers (e.g., PSMA), such constructs specifically inhibit VEGFR2 activation on such marker-positive cells, but do not activate VEGFR1 (if activity is removed) on the way to and on the target cells, thus eliminating, for example, the induction of an inflammatory response. This would provide a more selective and safer anti-angiogenic therapy for many tumor types compared to VEGF-A neutralization therapy.

[0234] In some embodiments, the modified signaling molecule has reduced affinity and / or activity (e.g., agonist activity) for VEGFR-2 and / or substantially reduced or eliminated affinity and / or activity for VEGFR-1. By targeting vascular endothelial cells, in some embodiments, such constructs promote angiogenesis without inducing inflammatory responses associated with VEGFR1. Thus, such constructs would have a targeted pro-angiogenic effect with a substantially reduced risk of side effects resulting from systemic activation of VEGFR2 and VEGFR1.

[0235] In one exemplary embodiment, the modified signaling molecule is a VEGF having the amino acid of SEQ ID NO: 95. 165 That is the case.

[0236] In another exemplary embodiment, the modified signaling molecule is a VEGF having the amino acid sequence of SEQ ID NO: 96. 165b That is the case.

[0237] In these embodiments, the modified signaling molecules have mutations at amino acid I83 (e.g., substitutional mutations at I83, e.g., I83K, I83R, or I83H). While not intended to be theoretically binding, such mutations are thought to result in reduced receptor binding affinity. See, for example, U.S. Patent No. 9,078,860. This entire content is incorporated herein by reference.

[0238] In some embodiments, the modified signaling molecules include, but are not limited to, human chorionic gonadotropins, gonadotropin-releasing hormone, androgens, estrogens, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growth hormone-releasing hormone, adrenocorticotropin-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, and sarcotropin These are modified hormones selected from a list of cerebrospinal hormones, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline, progesterone, insulin, glucagon, amylin, calcitriol, calciferol, atrial natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF), leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen.

[0239] In some embodiments, the modified signaling molecule is TNF-α. TNF is a pleiotropic cytokine with many diverse functions, including the regulation of cell proliferation, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and transport, inflammation, and septic shock. It binds to two distinct membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits a very broad expression pattern, while TNFR2 is selectively expressed on specific populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. In response to receptor activation, entirely separate biological pathways are activated, although some overlap exists. As a general principle, and without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), and TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFκB pathway). Administration of TNF is systemic toxic, which is primarily due to the involvement of TNFR1. However, it should be noted that TNFR2 activation, like TNFR1 activation, is associated with a variety of effects, and that TNF targeting and control of TNFR2 activity are important in the development of TNF-based therapeutics.

[0240] In some embodiments, the modified signaling molecule has reduced affinity and / or activity to TNFR1 and / or TNFR2. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to TNFR1 and / or TNFR2. TNFR1 is expressed in most tissues and is involved in cell death signaling, while TNFR2, in contrast, is involved in cell survival signaling. Therefore, in embodiments relating to cancer treatment, the modified signaling molecule has reduced affinity and / or activity to TNFR1 and / or substantially reduced or eliminated affinity and / or activity to TNFR2. In these embodiments, the chimeric protein or chimeric protein complex may target cells in which apoptosis is desirable, such as tumor cells or tumor vascular endothelial cells. For example, in embodiments relating to methods for promoting cell survival in neurogenesis for the treatment of neurodegenerative disorders, the modified signaling molecule has reduced affinity and / or activity to TNFR2 and / or substantially reduced or eliminated affinity and / or activity to TNFR1. In other words, the chimeric protein or chimeric protein complex of the present invention, in some embodiments, comprises a modified TNF-α substance that can prioritize either a death signal or a survival signal.

[0241] In some embodiments, the chimeric protein or chimeric protein complex has modified TNF having reduced affinity and / or activity to TNFR1 and / or substantially reduced or eliminated affinity and / or activity to TNFR2. Such chimeras are, in some embodiments, more potent apoptosis inducers than chimeras having only mutations that result in reduced affinity and / or activity to wild-type TNF and / or TNFR1. Such chimeras are used in some embodiments to induce tumor cell death or tumor vascular endothelial cell death (e.g., in cancer treatment). Also, in some embodiments, these chimeras are used, for example, via TNFR2 to induce T regThis avoids or reduces cell activation, thus further supporting TNFR1-mediated antitumor activity in vivo.

[0242] In some embodiments, the chimeric protein or chimeric protein complex has modified TNF having reduced affinity and / or activity for TNFR2 and / or substantially reduced or removed affinity and / or activity for TNFR1. Such chimeras are, in some embodiments, more potent activators of cell survival in certain cell types, which may be for specific therapeutic purposes in various diseases, including, but not limited to, stimulation of neurogenesis. Furthermore, such TNFR2-selective chimeras are also useful in the treatment of autoimmune diseases (e.g., Crohn's disease, diabetes, MS, colitis, etc., and many other diseases described herein). In some embodiments, the chimera targets autoreactive T cells. In some embodiments, the chimera targets T reg It promotes cell activation and indirect suppression of cytotoxic T cells.

[0243] In some embodiments, chimeric proteins induce the death of autoreactive T cells, for example, by activating TNFR2 and / or evading TNFR1 (e.g., by modified TNF having reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1). While we do not wish to be bound by theory, these autoreactive T cells have altered apoptosis / survival signals, for example, due to changes in NFκB pathway activity / signaling. In some embodiments, chimeric proteins induce the death of autoreactive T cells with lesions or alterations in the NFκB pathway, which underlie an imbalance in their cell death (apoptosis) / survival signaling properties and, optionally, altered sensitivity to certain death-inducing signals (e.g., TNFR2 activation).

[0244] In some embodiments, TNFR-2-based chimeras have additional therapeutic applications for diseases including, among others, autoimmune diseases, various cardiac diseases, demyelinating and neurodegenerative disorders, and infectious diseases.

[0245] In one embodiment, wild-type TNFα has the amino acid sequence of SEQ ID NO: 97.

[0246] In such embodiments, the modified TNFα substance has mutations at one or more amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147, resulting in a modified TNFα with reduced receptor binding affinity. See, for example, U.S. Patent No. 7,993,636, the entirety of which is incorporated herein by reference.

[0247] In some embodiments, the modified human TNF-α moiety has mutations at one or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, A145, and E146, as described in WO / 2015 / 007903 (the entire contents of which are incorporated herein by reference). In some embodiments, the modified human TNF-α moiety has substitution mutations selected from L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K, and S147D. In some embodiments, the human TNF-α moiety has mutations selected from Y87Q, Y87L, Y87A, Y87F, and Y87H. In another embodiment, the human TNF-α moiety has mutations selected from I97A, I97Q, and I97S. In yet another embodiment, the human TNF-α moiety has mutations selected from Y115A and Y115G. In some embodiments, the human TNF-α moiety has the E146K mutation. In some embodiments, the human TNF-α moiety has the Y87H and E146K mutations. In some embodiments, the human TNF-α moiety has the Y87H and A145R mutations. In some embodiments, the human TNF-α moiety has the R32W and S86T mutations. In some embodiments, the human TNF-α moiety has the R32W and E146K mutations. In some embodiments, the human TNF-α moiety has the L29S and R32W mutations. In some embodiments, the human TNF-α moiety has D143N and A145R mutations.In some embodiments, the human TNF-α moiety has A145T, E146D, and S147D mutations.

[0248] In some embodiments, as described in International Publication No. 2008 / 124086, the modified TNFα material comprises one or more mutations selected from N39Y, S147Y, and Y87H. The entirety of this patent is incorporated herein by reference.

[0249] In some embodiments, the modified human TNF-α moiety has mutations that provide receptor selectivity as described in PCT / IB2016 / 001668 (the entire contents of which are incorporated herein by reference). In some embodiments, the mutation to TNF is TNF-R1 selective. In some embodiments, the mutation to TNF that is TNF-R1 selective is located at one or more of the positions R32, S86, and E146. In some embodiments, the mutation to TNF that is TNF-R1 selective is located at one or more of the positions R32W, S86T, and E146K. In some embodiments, the mutation to TNF that is TNF-R1 selective is located at one or more of the positions R32W, R32W / S86T, R32W / E146K, and E146K. In some embodiments, the mutation to TNF is TNF-R2 selective. In some embodiments, the TNF mutation that is TNF-R2 selective is located at one or more of the positions A145, E146, and S147. In some embodiments, the TNF mutation that is TNF-R2 selective is located at one or more of the positions A145T, A145R, E146D, and S147D. In some embodiments, the TNF mutation that is TNF-R2 selective is located at one or more of the positions A145R, A145T / S147D, and A145T / E146D / S147D.

[0250] In one embodiment, the modified signaling molecule is TNF-β. TNFβ can form homotrimers or heterotrimers with LTβ (LTα1β2). In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2 and / or herpesvirus entry mediator (HEVM) and / or LTβR.

[0251] In one embodiment, wild-type TNFβ has the amino acid sequence of SEQ ID NO: 98.

[0252] In such embodiments, the modified TNFβ substance may contain mutations at one or more amino acid positions 106-113, resulting in a modified TNFβ with reduced receptor binding affinity to TNFR2. In one embodiment, the modified signaling substance has one or more substitutional mutations at amino acid positions 106-113. In exemplary embodiments, the substitutional mutations are selected from Q107E, Q107D, S106E, S106D, Q107R, Q107N, Q107E / S106E, Q107E / S106D, Q107D / S106E, and Q107D / S106D. In another embodiment, the modified signaling substance has approximately 1 to approximately 3 amino acid insertions at positions 106-113.

[0253] In some embodiments, the modified material is a TNF family member (e.g., TNF-alpha, TNF-beta), as described in WO 2015 / 007903 and PCT / IB2016 / 001668 (the full contents of which are incorporated herein by reference), and may be in the form of a single-chain trimmer.

[0254] In some embodiments, the modified material is a TNF family member (e.g., TNF-alpha, TNF-beta) which has reduced affinity and / or activity toward TNFR1, i.e., antagonist activity (e.g., innate antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520 (the entire contents of which are incorporated herein by reference)). In these embodiments, the modified material is a TNF family member (e.g., TNF-alpha, TNF-beta) which also optionally has substantially reduced or removed affinity and / or activity toward TNFR2. In some embodiments, the modified material is a TNF family member (e.g., TNF-alpha, TNF-beta) which has reduced affinity and / or activity toward TNFR2, i.e., antagonist activity (e.g., innate antagonist activity or antagonist activity as a result of one or more mutations, see, e.g., WO 2015 / 007520 (the entire contents of which are incorporated herein by reference)). In these embodiments, the modified material is a TNF family member (e.g., TNF-alpha, TNF-beta) which also optionally has substantially reduced or removed affinity and / or activity toward TNFR1. Constructs of such embodiments are used, for example, in methods for repressing the TNF response in a cell-specific manner. In some embodiments, the antagonist TNF family member (e.g., TNFα, TNFβ) is a single-chain trimer type, as described in International Publication No. 2015 / 007903.

[0255] In some embodiments, the modified signaling molecule is TRAIL. In some embodiments, the modified TRAIL molecule has reduced affinity and / or activity to DR4(TRAIL-RI) and / or DR5(TRAIL-RII) and / or DcR1 and / or DcR2. In some embodiments, the modified TRAIL molecule has reduced affinity and / or activity to DR4(TRAIL-RI) and / or DR5(TRAIL-RII) and / or DcR1 and / or DcR2.

[0256] In one embodiment, wild-type TRAIL has the amino acid sequence of SEQ ID NO: 99.

[0257] In such embodiments, the modified TRAIL material may contain mutations at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-1220, K224-A226, W231, E236-L239, E249-K251, T261-H264, and H270-E271 (Genbank acceptance number NP_003801, version 10 NP_003801.1, GI: 4507593, numbered based on human sequence; see above).

[0258] In some embodiments, the modified TRAIL material includes one or more mutations that substantially reduce its affinity and / or activity to TRAIL-R1. In such embodiments, the modified TRAIL material may specifically bind to TRIL-R2. Examples of mutations include mutations at one or more amino acid positions Y189, R191, Q193, H264, I266, and D267. For example, the mutations may be one or more of Y189Q, R191K, Q193R, H264R, I266L, and D267Q. In some embodiments, the modified TRAIL material includes the mutations Y189Q, R191K, Q193R, H264R, I266L, and D267Q.

[0259] In some embodiments, the modified TRAIL material includes one or more mutations that substantially reduce its affinity and / or activity to TRAIL-R2. In such embodiments, the modified TRAIL material may specifically bind to TRIL-R1. Examples of mutations include mutations at one or more amino acid positions G131, R149, S159, N199, K201, and S215. For example, the mutations may be one or more of G131R, R149I, S159R, N199R, K201H, and S215D. In some embodiments, the modified TRAIL material includes the mutations G131R, R149I, S159R, N199R, K201H, and S215D. Additional TRAIL mutations are described, for example, in Trebbing et al., (2014) Cell Death and Disease, 5:e1035, the entire disclosure of which is incorporated herein by reference.

[0260] In some embodiments, the modified signaling molecule is TGFα. In such embodiments, the modified TGFα substance has reduced affinity and / or activity toward the epidermal growth factor receptor (EGFR). In some embodiments, the modified TGFα substance has substantially reduced or eliminated affinity and / or activity toward the epidermal growth factor receptor (EGFR).

[0261] In some embodiments, the modified signaling molecule is TGFβ. In such embodiments, the modified signaling molecule has reduced affinity and / or activity to TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to TGFBR1 and / or TGFBR2. In some embodiments, the modified signaling molecule may have substantially reduced or eliminated affinity and / or activity to TGFBR3, which, while not intended to be bound by theory, may act as a ligand reservoir for the TGF beta receptor. In some embodiments, TGFβ prefers TGFBR1 over TGFBR2 or TGFBR2 over TGFBR1. Similarly, while not intended to be bound by theory, LAP may act as a ligand reservoir for the TGF beta receptor. In some embodiments, the modified signaling molecule has reduced affinity and / or activity to TGFBR1 and / or TGFBR2, and / or substantially reduced or eliminated affinity and / or activity to the latent related peptide (LAP). In some embodiments, such chimeras are used in Kamrachi-Engelmann disease or other diseases associated with inappropriate TGFβ signaling.

[0262] In some embodiments, the modified substance is a TGF family member (e.g., TGFα, TGFβ) which has reduced affinity and / or activity to one or more of TGFBR1, TGFBR2, and TGFBR3, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity resulting from one or more mutations, see, e.g., WO 2015 / 007520 (the entire content of which is incorporated herein by reference)). In these embodiments, the modified substance is a TGF family member (e.g., TGFα, TGFβ) which also likewise has substantially reduced or eliminated affinity and / or activity to one or more of TGFBR1, TGFBR2, and TGFBR3.

[0263] In some embodiments, the modified substance is a TGF family member (e.g., TGFα, TGFβ) which has reduced affinity and / or activity to TGFBR1 and / or TGFBR2, i.e., antagonist activity (e.g., natural antagonist activity or antagonist activity resulting from one or more mutations, see, e.g., WO 2015 / 007520 (the entire contents of which are incorporated herein by reference)). In these embodiments, the modified substance is a TGF family member (e.g., TGFα, TGFβ) which also likewise has substantially reduced or removed affinity and / or activity to TGFBR3, as may be the case.

[0264] In some embodiments, the modified signaling molecule is an interleukin. In some embodiments, the modified signaling molecule is IL-1. In some embodiments, the modified signaling molecule is IL1α or IL1β. In some embodiments, the modified signaling molecule has reduced affinity and / or activity to IL1R1 and / or IL1RAcP. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to IL1R1 and / or IL1RAcP. In some embodiments, the modified signaling molecule has reduced affinity and / or activity to IL1R2. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to IL1R2. In some embodiments, the modified IL1 substance avoids interaction with IL1R2 and thus substantially reduces its function as a decoy and / or sink to therapeutic agents.

[0265] In one embodiment, wild-type IL1β has the amino acid sequence of SEQ ID NO: 100.

[0266] IL-1 is a pro-inflammatory cytokine and an important immune system regulator. It is a potent activator of the CD4 T cell response, increasing the proportion of Th17 cells and increasing the proliferation of IFNγ and IL-4 producing cells. IL-1 is also CD8 + A potent regulator of T cells, antigen-specific CD8 + T cells promote proliferation, differentiation, migration to the periphery, and enhance memory. IL-1 receptors include L-1R1 and IL-1R2. Binding to IL-1R1 and signaling via IL-1R1 constitute a mechanism by which IL-1 mediates many of its biological (and pathological) activities. IL-1R2 can function as a decoy receptor, thereby reducing the availability of IL-1 for IL-1R1-mediated interactions and signaling.

[0267] In some embodiments, the modified IL-1 has reduced affinity and / or activity (e.g., agonist activity) toward IL-1R1. In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity toward IL-1R2. Such embodiments result in recoverable IL-1 / IL-1R1 signaling, as well as prevention of loss of therapeutic chimeras toward IL-R2 and consequently a reduction in the required IL-1 dose (e.g., for chimeras with wild-type or only attenuating mutations toward IL-R1). Such constructs are used, for example, in methods of treating cancer, including, for example, stimulating the immune system to initiate an anti-cancer response.

[0268] In some embodiments, the modified IL-1 has reduced affinity and / or activity toward IL-1R1 (e.g., antagonist activity, e.g., innate antagonist activity or antagonist activity as a result of one or more mutations, e.g., see WO 2015 / 007520 (the entire content of which is incorporated herein by reference)). In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity toward IL-1R2. In such embodiments, the IL-1 / IL-1R1 signaling is irreversible and also results in the prevention of loss of therapeutic chimeras toward IL-R2 and consequently a reduction in the required IL-1 dose (e.g., for chimeras having only wild-type or attenuating mutations toward IL-R1). Such constructs are used, for example, in methods of treating autoimmune diseases, including, for example, suppressing the immune system.

[0269] In such embodiments, the modified signaling molecule has a deletion of amino acids 52-54, which produces a modified human IL1β with reduced binding affinity and reduced bioactivity to type I IL1R. See, for example, International Publication No. 1994 / 000491. The entire contents of this patent are incorporated herein by reference. In some embodiments, modified human IL-1β is A117G / P118G, R120X, L122A, T125G / L126G, R127G, Q130X, Q131G, K132A, S137G / Q138Y, L145G, H146X, L145A / L147A, Q148X, Q148G / Q150G, Q150G / D151A, M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209X, K209A / K210A, K219X, E221X, E The sequences have one or more substitutional mutations selected from 221S / N224A, N224S / K225S, E244K, and N245Q (where X may be any change in amino acids, e.g., a non-conservative change), which exhibit reduced binding to IL-1R, as described, for example, in WO2015 / 007542 and WO / 2015 / 007536 (their entire contents are incorporated herein by reference) (Genbank acceptance number NP_000567, version NP-000567.1, Gl:10835145, numbered based on human IL-1β sequences). In some embodiments, modified human IL-1β may have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K. In some embodiments, modified human IL-1β includes mutations Q131G and Q148G. In some embodiments, modified human IL-1β includes mutations Q148G and K208E. In some embodiments, modified human IL-1β includes mutations R120G and Q131G. In some embodiments, modified human IL-1β includes mutations R120G and H146A. In one embodiment, the modified human IL-1β includes mutations R120G and H146N.In some embodiments, the modified human IL-1β contains mutations R120G and H146R. In some embodiments, the modified human IL-1β contains mutations R120G and H146E. In some embodiments, the modified human IL-1β contains mutations R120G and H146G. In some embodiments, the modified human IL-1β contains mutations R120G and K208E. In some embodiments, the modified human IL-1β contains mutations R120G, F162A, and Q164E.

[0270] In one embodiment, the modified signaling molecule is IL-2. In such embodiments, the modified signaling molecule has reduced affinity and / or activity for IL2Rα and / or IL2Rβ and / or IL2Rγ. In some embodiments, the modified signaling molecule has reduced affinity and / or activity for IL2Rβ and / or IL2Rγ. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for IL2Rα. Such embodiments may be suitable for the treatment of cancer, for example, when the modified IL2 is an agonist for IL2Rβ and / or IL2Rγ. For example, the construct of the present invention is CD8 having IL2 receptors β and γ. + Prioritizing the attenuated activation of T cells (which can exert antitumor effects), T cells possessing IL2 receptors α, β, and γ reg Prioritizing (the ability to impart immunosuppressive and tumor-promoting effects) is not the priority. Furthermore, in some embodiments, selectivity for IL2Rβ and / or IL2Rγ over IL2Rα avoids IL2 side effects such as pulmonary edema. Also, IL-2-based chimeras are useful in treating diseases (e.g., autoimmune diseases) if, for example, the modified IL-2 is an antagonist to IL-2Rβ and / or IL-2Rγ (e.g., natural antagonist activity or antagonist activity as a result of one or more mutations, see, for example, WO 2015 / 007520 (the entire content of which is incorporated herein by reference)). For example, the constructs of the present invention include CD8 having IL2 receptors β and γ. +Prioritizing the reduction of T cell suppression (and therefore suppression of the immune response), T cells possessing IL2 receptors α, β, and γ reg Prioritize not having IL2. Alternatively, in some embodiments, a chimera having IL2 is T reg Prioritizing the activation of CD8, and therefore immunosuppression, + They do not prioritize T cell activation. For example, these constructs are used to treat diseases in which immunosuppression is thought to be beneficial, such as autoimmune disorders.

[0271] In some embodiments, the chimeric protein or chimeric protein complex is CD8 + Having a targeting moiety directed to T cells as described herein, the modified IL-2 substance has reduced affinity and / or activity to IL-2Rβ and / or IL-2Rγ, and / or substantially reduced or removed affinity and / or activity to IL-2Rα. In some embodiments, these constructs are CD8 + It targets T cell activity, and normally, T reg It is inactive to cells (or has substantially reduced activity). In some embodiments, such constructs have an enhanced immunostimulatory effect compared to wild-type IL-2 (by not stimulating Tregs, although we do not wish to be bound by theory), while eliminating or reducing the systemic toxicity associated with IL-2.

[0272] In one embodiment, wild-type IL2 has the amino acid sequence of SEQ ID NO: 101.

[0273] In such embodiments, the modified IL2 substance has one or more mutations at the amino acid L72 position (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), the F42 position (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K), and the Y45 position (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K). While we do not wish to be bound by theory, these modified IL2 substances are thought to have reduced affinity for high-affinity IL2 receptors compared to wild-type IL2, while maintaining the same affinity for intermediate-affinity IL2 receptors. For example, see U.S. Patent Application Publication No. 2012 / 0244112. The entire contents of this patent are incorporated herein by reference.

[0274] In some embodiments, the modified IL-2 substance has one or more mutations in the amino acids R38, F42, Y45, and E62. For example, the modified IL-2 substance may comprise one or more of R38A, F42A, Y45A, and E62A. In some embodiments, the modified IL-2 substance may comprise a mutation in C125. For example, the mutation may be C125S. In such embodiments, the modified IL-2 substance may have substantially reduced affinity and / or activity for IL-2Rα, as described, for example, in Carmenate et al. (2013) The Journal of Immunology, 190:6230-6238 (this entire disclosure is incorporated herein by reference). In some embodiments, the modified IL-2 substance having mutations in R38, F42, Y45, and / or E62 can induce the proliferation of effector cells, including CD8+ T cells and NK cells, but not Treg cells. In some embodiments, modified IL-2 substances having mutations at R38, F42, Y45, and / or E62 are less toxic than wild-type IL-2 substances. Chimeric proteins or chimeric protein complexes containing modified IL-2 substances with substantially reduced affinity and / or activity for IL-2Rα may find applications, for example, in oncology.

[0275] In other embodiments, the modified IL-2 substance may have substantially reduced affinity and / or activity to IL-2Rβ, as described, for example, in WO2016 / 025385 (this entire disclosure is incorporated herein by reference). In such embodiments, the modified IL-2 substance may induce the proliferation of Treg cells but not effector cells such as CD8+ T cells and NK cells. Chimeric proteins or chimeric protein complexes containing the modified IL-2 substance having substantially reduced affinity and / or activity to IL-2Rβ may find applications, for example, in the treatment of autoimmune diseases. In some embodiments, the modified IL-2 substance may contain one or more mutations in amino acids N88, D20, and / or A126. For example, the modified IL-2 substance may contain one or more of N88R, N88I, N88G, D20H, Q126L, and Q126F.

[0276] In various embodiments, the modified IL-2 substance may contain mutations at D109 or C125. For example, the mutations may be D109C or C125S. In some embodiments, modified IL-2 having mutations at D109 or C125 may be used for binding to a PEG moiety.

[0277] In some embodiments, the modified signaling molecule is IL-3. In some embodiments, the modified signaling molecule has reduced affinity and / or activity to the IL3 receptor, and the IL3 receptor is a heterodimer having a distinctive alpha chain paired with a common beta (beta c or CD131) subunit. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to the IL3 receptor, and the IL3 receptor is a heterodimer having a distinctive alpha chain paired with a common beta (beta c or CD131) subunit.

[0278] In some embodiments, the modified signaling molecule is IL-4. In such embodiments, the modified signaling molecule has reduced affinity and / or activity to type 1 and / or type 2 IL4 receptors. In such embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to type 1 and / or type 2 IL4 receptors. The type 1 IL4 receptor consists of an IL4Rα subunit having a common γ chain and specifically binds to IL4. The type 2 IL4 receptor contains an IL4Rα subunit bound to a different subunit known as IL13Rα1. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to type 2 IL4 receptors.

[0279] In one embodiment, wild-type IL4 has the amino acid sequence of SEQ ID NO: 102.

[0280] In such embodiments, the modified IL4 substance has one or more mutations in the amino acids R121 (R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W), E122 (E122F), Y124 (Y124A, Y124Q, Y124R, Y124S, Y124T), and S125 (S125A). While we do not wish to be bound by theory, these modified IL4 substances are thought to maintain activity mediated by type I receptors but significantly reduce biological activity mediated by other receptors. See, for example, U.S. Patent No. 6,433,157. The entire contents of that patent are incorporated herein by reference.

[0281] In some embodiments, the modified signaling molecule is IL-6. IL-6 transmits signals via a cell surface type I cytokine receptor complex containing a ligand-bound IL6R chain (CD126) and the signaling component gp130. IL-6 can also bind to the soluble form of IL6R (sIL6R), which is the extracellular portion of IL6R. The sIL6R / IL6 complex is involved in neurite growth and neuronal survival and may therefore be important for nerve regeneration by remyelination. Accordingly, in some embodiments, the modified signaling molecule has reduced affinity and / or activity to IL6R / gp130 and / or sIL6R. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to IL6R / gp130 and / or sIL6R.

[0282] In one embodiment, wild-type IL6 has the amino acid sequence of SEQ ID NO: 103.

[0283] In such embodiments, the modified signaling molecule has one or more mutations in amino acids 58, 160, 163, 171, or 177. While not wishing to be bound by theory, these modified IL6 substances are thought to exhibit reduced binding affinity and reduced biological activity to IL6Rα. See, for example, International Publication 97 / 10338. The entire contents of this patent are incorporated herein by reference.

[0284] In one embodiment, the modified signaling molecule is IL-10. In such embodiments, the modified signaling molecule has reduced affinity and / or activity to IL10 receptor 1 and IL10 receptor 2. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to IL10 receptor 1 and IL10 receptor 2.

[0285] In one embodiment, the modified signaling molecule is IL-11. In such an embodiment, the modified signaling molecule has reduced affinity and / or activity for IL11Rα and / or IL11Rβ and / or gp130. In such an embodiment, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130.

[0286] In one embodiment, the modified signaling molecule is IL-12. In such an embodiment, the modified signaling molecule has reduced affinity and / or activity for IL12Rβ1 and / or IL12Rβ2. In such an embodiment, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2.

[0287] In one embodiment, the modified signaling molecule is IL-13. In such embodiments, the modified signaling molecule has reduced affinity and / or activity to the IL4 receptor (IL4Rα) and IL13Rα1. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to the IL4 receptor (IL4Rα) or IL13Rα1.

[0288] In one embodiment, wild-type IL13 has the amino acid sequence of SEQ ID NO: 104.

[0289] In such embodiments, the modified IL13 substance has one or more mutations in amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114. While not wishing to be bound by theory, these modified IL13 substances are thought to exhibit reduced biological activity. See, for example, International Publication 2002 / 018422. The entire contents of this patent are incorporated herein by reference.

[0290] In some embodiments, the modified signaling molecule is IL-18. In some embodiments, the modified signaling molecule has reduced affinity and / or activity for IL18Rα and / or IL18Rβ. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for IL18Rα and / or IL18Rβ. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity for IL18Rα type II, which is an isoform of IL18Rα lacking the TIR domain required for signaling.

[0291] In one embodiment, wild-type IL-18 has the amino acid sequence of SEQ ID NO: 105.

[0292] In such embodiments, the modified IL-18 substance may contain one or more mutations in amino acids or amino acid regions selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183 and Q190-N191, as described in WO / 2015 / 007542 (the entire contents of which are incorporated herein by reference).

[0293] In one embodiment, the modified signaling molecule is IL-33. In such embodiments, the modified signaling molecule has reduced affinity and / or activity to the ST2 receptor 1 and IL-1RAcP. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to the ST2 receptor and IL-1RAcP.

[0294] In one embodiment, wild-type IL33 has the amino acid sequence of SEQ ID NO: 106.

[0295] In such embodiments, as described in International Publication No. 2015 / 007542, the entire contents of which are incorporated herein by reference, the modified IL-33 substance may contain one or more mutations in amino acids or amino acid regions selected from I113-Y122, S127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227 and T260-E269 (Genbank acceptance number NP_254274, version 254274.1, Gl:15559209, numbered based on human sequence).

[0296] In one embodiment, the modified signaling molecule is epidermal growth factor (EGF). EGF is a family of highly potent growth factors. Members include EGF, HB-EGF, and others such as TGFα, amphiregulin, neuregulin, epiregulin, and betacerulin. EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB4. These can function as homodimer and / or heterodimer receptor subtypes. Different EGF family members exhibit different selectivity for various receptor subtypes. For example, EGF binds to ErbB1 / ErbB1, ErbB1 / ErbB2, ErbB4 / ErbB2, and several other heterodimer subtypes. HB-EGF has a similar pattern but binds to ErbB4 / 4. Positive or negative modulation of EGF (EGF-like) growth factor signaling is of great therapeutic interest. For example, inhibiting EGFR signaling is of interest in the treatment of various cancers in which EGFR signaling constitutes a major growth-promoting signal. Alternatively, stimulating EGFR signaling is of interest from a therapeutic perspective, for example, in wound healing (acute and chronic) and oral mucositis (but not limited to, a major side effect of various cancer therapies, including radiotherapy).

[0297] In some embodiments, the modified signaling molecule has reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4. Such embodiments are used, for example, in methods for treating wounds. In some embodiments, the modified signaling molecule binds to one or more of ErbB1, ErbB2, ErbB3, and ErbB4, and antagonistizes the activity of their receptors. In such embodiments, the modified signaling molecule has reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4, thereby antagonistizing in a way that attenuates the activity of their receptors. Such embodiments are used, for example, in the treatment of cancer. In one embodiment, the modified signaling molecule has reduced affinity and / or activity for ErbB1. ErbB1 is a therapeutic target for kinase inhibitors, but most have side effects because they are not very selective (e.g., gefitinib, erlotinib, afatinib, brigatinib, and icotinib). In some embodiments, attenuated antagonistic ErbB1 signaling is more on-target and has fewer side effects than other substances that target the EGF receptor.

[0298] In some embodiments, the modified signaling molecule has reduced affinity and / or activity to ErbB1 (e.g., antagonist activity, e.g., innate antagonist activity or antagonist activity as a result of one or more mutations, e.g., see WO 2015 / 007520 (the entire content of which is incorporated herein by reference)) and / or substantially reduced or eliminated affinity and / or activity to ErbB4 or other subtypes it may interact with. Specific targeting via the targeting moiety would achieve cell-selective suppression of ErbB1 / ErbB1 receptor activation (antagonism, e.g., innate antagonist activity or antagonist activity as a result of one or more mutations, e.g., see WO 2015 / 007520 (the entire content of which is incorporated herein by reference)) without involving other receptor subtypes that may be associated with inhibition-related side effects. Therefore, in contrast to EGFR kinase inhibitors that inhibit EGFR activity in all cell types throughout the body, such constructs would provide cell-selective anti-EGFR (ErbB1) drug action (e.g., tumor cells with activated EGFR signaling due to receptor amplification, overexpression, etc.) with reduced side effects.

[0299] In some embodiments, the modified signaling molecule has reduced affinity and / or activity (e.g., agonist activity) to ErbB4 and / or other subtypes with which it interacts. Selective activation of ErbB1 signaling is achieved by targeting specific target cells via the targeting moiety (e.g., epithelial cells). Such constructs are used in some embodiments for wound treatment with reduced side effects (promotion of wound healing), particularly for the treatment of chronic conditions and applications other than topical administration of therapeutic agents (e.g., systemic wound healing).

[0300] In some embodiments, the modified signaling molecule is insulin or an insulin analog. In some embodiments, the modified insulin or insulin analog has reduced affinity and / or activity toward the insulin receptor and / or the IGF1 or IGF2 receptor. In some embodiments, the modified insulin or insulin analog has reduced or eliminated affinity and / or activity toward the insulin receptor and / or the IGF1 or IGF2 receptor. The weakened response toward the insulin receptor allows for the control of conditions such as diabetes, obesity, and metabolic disorders, while simultaneously avoiding cancer-promoting effects by directing the signal away from the IGF1 or IGF2 receptor.

[0301] In some embodiments, the modified signaling molecule is insulin-like growth factor-I or insulin-like growth factor-II (IGF1 or IGF2). In some embodiments, the modified signaling molecule is IGF1. In such embodiments, the modified signaling molecule has reduced affinity and / or activity to the insulin receptor and / or the IGF1 receptor. In some embodiments, the modified signaling molecule binds to the IGF1 receptor and antagonizes the receptor's activity. In such embodiments, the modified signaling molecule has reduced affinity and / or activity to the IGF1 receptor, thereby enabling antagonization in a manner that attenuates the receptor's activity. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to the insulin receptor and / or the IGF1 receptor. In some embodiments, the modified signaling molecule has reduced affinity and / or activity to the IGF2 receptor, thereby enabling antagonization in a manner that attenuates the receptor's activity. In some embodiments, the modified signaling molecule has substantially reduced or eliminated affinity and / or activity to the insulin receptor and therefore does not interfere with insulin signaling. In various embodiments, this is applied to cancer treatment. In various embodiments, the substance can prevent IR isoform A from developing resistance to cancer treatment.

[0302] In some embodiments, the modified signaling molecule is EPO. In various embodiments, the modified EPO substance has reduced affinity and / or activity to the EPO receptor (EPOR) and / or ephrin receptor (EphR) compared to wild-type EPO or other EPO-based substances described herein. In some embodiments, the modified EPO substance has substantially reduced or eliminated affinity and / or activity to the EPO receptor (EPOR) and / or Eph receptor (EphR). Examples of EPO receptors include, but are not limited to, EPOR homodimers or EPOR / CD131 heterodimers. The beta common receptor (βcR) is also included in the EPO receptor. Examples of Eph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6. In some embodiments, the modified EPO protein comprises one or more mutations that cause the EPO protein to have reduced affinity for one or more different EPO receptors or Eph receptors (e.g., heterodimers and heterotrimers including, but not limited to, EPOR-EPHB4, EPOR-βcR-EPOR). Also provided are receptors of EP Patent Publication No. 2492355, including, but not limited to, NEPOR (the entirety of this patent is incorporated herein by reference).

[0303] In some embodiments, human EPO has the amino acid sequence of SEQ ID NO: 107 (the first 27 amino acids are a signal peptide).

[0304] In some embodiments, the human EPO protein is the mature form of EPO (with the signal peptide cleaved), which is a glycoprotein consisting of 166 amino acid residues having the sequence of SEQ ID NO: 108.

[0305] The structure of the human EPO protein is predicted to include four helix bundles, including helices A, B, C, and D. In various embodiments, the modified EPO protein contains one or more mutations located in four regions of the EPO protein that are important for biological activity, namely amino acid residues 10–20, 44–51, 96–108, and 142–156. In some embodiments, one or more mutations are located in residues 11–15, 44–51, 100–108, and 147–151. These residues are localized to helix A (Val11, Arg14, and Tyr15), helix C (Ser100, Arg103, Ser104, and Leu108), helix D (Asn147, Arg150, Gly151, and Leu155), and the A / B connecting loop (residues 42–51). In some embodiments, the modified EPO protein contains mutations in amino acid residues 41–52 and amino acid residues 147, 150, 151, and 155. While we do not wish to be bound by theory, mutations in these residues are thought to have a substantial effect on both receptor binding and in vitro bioactivity. In some embodiments, the modified EPO protein contains mutations in residues 11, 14, 15, 100, 103, 104, and 108. While we do not wish to be bound by theory, mutations in these residues are thought to have a moderate effect on receptor binding activity and a much greater effect on in vitro bioactivity. Examples of substitutions include, but are not limited to, one or more of the following: Val11Ser, Arg14Ala, Arg14Gln, Tyr15lle, Pro42Asn, Thr44lle, Lys45Asp, Val46Ala, Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104lle, Ser104Ala, Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala, and Leu155Ala.

[0306] In some embodiments, the modified EPO protein includes mutations that affect biological activity but not binding, such as those described in Eliot, et al. Mapping of the Active Site of Recombinant Human Erythropoietin January 15, 1997; Blood: 89(2) (the entire contents of which are incorporated herein by reference).

[0307] In some embodiments, the modified EPO protein contains one or more mutations involving surface residues of the EPO protein that are involved in receptor contact. While we do not wish to be bound by theory, these surface residue mutations are thought to have little impact on protein folding and therefore retain some biological activity. Examples of surface residues that can be mutagenesized include, but are not limited to, residues 147 and 150. In exemplary embodiments, the mutation is a substitution containing one or more of N147A, N147K, R150A, and R150E.

[0308] In some embodiments, the modified EPO protein contains one or more mutations at residues N59, E62, L67, and L70, and one or more mutations affecting disulfide bond formation. While we do not wish to be bound by theory, these modifications are expected to affect folding and / or be located in buried positions, and therefore are thought to indirectly affect biological activity.

[0309] In one embodiment, the modified EPO protein includes a K20E substitution that significantly reduces receptor binding. See, for example, Elliott, et al., (1997) Blood, 89:493-502. The entire contents of this document are incorporated herein by reference.

[0310] Additional EPO mutations that can be incorporated into the chimeric EPO protein of the present invention are disclosed, for example, in Elliott, et al., (1997) Blood, 89:493-502 (the entire contents of which are incorporated herein by reference) and Taylor et al., (2010) PEDS, 23(4):251-260 (the entire contents of which are incorporated herein by reference).

[0311] In one embodiment, the chimeric protein or chimeric protein complex of the present invention has (i) a targeting moiety comprising a domain recognizing PD-L1, and (ii) a targeting moiety directed toward tumor cells together with any of the modified or mutant signaling substances described herein. In another embodiment, the chimeric protein or chimeric protein complex of the present invention has a targeting moiety toward PD-L1 and a second targeting moiety toward another target on tumor cells.

[0312] In various embodiments, the signaling molecule is a toxin or toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Examples of toxins or toxic enzymes include, but are not limited to, diphtheria toxin, Pseudomonas toxin, anthrax toxin, ribosome inactivating proteins (RIPs) such as lysine and saporin, modesine, abrin, geronin, and pokeweed antiviral proteins. Additional toxins include those disclosed in Mathew et al., (2009) Cancer Sci 100(8):1359-65, the entire disclosure of which is incorporated herein by reference. In such embodiments, the chimeric protein or chimeric protein complex of the present invention may be used to induce cell-type-specific cell death. In such embodiments, the toxin may be modified, for example, by mutagenesis, to reduce the affinity and / or activity of the toxin in order to weaken its effect, as described herein with respect to other signaling molecules.

[0313] Linker and functional group In various embodiments, the chimeric protein or chimeric protein complex of the present invention may comprise one or more functional groups, residues, or moieties. In various embodiments, one or more functional groups, residues, or moieties may be bound to or genetically fused to one of the signaling molecules or targeting moieties (e.g., PD-L1) described herein. In some embodiments, such functional groups, residues, or moieties may confer one or more desirable properties or functionalities to the chimeric protein or chimeric protein complex of the present invention. Examples of such functional groups and techniques for introducing them into the chimeric protein or chimeric protein complex of the present invention are known in the art; see, for example, Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

[0314] In various embodiments, the chimeric protein or chimeric protein complex of the present invention may be complexed and / or fused with another substance to extend its half-life or otherwise improve its pharmacodynamic and pharmacokinetic properties. In some embodiments, the chimeric protein or chimeric protein complex of the present invention may be fused or complexed with one or more of the following: PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, etc. In some embodiments, the chimeric protein or chimeric protein complex of the present invention may be fused or complexed with an antibody or an antibody fragment such as an Fc fragment. For example, the chimeric protein or chimeric protein complex may be fused to either the N-terminus or C-terminus of the Fc domain of human immunoglobulin (Ig) G. In various embodiments, each individual chimeric protein or chimeric protein complex is fused to one or more of the substances described in BioDrugs (2015) 29:215-239 (the entire contents of which are incorporated herein by reference).

[0315] In some embodiments, the functional group, residue, or moiety comprises a suitable pharmaceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly(ethylene glycol) or mPEG). In some embodiments, the binding of the PEG moiety extends the half-life and / or reduces the immunogenicity of the PD-L1 binding protein. Any suitable form of pegy used in the art may be used for antibodies and antibody fragments (including, but not limited to, single-domain antibodies such as VHH). See, for example, Chapman, Nat. Biotechnol., 54, 531-545 (2002), Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003), and WO04 / 060965 (the entire contents of which are incorporated herein by reference). Various reagents for protein pegylation are commercially available, for example, from Nektar Therapeutics, USA. In some embodiments, site-specific pegylation via cysteine ​​residues is used in particular (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 (2003), the entire contents of which are incorporated herein by reference). For example, for this purpose, PEG may attach to naturally occurring cysteine ​​residues in the chimeric protein or chimeric protein complex of the present invention. In some embodiments, the chimeric protein or chimeric protein complex of the present invention may be modified to appropriately introduce one or more cysteine ​​residues for PEG binding, or an amino acid sequence containing one or more cysteine ​​residues for PEG binding may be fused to the amino and / or carboxyl termini of the chimeric protein or chimeric protein complex of the present invention using techniques known in the art.

[0316] In some embodiments, the functional group, residue, or moiety includes N-linked or O-linked glycosylation. In some embodiments, N-linked or O-linked glycosylation is introduced as part of a post-translational modification.

[0317] In some embodiments, a functional group, residue, or moiety comprises one or more detectable labels or other signal-generating groups or moieties. Suitable labels and techniques for binding, using, and detecting them are known in the art and are not limited to, but include, fluorescent labels (fluorescein, isothiocyanates, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine, as well as fluorescent metals, e.g., Eu or other metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (luminol, isoluminol, ceromatic acridinium esters, imidazole, acridinium salts, oxalate esters, dioxetane or GFP and their analogues, etc.), radioisotopes, metals, metal chelates or metal cations, and This includes other metals or metal cations, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase) that are particularly suitable for use in vivo, in vitro, or in situ diagnostics and imaging. The VHH and polypeptides of the present invention, labeled in this manner, can be used, for example, in in vitro, in vivo, or insight assays (immunoassays themselves known as ELISA, RIA, EIA, and other "sandwich methods"), as well as for in vivo diagnostic and imaging purposes, depending on the selection of specific labels.

[0318] In some embodiments, the functional group, residue, or portion comprises a tag bound to or genetically fused to the chimeric protein or chimeric protein complex. In some embodiments, the chimeric protein or chimeric protein complex of the present invention may comprise a single tag or multiple tags. For example, the tag is a peptide, sugar, or DNA molecule that does not inhibit or interfere with the binding of the chimeric protein or chimeric protein complex of the present invention to any other target antigen, such as PD-L1 or a tumor antigen. In various embodiments, the tag is at least about: 3–5 amino acid length, 5–8 amino acid length, 8–12 amino acid length, 12–15 amino acid length, or 15–20 amino acid length. Examples of tags are described, for example, in U.S. Patent Application Publication 2013 / 0058962. In some embodiments, the tag is an affinity tag, such as a glutathione-S-transferase (GST) and a histidine (His) tag. In some embodiments, the chimeric protein or chimeric protein complex of the present invention comprises a His tag.

[0319] In some embodiments, the functional group, residue, or moiety includes, for example, a chelating group for chelating a metal or metal cation. Preferred chelating groups include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0320] In some embodiments, the functional group, residue, or portion comprises a functional group that is one half of a specific binding pair, such as a biotin-(strept)avidin binding pair. Using such a functional group, the chimeric protein or chimeric protein complex of the present invention can be linked to another protein, polypeptide, or chemical compound linked to the other half of the binding pair, i.e., via the formation of a binding pair. For example, the chimeric protein or chimeric protein complex of the present invention can be linked to another protein, polypeptide, compound, or carrier that is complexed with biotin and complexed with avidin or streptavidin. For example, such linked chimeric protein or chimeric protein complex of the present invention can be used with a reporter in a diagnostic system in which a detectable signaling agent is complexed with avidin or streptavidin. For example, using such a binding pair, the chimeric protein or chimeric protein complex can be linked to a carrier, including a carrier suitable for pharmaceutical purposes. One non-limiting example is the liposomal formulation described in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Furthermore, such binding pairs may be used to link therapeutically active agents to the chimeric protein or chimeric protein complex of the present invention.

[0321] In some embodiments, the chimeric protein or chimeric protein complex of the present invention optionally includes one or more linkers. In some embodiments, the chimeric protein or chimeric protein complex of the present invention includes a targeting moiety and a linker connecting a signaling molecule. In some embodiments, the chimeric protein or chimeric protein complex of the present invention includes a linker within the signaling molecule (for example, in the case of single-chain TNF, it may include two linkers to obtain a trimer).

[0322] In some embodiments, vectors encoding the chimeric protein or chimeric protein complex of the present invention, linked as a single nucleotide sequence to one of the linkers described herein, are provided and can be used to prepare such chimeric protein or chimeric protein complex.

[0323] In some embodiments, the length of the linker allows for the efficient binding of targeting moieties and signaling molecules to their receptors. For example, in some embodiments, the length of the linker allows for the efficient binding of one of the targeting moieties and a signaling molecule to a receptor on the same cell, as well as the efficient binding of another targeting moiety to another cell. Examples of cell pairs are provided elsewhere in this specification.

[0324] In some embodiments, the linker length is equal to at least the shortest distance between one targeting region and the receptor binding site of a signaling molecule on the same cell. In some embodiments, the linker length is at least 2, 3, 4, 5, 10, 20, 25, 50, or 100 times, or more, the shortest distance between one targeting region and the receptor binding site of a signaling molecule on the same cell.

[0325] As described herein, the linker length allows for the effective binding of a targeting moiety and one signaling molecule to a receptor on the same cell, and the binding is sequential, for example, targeting moiety / receptor binding precedes signaling molecule / receptor binding.

[0326] In some embodiments, two linkers are present in a single chimera, each linking a signaling molecule to a targeting region. In various embodiments, the linkers have a length that allows for the formation of regions containing disease cells and effector cells without steric hindrance that could interfere with the regulation of either cell.

[0327] The present invention intends to utilize a variety of linker sequences. In various embodiments, the linker may be derived from a native multidomain protein or be an empirical linker described, for example, Chichili et al., (2013), Protein Sci. 22(2):153-167; Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369. The entire contents of these documents are incorporated herein by reference. In some embodiments, the linker may be designed using a linker design database, as well as computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et al., (2000), Protein Eng. 13(5):309-312 (the entire contents of these are incorporated herein by reference). In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve folding and / or stability, to improve expression, to improve pharmacokinetics, and / or to improve the biological activity of the chimeric protein or chimeric protein complex of the present invention.

[0328] In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids long. For example, the linker may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In some embodiments, the linker is a polypeptide. In some embodiments, the linker is more than about 100 amino acids long. For example, the linker may be about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acid lengths. In some embodiments, the linker is flexible. In other embodiments, the linker is rigid.

[0329] In some embodiments, one linker connects two targeting moieties and has a short length, while another linker connects a targeting moiety and a signaling molecule and has a longer length than the linker connecting the two targeting moieties. For example, the difference in amino acid length between the linker connecting two targeting moieties and the linker connecting the targeting moieties and the signaling molecule may be about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acid lengths.

[0330] In various embodiments, the linker is substantially composed of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). For example, in some embodiments, the linker is (Gly4Ser) n The formula is such that n is approximately 1 to approximately 8, for example, 1, 2, 3, 4, 5, 6, 7, or 8 (sequences 109 to 116). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (sequence 117). Examples of additional linkers, but not limited to, the sequence:LE,GGGGS (sequence 109), (GGGGS) n (n=1~4)(Sequence IDs 109~112), (Gly)8(Sequence ID 118), (Gly)6(Sequence ID 119), (EAAAK) n (n=1~3)(Sequence IDs 120~122), A(EAAAK) n A(n=2~5)(sequences 123~126), AEAAAKEAAAKA(sequence 123), A(EAAAK)4ALEA(EAAAK)4A(sequence 127), PAPAP(sequence 128), KESGSVSSEQLAQFRSLD(sequence 129), EGKSSGSGSESKST(sequence 130), GSAGSAAGSGEF(sequence 131), and (XP) n Examples of linkers include one having (GGS), where X represents any amino acid, e.g., Ala, Lys, or Glu. In various embodiments, the linker is (GGS) n (n=1~20) (Sequence IDs 132~151). In some embodiments, the linker is G. In some embodiments, the linker is AAA. In some embodiments, the linker is (GGGGS). n (n=5~20) (Sequence codes 113~116 and 152~163)

[0331] In some embodiments, the linker is one or more of GGGSE (SEQ ID NO: 164), GSESG (SEQ ID NO: 165), GSEGS (SEQ ID NO: 166), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 167), and G, S, and E linkers randomly arranged at intervals of four amino acids.

[0332] In some embodiments, the linker is a hinge region of the antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region found in IgG, IgA, IgD, and IgE class antibodies functions as a flexible spacer, allowing the Fab region to move freely in space. In contrast to the constant region, the hinge domain is structurally diverse and varies in both sequence and length within the immunoglobulin class and subclass. For example, the length and flexibility of the hinge region vary within the IgG subclass. The hinge region of IgG1 contains amino acids 216-231, and because it is freely flexible, the Fab fragment can rotate around its axis of symmetry and move within a sphere centered on the first two interhelic disulfide bridges. IgG2 has a shorter hinge than IgG1, containing 12 amino acid residues and 4 disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by additional interhelic disulfide bridges. These properties limit the flexibility of the IgG2 molecule. Unlike the other subclasses, IgG3 forms a less flexible polyproline double helix due to its distinctive extended hinge region (about four times the length of the IgG1 hinge) containing 62 amino acids (including 21 prolines and 11 cysteines). In IgG3, the Fab fragment is relatively far from the Fc fragment, giving the molecule greater flexibility. The elongated hinge of IgG3 is also the reason for its higher molecular weight compared to other subclasses. The hinge of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge has been reported to decrease in the following order: IgG3 > IgG1 > IgG4 > IgG2.

[0333] Crystallographic investigations reveal that the immunoglobulin hinge region can be functionally further subdivided into three areas: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region is C H1 The first residue in the hinge that restricts movement from the carboxyl terminus, usually the amino acid of the first cysteine ​​residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the flexibility of the antibody segment. The core hinge region contains an interchain disulfide bridge, and the lower hinge region contains a C H2 It connects to the amino terminus of the domain, C H2 The residues within are included (ibid.). The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 168), which, when dimerized by disulfide bond formation, is thought to generate a cyclic octapeptide, which functions as a pivot axis and thus confers flexibility. In various embodiments, the linker of the present invention comprises one, two, or three upper hinge regions, a core region, and a lower hinge region of any antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge regions may also include one or more glycosylation sites, which include many structurally different types of carbohydrate attachment sites. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, which is considered an advantageous property for secretory immunoglobulins. In various embodiments, the linker of the present invention comprises one or more glycosylation sites. In various embodiments, the linker is the hinge-CH2-CH3 domain of a human IgG4 antibody.

[0334] If desired, the chimeric protein or chimeric protein complex of the present invention is C H 2 and C HOne or both of the three domains, and optionally including a hinge region, can be ligated to the antibody Fc region. For example, such polypeptides can be prepared using a vector encoding the chimeric protein of the present invention, ligated to the Fc region as a single nucleotide sequence.

[0335] In some embodiments, the linker is a synthetic linker such as PEG.

[0336] In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve folding and / or stability, to improve expression, to improve pharmacokinetics, and / or to improve the biological activity of the chimeric protein or chimeric protein complex of the present invention. In another example, the linker may function to direct the chimeric protein or chimeric protein complex to a specific cell type or location.

[0337] Chimeric protein complex having an Fc domain In some embodiments, the present invention relates to a chimeric protein complex comprising one or more fragment crystallizable domains (Fc domains). In some embodiments, the Fc domain has one or more mutations that reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain.

[0338] In various embodiments, the present invention comprises a chimeric protein complex comprising one or more targeting substances, one or more signaling substances, and one or more Fc domains. In one embodiment, the chimeric protein complex comprises PD-L1 and at least one targeting moiety that specifically binds to at least one Fc domain. In another embodiment, the chimeric protein complex comprises at least one targeting moiety that specifically binds to PD-L1, at least one signaling substance that is tumor necrosis factor (TNF), and at least one Fc domain. In various embodiments, the TNF signaling substance may be modified to reduce its activity. In some embodiments, the PD-L1 targeted chimeric protein complex can recruit immune cells directly or indirectly to a site of action (a non-limiting example being the tumor microenvironment).

[0339] In some embodiments, the present invention relates to an Fc-based chimeric protein complex, which comprises (A) a targeting moiety, (a) three complementarity-determining regions (CDR1, CDR2, and CDR3), where (i) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 2 or 5, (ii) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (iii) CDR3 comprises an amino acid sequence selected from either SEQ ID NO: 4 or 7, or (b) comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, and (a) or (b) is The invention comprises: a targeting moiety further comprising one or more mutations at positions D54 and G55 numbered relative to Sequence ID No. 1; (B) a signaling molecule which is a) a wild-type signaling molecule, or b) a modified signaling molecule which has one or more mutations that confer improved safety to the wild-type signaling molecule; and (C) an Fc domain which optionally has one or more mutations that reduce or eliminate the effector function of one or more Fc domains, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain.

[0340] In this embodiment, the PD-L1 targeting portion, which includes the recognition domain, further comprises one or more mutations at positions Q1, Q5, A14, A63, T74, K76, S79, K86, and Q110.

[0341] In the embodiments, the mutation is a substitution, which is optionally selected from arginine (R) and lysine (K), an aromatic, polar, and positively charged hydrophilic residue including histidine (H), a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0342] In embodiments, the mutation is selected from one or more of the following: a hydrophobic, aliphatic amino acid at position D54, optionally D54G, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); a polar, positively charged hydrophilic residue at position D54, optionally D54K, selected from arginine (R) and lysine (K); a polar, neutrally charged hydrophilic residue at position G54T, optionally D54T, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); and a polar, positively charged hydrophilic residue at position G55, optionally G55R, selected from arginine (R) and lysine (K).

[0343] In the embodiment, the mutation is optionally Q1D, a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E) at position Q1; optionally Q5V, a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q5; optionally A14P, a polar and neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C) at position A14; optionally A63V, a hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position A63; optionally T74S, asparagine (N) at position T74, One or more of the following are selected: a polar and neutrally charged hydrophilic residue selected from glutamine (Q), serine (S), proline (P), and cysteine ​​(C); a polar and neutrally charged hydrophilic residue at position K76, optionally K76N, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C); a hydrophobic, aromatic amino acid selected at position S79, optionally S79Y, selected from phenylalanine (F), tryptophan (W), and tyrosine (Y) at position S79; a hydrophobic, aliphatic amino acid selected at position Q110, optionally Q110L, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V). In the embodiment, the mutation is one or more of Q1D, Q5V, A14P, A63V, T74S, S79Y, K86R, and Q110L, or optionally selected from all of Q1D, Q5V, A14P, D54G, T74S, K76N, S79Y, K86R, and Q110L.

[0344] In some embodiments, the present invention relates to an Fc-based chimeric protein complex, which is (A) The targeting portion, (a) Three complementarity determination regions (CDR1, CDR2, and CDR3), (i) CDR1 contains an amino acid sequence selected from either SEQ ID NO: 2 or 5, (ii) CDR2 contains an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (iii) CDR3 contains three complementarity-determining regions, each containing an amino acid sequence selected from either SEQ ID NO: 4 or 7, or (b) A targeting moiety comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, wherein (a) or (b) further comprises one or more mutations at positions D54, G55, K76, and S79 numbered relative to SEQ ID NO: 1, (B) A signaling substance, a) Wild-type signaling molecules, or b) A signaling molecule that is a modified signaling molecule having one or more mutations that confer improved safety to a wild-type signaling molecule, (C)Fc domain comprising an Fc domain having one or more mutations that optionally reduce or eliminate the effector function of one or more Fc domains, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain.

[0345] In some embodiments, the Fc-based chimeric protein complex includes a targeting moiety containing one or more mutations at positions T74, K86, and Q110 relative to SEQ ID NO: 1. In some embodiments, the Fc-based chimeric protein complex has a mutation which is a substitution, and optionally the substitution is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), an aromatic, polar, positively charged hydrophilic residue including histidine (H), a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0346] In some embodiments, the Fc-based chimeric protein complex has mutations selected from one or more of the following: • Optionally, D54G is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position D54, or optionally, D54K is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), or optionally, D54T is a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). • A polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K) at position G55, which is optionally G55R. • A polar and neutrally charged hydrophilic residue at position T74, selected from asparagine (N), glutamine (Q), serine (S), proline (P), and cysteine ​​(C), which is optionally T74S. • A polar and neutrally charged hydrophilic residue at position K76, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), which is optionally K76N. • A hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y) at position S79, optionally being S79Y. · Arginine (R) at position K86, which is K86R, and • A hydrophobic, aliphatic amino acid at position Q110, optionally Q110L, selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0347] In some embodiments, the Fc-based chimeric protein complex of the present invention is (A) The targeting portion, (a) Three complementarity determination regions (CDR1, CDR2, and CDR3), (i) CDR1 contains an amino acid sequence selected from either SEQ ID NO: 27 or 30, (ii) CDR2 contains an amino acid sequence selected from either SEQ ID NO: 28 or 31, and (iii) CDR3 contains three complementarity-determining regions, each containing an amino acid sequence selected from either SEQ ID NO: 29 or 32, or (b) A targeting moiety comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, wherein (a) or (b) further comprises one or more mutations at positions N32, D33, and M97 numbered relative to SEQ ID NO: 26, (B) A signaling molecule, which is a) a wild-type signaling molecule, or b) a modified signaling molecule having one or more mutations that confer improved safety to a wild-type signaling molecule, (C)Fc domain comprising an Fc domain having one or more mutations that optionally reduce or eliminate the effector function of one or more Fc domains, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain.

[0348] In some embodiments, the Fc-based chimeric protein complex of the present invention has a mutation which is a substitution for SEQ ID NO: 26. In some embodiments, the Fc-based chimeric protein complex includes substitutions of hydrophilic amino acid residues which are polar and positively charged hydrophilic residues selected from arginine (R) and lysine (K), or aromatic, polar and positively charged hydrophilic residues which include histidine (H). In some embodiments, the substitution is a hydrophilic amino acid residue which is a polar and neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). In some embodiments, the substitution is a hydrophilic amino acid residue which is a polar and negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E). In some embodiments, the substitution is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0349] In some embodiments, the substitution at position N32 in SEQ ID NO: 26 is a positive hydrophilic residue selected from arginine (R) and lysine (K). In some embodiments, the substitution at position N32 in SEQ ID NO: 26 is a polar and neutral hydrophilic residue selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). In some embodiments, the substitution at position N32 in SEQ ID NO: 26 is N32Q or N32R.

[0350] In some embodiments, the substitution at position D33 in SEQ ID NO: 26 is D33H. In other embodiments, the substitution at position M97 in SEQ ID NO: 26 is an aliphatic hydrophobic residue selected from glycine (G), leucine (L), isoleucine (I), and valine (V). In some embodiments, the substitution at position M97 in SEQ ID NO: 26 is M97I, M97L, or M97V.

[0351] In the embodiment, the PD-L1 targeting moiety including the recognition domain includes one or more of the following mutations (relative to SEQ ID NO: 26): Q1D, Q5V, A14P, A62S, A74S, M77T, M78V, S79Y, K86R, and Q109L, and optionally further includes all of Q1D, Q5V, A14P, D33H, A62S, A74S, M77T, M78V, K86R, and M97V.

[0352] In some embodiments, the present invention relates to a PD-L1-targeted chimeric protein complex having at least one targeting moiety that specifically binds to PD-L1, a signaling molecule that is at least one interferon (IFN) or a modified thereof, and at least one Fc domain. In various embodiments, the IFN signaling molecule may be modified to reduce its activity. In one embodiment, the interferon is IFN-γ or a modified thereof.

[0353] The fragment crystallizable domain (Fc domain) is the tail region of antibodies that interacts with Fc receptors located on the cell surface of cells involved in the immune system, such as B lymphocytes, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. In IgG, IgA, and IgD antibody isotypes, the Fc domain consists of two identical protein fragments derived from the second and third constant domains of the antibody's two heavy chains. In IgM and IgE antibody isotypes, the Fc domain consists of three heavy chain constant domains (C) in each polypeptide chain. H Includes domains 2-4).

[0354] In some embodiments, the Fc-based chimeric protein complex of the present technology includes an Fc domain. In some embodiments, the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from IgG1, IgG2, IgG3, or IgG4.

[0355] In some embodiments, the Fc domain is selected from human IgG, IgA, IgD, IgM, or IgE. In some embodiments, the Fc domain is selected from human IgG1, IgG2, IgG3, or IgG4.

[0356] In some embodiments, the Fc domain of the Fc-based chimeric protein complex includes the CH2 and CH3 regions of IgG. In some embodiments, IgG is human IgG. In some embodiments, human IgG is selected from IgG1, IgG2, IgG3, or IgG4.

[0357] In some embodiments, the Fc domain contains one or more mutations. In some embodiments, the mutations to the Fc domain reduce or eliminate the effector function of the Fc domain. In some embodiments, the mutant Fc domain has reduced affinity or binding to a target receptor. For example, in some embodiments, the mutations to the Fc domain reduce or eliminate the binding of the Fc domain to FcγR. In some embodiments, FcγR is selected from FcγRI;FcγRIIa,131R / R;FcγRIIa,131H / H,FcγRIIb; and FcγRIII. In some embodiments, the mutations to the Fc domain reduce or eliminate binding to complement proteins such as C1q. In some embodiments, the mutations to the Fc domain reduce or eliminate binding to both FcγR and complement proteins such as C1q.

[0358] In some embodiments, the Fc domain contains LALA mutations that reduce or eliminate the effector function of the Fc domain. For example, in some embodiments, the LALA mutations include L234A and L235A substitutions in human IgG (e.g., IgG1) (numbering is based on the numbering of CH2 residues commonly used for human IgG1 in accordance with EU regulations (Edelman et al., PNAS, 1969;63(1)78-85)).

[0359] In some embodiments, the Fc domain of human IgG includes a mutation at position 46 to reduce or eliminate the effector function of the Fc domain. For example, in some embodiments, the mutation is selected from L234A, L234F, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, P329A, P331G, and P331S.

[0360] In some embodiments, the Fc domain includes FALA mutations that reduce or eliminate the effector function of the Fc domain. For example, in some embodiments, the FALA mutations include F234A and L235A substitutions in human IgG4.

[0361] In some embodiments, the Fc domain of human IgG4 contains mutations at one or more of the following positions: F234, L235, K322, D265, and P329, which reduce or eliminate the effector function of the Fc domain. For example, in some embodiments, the mutations are selected from F234A, L235A, L235E, L235Q, K322A, K322Q, D265A, P329G, and P329A.

[0362] In some embodiments, mutations in the Fc domain stabilize the hinge region of the Fc domain. For example, in some embodiments, the Fc domain includes a mutation at the S228 position in human IgG, which stabilizes the hinge region. In some embodiments, the mutation is S228P.

[0363] In some embodiments, mutations in the Fc domain promote chain pairing of the Fc domain. In some embodiments, chain pairing is promoted by ion pairing (also known as charge pairing, ionic bonding, or charge residue pairing).

[0364] In some embodiments, the Fc domain contains a mutation in one of the following amino acid residues of IgG to promote ion pairing: D356, E357, L368, K370, K392, D399, and K409.

[0365] For example, in some embodiments, the human IgG Fc domain contains one of the mutation combinations in Table 1 to promote ion pairing. [Table 1]

[0366] In some embodiments, chain pairing is facilitated by a knob-in-hole mutation. In some embodiments, the Fc domain contains one or more mutations that enable knob-in-hole interactions within the Fc domain. In some embodiments, the first Fc chain is engineered to express a “knob,” and the second Fc chain is engineered to express a complementary “hole.” For example, in some embodiments, the human IgG Fc domain contains the mutations in Table 2 that enable knob-in-hole interactions. [Table 2]

[0367] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of the present technology includes any combination of the mutations disclosed above. For example, in some embodiments, the Fc domain includes mutations that promote ion pairing and / or knob-in-hole interaction. For example, in some embodiments, the Fc domain includes mutations having one or more of the following properties: promoting ion pairing, inducing knob-in-hole interaction, reducing or eliminating the effector function of the Fc domain, and resulting in Fc stabilization (e.g., hinge).

[0368] For example, in some embodiments, the human IgG Fc domain includes mutations disclosed in Table 3, which promote ion pairing in the Fc domain and / or promote knob-in-hole interactions. [Table 3-1] [Table 3-2]

[0369] For example, in some embodiments, the human IgG Fc domain includes mutations disclosed in Table 4, which promote ion-pairing of the Fc domain and / or promote knob-in-hole interactions, or a combination thereof. In some embodiments, "Chain 1" and "Chain 2" in Table 4 are interchangeable (for example, Chain 1 may have Y407T and Chain 2 may have T366Y). [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0370] For example, in some embodiments, the human IgG Fc domain contains mutations disclosed in Table 5 that reduce or eliminate FcγR and / or complement binding in the Fc domain. In some embodiments, the mutations in Table 5 are present in both strands. [Table 5-1] [Table 5-2] [Table 5-3]

[0371] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of this technology is homodimer, i.e., the Fc region in the chimeric protein complex contains two identical protein fragments.

[0372] In some embodiments, the Fc domain in the Fc-based chimeric protein complex of this technology is a heterodimer, i.e., the Fc domain contains two non-identical protein fragments.

[0373] In some embodiments, the heterodimer Fc domain is modified using ion-pairing and / or knob-in-hole mutations as described herein. In some embodiments, the heterodimer Fc-based chimeric protein complex has a trans orientation / structure. In the trans orientation / structure, the targeting moiety and signaling molecule are not located on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention, in some embodiments.

[0374] In some embodiments, the Fc domain includes or begins with the core hinge region of wild-type human IgG1, which contains the sequence Cys-Pro-Pro-Cys. In some embodiments, the Fc domain also includes the upper hinge, or a portion thereof (e.g., DKTHTCPPC (see International Publication No. 2009053368), EPKSCDKTHTCPPC, or EPKSSDKTHTCPPC (see Lo et al., Protein Engineering vol.11 no.6 pp.495-500, 1998)).

[0375] Fc-based chimeric protein complex The Fc-based chimeric protein complex of this technology comprises at least one Fc domain as disclosed herein, at least one signaling molecule as disclosed herein, and at least one targeting moiety (TM) as disclosed herein.

[0376] It is understood that the Fc-based chimeric protein complex of the present invention may contain two fusion proteins, each containing an Fc domain.

[0377] In some embodiments, the Fc-based chimeric protein complex is a heterodimer. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a trans orientation / structure. In some embodiments, the heterodimeric Fc-based chimeric protein complex has a cis orientation / structure.

[0378] In some embodiments, the heterodimer Fc domain is modified using ion-pairing and / or knob-in-hole mutations described herein. In some embodiments, the heterodimer Fc-based chimeric protein complex has a trans orientation.

[0379] In trans orientation, the targeting moiety and signaling molecule are not found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention in some embodiments. In trans orientation, the targeting moiety and signaling molecule are found on separate polypeptide chains in the Fc-based chimeric protein complex of the present invention in some embodiments. In cis orientation, the targeting moiety and signaling molecule are found on the same polypeptide chain in the Fc-based chimeric protein complex of the present invention in some embodiments.

[0380] In some embodiments in which two or more targeting moieties are present in the heterodimer protein complex described herein, one targeting moiety may be trans-oriented (towards the signaling molecule), while another targeting moiety may be cis-oriented (towards the signaling molecule). In some embodiments, the signaling molecule and the targeting moiety are located on the same end / side (N-terminus or C-terminus) of the Fc domain. In some embodiments, the signaling molecule and the targeting moiety are located on different sides / terminuses (N-terminus or C-terminus) of the Fc domain.

[0381] In some embodiments described herein, where two or more targeting moieties are present in the heterodimer protein complex, the targeting moieties may be located on the same Fc chain or on two different Fc chains in the heterodimer protein complex (in the latter case, the targeting moieties should be trans to each other because they are located on different Fc chains). In some embodiments, where two or more targeting moieties are present on the same Fc chain, the targeting moieties may be located on the same or different side / end of the Fc chain (N-terminus or C-terminus).

[0382] In some embodiments described herein, where two or more targeting moieties are present in the heterodimer protein complex, the targeting moieties are located on the same Fc chain or on two different Fc chains in the heterodimer protein complex (in the latter case, the targeting moieties should be trans to each other because they are located on different Fc chains). In some embodiments, where two or more signaling molecules are present on the same Fc chain, the signaling molecules may be located on the same or different sides / ends of the Fc chain (N-terminus or C-terminus).

[0383] In some embodiments in which two or more signaling molecules are present in the heterodimer protein complex described herein, one signaling molecule may be trans-oriented (relative to the targeting moiety), while another signaling molecule may be cis-oriented (relative to the targeting moiety).

[0384] In some embodiments, the heterodimer Fc-based chimeric protein complex does not contain a signaling molecule and a targeting moiety on a single polypeptide.

[0385] In some embodiments, Fc-based chimeric proteins have an improved in vivo half-life compared to chimeric proteins lacking Fc or chimeric proteins that are not heterodimer complexes. In some embodiments, Fc-based chimeric proteins have improved solubility, stability, and other pharmacological properties compared to chimeric proteins lacking Fc or chimeric proteins that are not heterodimer complexes.

[0386] A heterodimer Fc-based chimeric protein complex is composed of two different polypeptides. In some embodiments described herein, the targeting domain resides on a polypeptide different from the signaling molecule, and therefore the protein contains only one targeting domain copy and, similarly, only one signaling molecule. Furthermore, in some embodiments, only one targeting domain (e.g., VHH) can avoid crosslinking of antigens on the cell surface (which may induce undesirable effects). Furthermore, in embodiments, one signaling molecule can mitigate molecular "clustering" and, depending on the targeting domain, potential interference with avidity-mediated recovery of effector function. Furthermore, in some embodiments, the heterodimer Fc-based chimeric protein complex can have two targeting moieties, which can be located on two different polypeptides. For example, in some embodiments, the C-terminuses of both targeting moieties (e.g., VHH) can be masked to avoid potential autoantibodies or existing antibodies (e.g., VHH autoantibodies or existing antibodies). Furthermore, in some embodiments, heterodimer Fc-based chimeric protein complexes having a targeting domain on a polypeptide different from the signaling molecule may prioritize "crosslinking" between two cell types (e.g., tumor cells and immune cells). In addition, in some embodiments, the heterodimer Fc-based chimeric protein complexes each have two signaling molecules on different polypeptides, enabling more complex effector reactions.

[0387] Furthermore, in some embodiments, a heterodimer Fc-based chimeric protein complex having a targeting domain on a polypeptide different from the signaling molecule, and having diversity in the combination of the targeting moiety and the signaling molecule. For example, in some embodiments, a polypeptide having any of the targeting moieties described herein can be combined "as a ready-made product" with a polypeptide having any of the signaling molecules described herein to enable the rapid generation of various combinations of targeting moieties and signaling molecules in a single Fc-based chimeric protein complex.

[0388] In some embodiments, the Fc-based chimeric protein complex includes one or more linkers. In some embodiments, the Fc-based chimeric protein complex includes linkers connecting the Fc domain, a signaling molecule, and a targeting moiety(s). In some embodiments, the Fc-based chimeric protein complex includes linkers connecting each signaling molecule and a targeting moiety (or, in the case of two or more targeting moieties, the signaling molecules). In some embodiments, the Fc-based chimeric protein complex includes linkers connecting each signaling molecule to the Fc domain. In some embodiments, the Fc-based chimeric protein complex includes linkers ligating each targeting moiety to the Fc domain. In some embodiments, the Fc-based chimeric protein complex includes linkers ligating a targeting molecule to another targeting moiety. In some embodiments, the Fc-based chimeric protein complex includes linkers ligating one signaling molecule to another signaling molecule.

[0389] In some embodiments, the Fc-based chimeric protein complex includes two or more targeting regions. In such embodiments, the targeting regions may be the same or different.

[0390] In some embodiments, the Fc-based chimeric protein complex comprises two or more signaling molecules. In such embodiments, the signaling molecules may be the same targeting moiety or different targeting moieties.

[0391] For example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, at least two signaling molecules (SAs), and at least two targeting moieties (TMs), the Fc domain, signaling molecules, and targeting moieties being selected from any of the Fc domains, signaling molecules, and targeting moieties disclosed herein. In some embodiments, the Fc domain is a homodimer.

[0392] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 9A-F, 10A-H, 11A-H, 12A-D, 13A-F, 14A-J, 15A-D, 16A-F, 17A-J, 18A-F, 19A-L, 20A-L, 21A-F, 22A-L, 23A-L, 24A-J, 25A-J, 26A-F, and 27A-F.

[0393] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 9A to F.

[0394] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 10A to H.

[0395] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 11A to H.

[0396] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 12A to D.

[0397] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 13A to F.

[0398] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 14A to J.

[0399] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 15A to D.

[0400] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 16A to F.

[0401] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 17A to J.

[0402] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 18A to F.

[0403] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 19A to L.

[0404] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 20A to L.

[0405] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 21A to F.

[0406] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 22A to L.

[0407] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 23A-L.

[0408] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 24A to J.

[0409] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 25A to J.

[0410] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 26A to F.

[0411] In various embodiments, the Fc-based chimeric protein complex takes the form of one of the schematic diagrams shown in Figures 27A to F.

[0412] In some embodiments, the signaling molecule is ligated to a targeting moiety, and the targeting moiety is ligated to an Fc domain at the same end (see Figures 9A-F). In some embodiments, the Fc domain is homodimer.

[0413] In some embodiments, the signaling molecule and the targeting moiety are linked to the Fc domain, and the targeting moiety and the signaling molecule are linked at the same end (see Figures 9A-F). In some embodiments, the Fc domain is homodimer.

[0414] In some embodiments, the targeting portion is linked to a signaling molecule, and the signaling molecule is linked to an Fc domain at the same end (see Figures 9A-F). In some embodiments, the Fc domain is homodimer.

[0415] In some embodiments, the homodimer Fc-based chimeric protein complex contains two or more targeting moieties. In some embodiments, there are four targeting moieties and two signaling molecules, where the targeting moieties are linked to the Fc domain and the signaling molecules are linked to the targeting moieties at the same end (see Figures 10A-H). In some embodiments, the Fc domain is homodimer. In some embodiments with four targeting moieties and two signaling molecules, two targeting moieties are linked to the Fc domain and two targeting moieties are linked to the signaling molecules, and these are linked to the Fc domain at the same end (see Figures 10A-H). In some embodiments, the Fc domain is homodimer. In some embodiments with four targeting moieties and two signaling molecules, two targeting moieties are linked to each other, with one of the targeting moieties from each pair linked to the Fc domain at the same end and the signaling molecules linked to the Fc domain at the same end (see Figures 10A-H). In some embodiments, the Fc domain is homodimer. In some embodiments where there are four targeting moieties and two signaling molecules, the two targeting moieties are linked to each other, with one of the targeting moieties from each pair linked to a signaling molecule, and the other targeting moiety from this pair linked to the Fc domain, and the targeting moieties linked to the Fc domain are linked at the same end (see Figures 10A-H). In some embodiments, the Fc domain is homodimer.

[0416] In some embodiments, the homodimer Fc-based chimeric protein complex contains two or more signaling molecules. In some embodiments where there are four signaling molecules and two signaling molecules, the two signaling molecules are linked to each other, with one of the signaling molecules from the pair linked to the Fc domain at the same end, and the targeting moiety linked to the Fc domain at the same end (see Figures 11A-H). In some embodiments, the Fc domain is homodimer. In some embodiments where there are four signaling molecules and two signaling molecules, the two signaling molecules are linked to the Fc domain at the same end, with the two signaling molecules each linked to the targeting moiety, and the targeting moiety linked to the Fc domain at the same end (see Figures 11A-H). In some embodiments, the Fc domain is homodimer. In some embodiments where there are four signaling molecules and two signaling molecules, the two signaling molecules are linked to each other, with one of the signaling molecules from the pair linked to the targeting moiety, and the targeting moiety linked to the Fc domain at the same end (see Figures 11A-H). In some embodiments, the Fc domain is a homodimer.

[0417] For example, in some embodiments, the Fc-based chimeric protein complex comprises an Fc domain, the Fc domain comprising ion-pairing mutations and / or knob-in-hole mutations, at least one signaling molecule, and at least one targeting moiety, wherein the ion-pairing motif and / or knob-in-hole motif, signaling molecule, and targeting moiety are selected from any of the ion-pairing motif and / or knob-in-hole motif, signaling molecule, and targeting moiety disclosed herein. In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain comprises mutations that reduce or eliminate its effector function.

[0418] In some embodiments, the signaling molecule is ligated to a targeting moiety, which is ligated to an Fc domain (see Figures 18A-F and 19A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains mutations that reduce or eliminate its effector function.

[0419] In some embodiments, the signaling molecule and the targeting moiety are ligated to the Fc domain (see Figures 12A-D, 13A-D, 18A-F, and 19A-F). In some embodiments, the targeting moiety and the signaling molecule are ligated to different Fc chains on the same end (see Figures 12A-D and 15A-D). In some embodiments, the targeting moiety and the signaling molecule are ligated to different Fc chains on different ends (see Figures 12A-D and 15A-D). In some embodiments, the targeting moiety and the signaling molecule are ligated to the same Fc chain (see Figures 18A-F and 19A-F). In some embodiments, the Fc domain is a heterodimer. In some embodiments, the Fc domain contains mutations tha...

Claims

1. A PD-L1 targeting portion comprising one or more recognition domains, wherein the one or more recognition domains (i) Three complementarity determination regions (CDR1, CDR2, and CDR3), (a) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 2 or 5, (b) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (c) CDR3 contains an amino acid sequence selected from either SEQ ID NO: 4 or 7, or the three complementarity-determining regions, (ii) Containing an amino acid sequence having at least 90% sequence identity with Sequence ID No. 1, The PD-L1 targeting portion, wherein (i) or (ii) further comprises one or more mutations at positions D54 and G55 numbered relative to sequence number 1.

2. The PD-L1 targeting portion according to claim 1, further comprising one or more mutations at positions Q1, Q5, A14, A63, T74, K76, S79, K86, and Q110.

3. The PD-L1 targeting moiety according to claim 1 or 2, wherein the mutation is a substitution, and optionally the substitution is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), an aromatic, polar, positively charged hydrophilic residue including histidine (H), a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

4. The aforementioned mutation, Optionally, D54G is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position D54, or optionally, D54K is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), or optionally, D54T is a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), and The PD-L1 targeting moiety according to any one of claims 1 to 3, which is optionally G55R, and is selected from one or more polar and positively charged hydrophilic residues selected from arginine (R) and lysine (K) at position G55.

5. The aforementioned mutation, A polar, negatively charged hydrophilic residue at position Q1, which is optionally Q1D, selected from aspartate (D) and glutamate (E). A hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q5, which is optionally Q5V. A polar and neutrally charged hydrophilic residue at position A14, which is optionally A14P, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). A hydrophobic, aliphatic amino acid at position A63, which is optionally A63V, selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V). A polar and neutrally charged hydrophilic residue at position T74, which is optionally T74S, selected from asparagine (N), glutamine (Q), serine (S), proline (P), and cysteine ​​(C). A polar and neutrally charged hydrophilic residue at position K76, which is optionally K76N, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). A hydrophobic, aromatic amino acid at position S79, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y), is S79Y. The PD-L1 targeting moiety according to any one of claims 1 to 4, comprising K86R, which is arginine (R) at position K86, and optionally Q110L, which is one or more hydrophobic, aliphatic amino acids selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q110.

6. The PD-L1 targeting portion according to any one of claims 1 to 5, wherein the mutation is one or more of Q1D, Q5V, A14P, A63V, T74S, K76N, S79Y, K86R, and Q110L, or optionally selected from all of Q1D, Q5V, A14P, D54G, T74S, K76N, S79Y, K86R, and Q110L.

7. The targeting portion is a full-length antibody, a single-domain antibody, a recombinant heavy chain-only antibody (VHH), a single-chain antibody, a shark heavy chain-only antibody (VNAR), a microprotein, darpin, anticarin, adonectin, an aptamer, Fv, Fab, Fab', F(ab'). ２ The targeting portion is a peptide mimetic molecule, a native ligand for a receptor, or a synthetic molecule, and optionally the targeting portion is a variable domain heavy chain antibody (V Ｈ H) or humanized (V Ｈ The PD-L1 targeting portion according to any one of claims 1 to 6, including H).

8. The PD-L1 targeting portion according to any of the prior claims, wherein the targeting portion recognizes PD-L1, binds to it, and substantially functionally modulates its activity, or does not substantially functionally modulate its activity.

9. The PD-L1 targeting portion according to any of the prior claims, wherein the targeting portion recognizes and / or binds to the target without substantially neutralizing the activity of the target, or the targeting portion recognizes and / or binds to the target and substantially neutralizes the activity of the target.

10. The PD-L1 targeting portion according to any one of the prior claims, wherein the targeting portion includes one or more additional recognition domains.

11. The PD-L1 targeting moiety according to claim 10, wherein the one or more additional recognition domains bind to CD8, CD13, CD20, NKp46, Clec9A, Clec4c, PD-1, PD-L1, PD-L2, SIRP1α, FAP, XCR1, tenascin CA1, Flt3, or ECM protein.

12. The PD-L1 targeting portion according to any one of the prior claims, wherein the targeting portion recognizes a tumor antigen and optionally modulates it functionally.

13. The PD-L1 targeting portion according to any one of the prior claims, wherein the targeting portion recognizes an antigen on an immune cell and optionally modulates it functionally.

14. The PD-L1 targeting portion according to claim 13, wherein the immune cells are selected from T cells, B cells, dendritic cells, macrophages, neutrophils, NK cells, and NKT cells.

15. The PD-L1 targeting portion according to any of the prior claims, wherein the targeting portion recruits cytotoxic T cells to tumor cells or the tumor environment.

16. The PD-L1 targeting moiety according to any of the prior claims, further comprising one or more (a) wild-type signaling molecules, or (b) modified signaling molecules having reduced affinity or activity to the receptor of the wild-type signaling molecule.

17. The PD-L1 targeting portion according to claim 16, wherein the targeting portion restores the affinity or activity of the modified signaling molecule to the receptor of the signaling molecule.

18. The PD-L1 targeting portion according to claim 16, wherein the modification in the modified signaling molecule enables a reduction in activity.

19. The PD-L1 targeting portion according to claim 16, wherein the agonist or antagonist activity of the modified signaling molecule is attenuated.

20. The PD-L1 targeting portion according to claims 16 to 19, wherein the signaling molecule is selected from one or more of interferon, interleukin, and tumor necrosis factor, and any of these is optionally modified or mutated.

21. The PD-L1 targeting portion according to claim 20, wherein the signaling molecule is selected from human: IFNα2, IFNα1, IFNβ, IFNγ, consensus interferon, TNF, TNFR, TGF-α, TGF-β, VEGF, EGF, PDGF, FGF, TRAIL, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-33, IGF-1, or EPO.

22. The human IFNα2 is R33A, T106X ３ , R120E, R144X １ , A145X ２ , M148A, R149A, and L153A, and includes one or more mutations with respect to the amino acid sequence of SEQ ID NO: 81 or 82, where X １ is selected from A, S, T, Y, L, and I, and X ２ is selected from G, H, Y, K, and D, and X ３ is selected from A and E, the PD-L1 targeting moiety according to claim 21.

23. The aforementioned human IFNα1 is A146G, C86X １ , and M149X ２ Selected from, and containing one or more mutations with respect to the amino acid sequence of SEQ ID NO: 83, X １ However, selected from A, Y, and S, X ２ The PD-L1 targeting portion according to claim 21, selected from V and A.

24. The PD-L1 targeting moiety according to claim 21, wherein the human IFNβ comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, and R152G with respect to the amino acid sequence of SEQ ID NO:

84.

25. The aforementioned human IL-1β has the following amino acid sequences with respect to SEQ ID NO: 100: A117G / P118G, R120G, R120A, L122A, T125G / L126G, R127G, Q130A, Q130W, Q131G, K132A, S137G / Q138Y, L145G, H146A, H146G, H146E, H146N, H146R, L145A / L147A, Q148E, Q148G, Q148L, Q148G / Q150G, Q150G / D151A The PD-L1 targeting portion according to claim 21, comprising one or more mutations selected from M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209A, K209D, K209A / K210A, K219S, K219Q, E221S, E221K, E221S / N224A, N224S / K225S, E244K, and N245Q.

26. The PD-L1 targeting moiety according to claim 21, wherein the human IL-2 comprises one or more mutations selected from R38A, F42A, Y45A, E62A, N88R, N88I, N88G, D20H, Q126L, Q126F, D109, and C125 with respect to the amino acid sequence of SEQ ID NO:

101.

27. The PD-L1 targeting portion according to claim 21, wherein the human TNFα comprises one or more mutations selected from R32G, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, Y870, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145T, and Y87Q / I97A with respect to the amino acid sequence of SEQ ID NO:

97.

28. The PD-L1 targeting portion according to any of the prior claims, wherein the PD-L1 targeting portion binds to PD-L1 with improved affinity compared to the PD-L1 targeting portion of Sequence ID No.

1.

29. Fc-based chimeric protein complex, (A) The targeting part, (a) Three complementarity-determining regions (CDR1, CDR2, and CDR3), (i) CDR1 contains an amino acid sequence selected from either SEQ ID NO: 2 or 5, (ii) CDR2 contains an amino acid sequence selected from either SEQ ID NO: 3 or 6, and (iii) CDR3 contains an amino acid sequence selected from either SEQ ID NO: 4 or 7, or the three complementarity-determining regions, (b) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1, (a) or (b) further includes one or more mutations at positions D54 and G55 numbered relative to sequence number 1, the targeting portion, (B) A signaling molecule, a) Wild-type signaling molecules, or b) A modified signaling molecule having one or more mutations that confer improved safety to the wild-type signaling molecule, (C) The Fc-based chimeric protein complex comprising an Fc domain having one or more mutations that optionally reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain.

30. The Fc-based chimeric protein complex according to claim 29, wherein the targeting portion further comprises one or more mutations at positions Q1, Q5, A14, A63, T74, K76, S79, K86, and Q110.

31. The Fc-based chimeric protein complex according to claim 29 or 30, wherein the mutation is a substitution, and optionally the substitution is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), an aromatic, polar, positively charged hydrophilic residue including histidine (H), a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E), or a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

32. The aforementioned mutation, Optionally, D54G is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position D54, or optionally, D54K is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), or optionally, D54T is a polar, neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C), and An Fc-based chimeric protein complex according to any one of claims 29 to 31, wherein one or more polar and positively charged hydrophilic residues selected from arginine (R) and lysine (K) at position G55, which are optionally G55R.

33. The aforementioned mutation, A polar, negatively charged hydrophilic residue at position Q1, which is optionally Q1D, selected from aspartate (D) and glutamate (E). A hydrophobic, aliphatic amino acid selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q5, which is optionally Q5V. A polar and neutrally charged hydrophilic residue at position A14, which is optionally A14P, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). A hydrophobic, aliphatic amino acid at position A63, which is optionally A63V, selected from glycine (G), leucine (L), isoleucine (I), methionine (M), and valine (V). A polar and neutrally charged hydrophilic residue at position T74, which is optionally T74S, selected from asparagine (N), glutamine (Q), serine (S), proline (P), and cysteine ​​(C). A polar and neutrally charged hydrophilic residue at position K76, which is optionally K76N, selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C). A hydrophobic, aromatic amino acid at position S79, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y), is S79Y. K86R is arginine (R) at position K86, and An Fc-based chimeric protein complex according to any one of claims 29 to 32, optionally comprising one or more hydrophobic, aliphatic amino acids selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V) at position Q110, wherein Q110L is optionally selected.

34. The Fc-based chimeric protein complex according to any one of claims 29 to 33, wherein the mutation is one or more of Q1D, Q5V, A14P, A63V, T74S, K76N, S79Y, K86R, and Q110L, or optionally selected from all of Q1D, Q5V, A14P, D54G, T74S, K76N, S79Y, K86R, and Q110L.

35. The targeting portion is a full-length antibody, a single-domain antibody, a recombinant heavy chain-only antibody (VHH), a single-chain antibody, a shark heavy chain-only antibody (VNAR), a microprotein, darpin, anticarin, adonectin, an aptamer, Fv, Fab, Fab', F(ab'). ２ The targeting portion is a peptide mimetic molecule, a native ligand for a receptor, or a synthetic molecule, and optionally the targeting portion is a variable domain heavy chain antibody (V Ｈ H) or humanized (V Ｈ An Fc-based chimeric protein complex according to any one of claims 29 to 34, comprising H).

36. The Fc-based chimeric protein complex according to any one of claims 29 to 35, wherein the targeting portion recognizes PD-L1, binds to it, and substantially functionally modulates its activity, or does not substantially functionally modulate its activity.

37. The Fc-based chimeric protein complex according to any one of claims 29 to 35, wherein the targeting portion recognizes and / or binds to the target without substantially neutralizing the activity of the target, or the targeting portion recognizes and / or binds to the target and substantially neutralizes the activity of the target.

38. An Fc-based chimeric protein complex according to any one of claims 29 to 37, further comprising one or more additional targeting moieties.

39. The Fc-based chimeric protein complex according to claim 38, wherein the one or more additional targeting portions bind to CD8, CD13, CD20, NKp46, Clec9A, Clec4c, PD-1, PD-L1, PD-L2, SIRP1α, FAP, XCR1, tenascin CA1, Flt3, or ECM protein.

40. The Fc-based chimeric protein complex according to any one of claims 29 to 39, wherein the targeting portion recognizes a tumor antigen and optionally modulates it functionally.

41. The Fc-based chimeric protein complex according to any one of claims 29 to 40, wherein the targeting portion recognizes an antigen on an immune cell and optionally modulates it functionally.

42. The Fc-based chimeric protein complex according to claim 41, wherein the immune cells are selected from T cells, B cells, dendritic cells, macrophages, neutrophils, NK cells, and NKT cells.

43. The Fc-based chimeric protein complex according to any one of claims 29 to 42, wherein the targeting portion recruits cytotoxic T cells to tumor cells or the tumor environment.

44. The Fc-based chimeric protein complex according to claim 29, further comprising one or more linkers.

45. The Fc-based chimeric protein complex according to claim 44, wherein the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE.

46. The Fc-based chimeric protein complex according to claim 45, wherein the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

47. The Fc-based chimeric protein complex according to claim 46, wherein the Fc domain is selected from human IgG, IgA, IgD, IgM, or IgE.

48. The Fc-based chimeric protein complex according to claim 47, wherein human IgG is selected from human IgG1, IgG2, IgG3, or IgG4.

49. The Fc-based chimeric protein complex according to any one of claims 29 to 48, wherein the signaling molecule is a modified signaling molecule and has reduced affinity or activity for the receptor of the signaling molecule compared to a wild-type signaling molecule.

50. The Fc-based chimeric protein complex according to claim 49, wherein the signaling substance is a modified signaling substance, and the targeting portion restores the affinity or activity of the modified signaling substance to the receptor of the signaling substance.

51. The Fc-based chimeric protein complex according to any one of claims 29 to 50, wherein the Fc chain pair formation is promoted by ion pair formation and / or knob-in-hole pair formation.

52. The Fc-based chimeric protein complex according to any one of claims 29 to 51, wherein the one or more mutations in the Fc domain result in ion pairing between the Fc chains in the Fc domain.

53. The Fc-based chimeric protein complex according to any one of claims 29 to 52, wherein the one or more mutations in the Fc domain result in the formation of a knob-in-hole pair in the Fc domain.

54. The Fc-based chimeric protein complex according to any one of claims 29 to 53, wherein the one or more mutations in the Fc domain result in a reduction or removal of the effector function of the Fc domain.

55. The Fc-based chimeric protein complex according to any one of claims 29 to 54, wherein the complex is a homodimer or a heterodimer.

56. The Fc-based chimeric protein complex according to any one of claims 29 to 55, wherein the Fc-based chimeric protein complex has the structure and / or orientation shown in any one of Figures 9A to F, 10A to H, 11A to H, 12A to D, 13A to F, 14A to J, 15A to D, 16A to F, 17A to J, 18A to F, 19A to L, 20A to L, 21A to F, 22A to L, 23A to L, 24A to J, 25A to J, 26A to F, and 27A to F.

57. The Fc-based chimeric protein complex according to claim 56, wherein the Fc-based chimeric protein complex has the structure and / or orientation shown in Figure 15B.

58. The Fc-based chimeric protein complex according to any one of claims 29 to 57, wherein the Fc-based chimeric protein complex has a trans orientation / structure with respect to any targeting moieties and signaling molecules to each other and / or any targeting moieties and / or any signaling molecules to each other.

59. The Fc-based chimeric protein complex according to any one of claims 29 to 58, wherein the Fc-based chimeric protein complex has a cis orientation / structure with respect to any targeting moieties and signaling molecules to each other and / or any targeting moieties and / or any signaling molecules to each other.

60. The Fc-based chimeric protein complex according to any one of claims 29 to 59, wherein the Fc comprises L234A, L235A, and one additional mutation selected from the substitutions K322A, K322Q, D265A, P329G, and P331S in human IgG1, and the numbering is in accordance with EU regulations.

61. The Fc-based chimeric protein complex according to any one of claims 29 to 60, wherein the Fc contains an S228P substitution in human IgG4, and the numbering is based on EU regulations.

62. The Fc-based chimeric protein complex according to any one of claims 29 to 61, wherein the modified signaling molecule has reduced affinity or activity for the receptor of the signaling molecule compared to the wild-type signaling molecule.

63. The Fc-based chimeric protein complex according to claims 29 to 61, wherein the targeting portion restores the affinity or activity of the modified signaling molecule to the receptor of the signaling molecule.

64. The Fc-based chimeric protein complex according to claim 63, wherein the modification in the modified signaling molecule enables a reduction in activity.

65. The Fc-based chimeric protein complex according to claim 63, wherein the agonist or antagonist activity of the modified signaling molecule is attenuated.

66. The Fc-based chimeric protein complex according to claims 29 to 65, wherein the signaling molecule is selected from one or more of interferon, interleukin, and tumor necrosis factor, and any of these is optionally modified or mutated.

67. The Fc-based chimeric protein complex according to claim 66, wherein the signaling molecule is selected from human: IFNα2, IFNα1, IFNβ, IFNγ, consensus interferon, TNF, TNFR, TGF-α, TGF-β, VEGF, EGF, PDGF, FGF, TRAIL, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-33, IGF-1, or EPO.

68. The aforementioned human IFNα2 is R33A, T106X ３ , R120E, R144X １ A145X ２ Selected from M148A, R149A, and L153A, and containing one or more mutations with respect to the amino acid sequence of SEQ ID NO: 81 or 82, X １ However, it is selected from A, S, T, Y, L, and I, X ２ However, selected from G, H, Y, K, and D, X ３ The Fc-based chimeric protein complex according to claim 67, selected from A and E.

69. The aforementioned human IFNα1 is A146G, C86X １ , and M149X ２ Selected from, and containing one or more mutations with respect to the amino acid sequence of SEQ ID NO: 83, X １ However, selected from A, Y, and S, X ２ The Fc-based chimeric protein complex according to claim 67, selected from V and A.

70. The Fc-based chimeric protein complex according to claim 67, wherein the human IFNβ comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, and R152G with respect to the amino acid sequence of SEQ ID NO:

84.

71. The aforementioned human IL-1β has the following amino acid sequence with respect to SEQ ID NO: 100: A117G / P118G, R120G, R120A, L122A, T125G / L126G, R127G, Q130A, Q130W, Q131G, K132A, S137G / Q138Y, L145G, H146A, H146G, H146E, H146N, H146R, L145A / L147A, Q148E, Q148G, Q148L, Q148G / Q150G, Q150G / D151A, The Fc-based chimeric protein complex according to claim 67, comprising one or more mutations selected from M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209A, K209D, K209A / K210A, K219S, K219Q, E221S, E221K, E221S / N224A, N224S / K225S, E244K, and N245Q.

72. The Fc-based chimeric protein complex according to claim 67, wherein the human IL-2 comprises one or more mutations selected from R38A, F42A, Y45A, E62A, N88R, N88I, N88G, D20H, Q126L, Q126F, D109, and C125 with respect to the amino acid sequence of SEQ ID NO:

101.

73. The Fc-based chimeric protein complex according to claim 67, wherein the human TNFα comprises one or more mutations selected from R32G, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, Y870, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145T, and Y87Q / I97A with respect to the amino acid sequence of SEQ ID NO:

97.

74. The Fc-based chimeric protein complex according to claim 67, wherein the signaling molecule is a modified IFNα2 having the R149A mutation with respect to the amino acid sequence of SEQ ID NO: 81 or 82, at optional selection.

75. A PD-L1 targeting portion including a recognition domain, wherein the recognition domain is (i) Three complementarity determination regions (CDR1, CDR2, and CDR3), (a) CDR1 comprises an amino acid sequence selected from either SEQ ID NO: 27 or 30, (b) CDR2 comprises an amino acid sequence selected from either SEQ ID NO: 28 or 31, and (c) CDR3 contains an amino acid sequence selected from either SEQ ID NO: 29 or 32, or the three complementarity-determining regions, (ii) Containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, The PD-L1 targeting portion, wherein (i) or (ii) further comprises one or more mutations at positions N32, D33, and M97 numbered relative to sequence number 26.

76. The PD-L1 targeting portion according to claim 75, wherein the mutation is a substitution.

77. The PD-L1 targeting moiety according to claim 75, wherein the substitution is a polar, positively charged hydrophilic residue selected from arginine (R) and lysine (K), or an aromatic, polar, positively charged hydrophilic residue containing histidine (H).

78. The PD-L1 targeting moiety according to claim 75, wherein the substitution is a polar and neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C).

79. The PD-L1 targeting moiety according to claim 75, wherein the substitution is a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E).

80. The PD-L1 targeting moiety according to claim 75, wherein the substitution is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

81. The PD-L1 targeting moiety according to claim 75, wherein the substitution at position N32 is a positive hydrophilic residue selected from arginine (R) and lysine (K).

82. The PD-L1 targeting moiety according to claim 75, wherein the substitution at position N32 is a polar and neutral hydrophilic residue selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C).

83. The PD-L1 targeting portion according to claim 81 or 82, wherein the substitution of position N32 is N32Q or N32R.

84. The PD-L1 targeting portion according to claim 75, wherein the substitution of position D33 is D33H.

85. The PD-L1 targeting moiety according to claim 75, wherein the substitution at position M97 is an aliphatic hydrophobic residue selected from glycine (G), leucine (L), isoleucine (I), and valine (V).

86. The PD-L1 targeting portion according to claim 85, wherein the substitution of position M97 is M97I, M97L, or M97V.

87. The PD-L1 targeting portion according to any one of claims 75 to 86, comprising one or more of the following mutations: Q1D, Q5V, A14P, A62S, A74S, M77T, M78V, K86R, and Q109L, and optionally further comprising all of Q1D, Q5V, A14P, D33H, A62S, A74S, M77T, M78V, K86R, M97V, and Q109L.

88. The targeting portion is a full-length antibody, a single-domain antibody, a recombinant heavy chain-only antibody (VHH), a single-chain antibody, a shark heavy chain-only antibody (VNAR), a microprotein, darpin, anticarin, adonectin, an aptamer, Fv, Fab, Fab', F(ab'). ２ The PD-L1 targeting moiety according to any one of claims 75 to 87, which is a peptide-mimicking molecule, a natural ligand for a receptor, or a synthetic molecule.

89. The targeting portion is a variable domain heavy chain antibody (V Ｈ H) or humanized V Ｈ The PD-L1 targeting portion according to claim 88, including H.

90. The PD-L1 targeting portion according to any one of claims 75 to 89, wherein the targeting portion recognizes PD-L1, binds to it, substantially functionally modulates its activity, or does not substantially functionally modulate its activity.

91. The PD-L1 targeting portion according to any one of claims 75 to 90, wherein the targeting portion recognizes and / or binds to the target without substantially neutralizing the activity of the target, or the targeting portion recognizes and / or binds to the target and substantially neutralizes the activity of the target.

92. The PD-L1 targeting portion according to any one of claims 75 to 91, wherein the targeting portion includes one or more additional recognition domains.

93. The PD-L1 targeting moiety according to claim 92, wherein the one or more additional recognition domains bind to CD8, CD13, CD20, NKp46, Clec9A, Clec4c, PD-1, PD-L1, PD-L2, SIRP1α, FAP, XCR1, tenascin CA1, Flt3, or ECM protein.

94. The PD-L1 targeting portion according to any one of claims 75 to 93, wherein the recognition domain recognizes a tumor antigen and optionally modulates it functionally.

95. The PD-L1 targeting portion according to any one of claims 75 to 94, wherein the targeting portion recognizes an antigen on an immune cell and optionally modulates it functionally.

96. The PD-L1 targeting portion according to claim 95, wherein the immune cells are selected from T cells, B cells, dendritic cells, macrophages, neutrophils, NK cells, and NKT cells.

97. The PD-L1 targeting portion according to any one of claims 75 to 96, wherein the targeting portion recruits cytotoxic T cells to tumor cells or the tumor environment.

98. The PD-L1 targeting moiety according to any one of claims 75 to 97, further comprising one or more (a) wild-type signaling molecules, or (b) modified signaling molecules having reduced affinity or activity to the receptor of the wild-type signaling molecule.

99. The PD-L1 targeting portion according to claim 98, wherein the targeting portion restores the affinity or activity of the modified signaling molecule to the receptor of the signaling molecule.

100. The PD-L1 targeting portion according to claim 98, wherein the modification in the modified signaling molecule enables a reduction in activity.

101. The PD-L1 targeting portion according to claim 98, wherein the agonist or antagonist activity of the modified signaling molecule is attenuated.

102. The PD-L1 targeting moiety according to claims 98 to 101, wherein the signaling molecule is selected from one or more of interferon, interleukin, and tumor necrosis factor, and any of these is optionally modified or mutated.

103. The PD-L1 targeting portion according to claim 102, wherein the signaling molecule is selected from human: IFNα2, IFNα1, IFNβ, IFNγ, consensus interferon, TNF, TNFR, TGF-α, TGF-β, VEGF, EGF, PDGF, FGF, TRAIL, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-33, IGF-1, or EPO.

104. The aforementioned human IFNα2 is R33A, T106X ３ , R120E, R144X １ A145X ２ Selected from M148A, R149A, and L153A, and containing one or more mutations with respect to the amino acid sequence of SEQ ID NO: 81 or 82, X １ However, it is selected from A, S, T, Y, L, and I, X ２ However, selected from G, H, Y, K, and D, X ３ The PD-L1 targeting portion according to claim 103, selected from A and E.

105. The aforementioned human IFNα1 is A146G, C86X １ , and M149X ２ Selected from, and containing one or more mutations with respect to the amino acid sequence of SEQ ID NO: 83, X １ However, selected from A, Y, and S, X ２ The PD-L1 targeting portion according to claim 103, selected from V and A.

106. The PD-L1 targeting moiety according to claim 103, wherein the human IFNβ comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, and R152G with respect to the amino acid sequence of SEQ ID NO:

84.

107. The aforementioned human IL-1β has the following amino acid sequences with respect to SEQ ID NO: 100: A117G / P118G, R120G, R120A, L122A, T125G / L126G, R127G, Q130A, Q130W, Q131G, K132A, S137G / Q138Y, L145G, H146A, H146G, H146E, H146N, H146R, L145A / L147A, Q148E, Q148G, Q148L, Q148G / Q150G, Q150G / D151A The PD-L1 targeting portion according to claim 103, comprising one or more mutations selected from M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209A, K209D, K209A / K210A, K219S, K219Q, E221S, E221K, E221S / N224A, N224S / K225S, E244K, and N245Q.

108. The PD-L1 targeting moiety according to claim 103, wherein the human IL-2 comprises one or more mutations selected from R38A, F42A, Y45A, E62A, N88R, N88I, N88G, D20H, Q126L, Q126F, D109, and C125 with respect to the amino acid sequence of SEQ ID NO:

101.

109. The PD-L1 targeting moiety according to claim 103, wherein the human TNFα comprises one or more mutations selected from R32G, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, Y870, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145T, and Y87Q / I97A with respect to the amino acid sequence of SEQ ID NO:

97.

110. The PD-L1 targeting portion according to any one of claims 75 to 109, wherein the PD-L1 targeting portion binds to PD-L1 with improved affinity compared to the PD-L1 targeting portion of Sequence ID No.

26.

111. Fc-based chimeric protein complex, (A) The targeting part, (a) Three complementarity-determining regions (CDR1, CDR2, and CDR3), (i) CDR1 contains an amino acid sequence selected from either SEQ ID NO: 27 or 30, (ii) CDR2 contains an amino acid sequence selected from either SEQ ID NO: 28 or 31, and (iii) CDR3 contains an amino acid sequence selected from either SEQ ID NO: 29 or 32, or the three complementarity-determining regions, (b) comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 26, (a) or (b) further includes one or more mutations at positions N32, D33, and M97 numbered with respect to sequence number 26, the targeting portion, (B) A signaling molecule, a) Wild-type signaling molecules, or b) A modified signaling molecule having one or more mutations that confer improved safety to the wild-type signaling molecule, (C) The Fc-based chimeric protein complex comprising an Fc domain having one or more mutations that optionally reduce or eliminate one or more effector functions of the Fc domain, promote Fc chain pair formation in the Fc domain, and / or stabilize a hinge region in the Fc domain.

112. The Fc-based chimeric protein complex according to claim 111, wherein the mutation is a substitution.

113. The Fc-based chimeric protein complex according to claim 111, wherein the substitution is a hydrophilic amino acid residue which is a polar and positively charged hydrophilic residue selected from arginine (R) and lysine (K), or an aromatic, polar and positively charged hydrophilic residue which contains histidine (H).

114. The Fc-based chimeric protein complex according to claim 111, wherein the substitution is a hydrophilic amino acid residue, which is a polar and neutrally charged hydrophilic residue selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C).

115. The Fc-based chimeric protein complex according to claim 111, wherein the substitution is a polar, negatively charged hydrophilic residue selected from aspartate (D) and glutamate (E).

116. The Fc-based chimeric protein complex according to claim 111, wherein the substitution is a hydrophobic, aliphatic amino acid selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), or a hydrophobic, aromatic amino acid selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).

117. The Fc-based chimeric protein complex according to claim 111, wherein the substitution at position N32 is a positive hydrophilic residue selected from arginine (R) and lysine (K).

118. The Fc-based chimeric protein complex according to claim 111, wherein the substitution at position N32 is a polar and neutral hydrophilic residue selected from glutamine (Q), serine (S), threonine (T), proline (P), and cysteine ​​(C).

119. The Fc-based chimeric protein complex according to claim 111, wherein the substitution at position N32 is N32Q or N32R.

120. The Fc-based chimeric protein complex according to claim 111, wherein the substitution at position D33 is D33H.

121. The Fc-based chimeric protein complex according to claim 111, wherein the substitution at position M97 is an aliphatic hydrophobic residue selected from glycine (G), leucine (L), isoleucine (I), and valine (V).

122. The Fc-based chimeric protein complex according to claim 111, wherein the substitution at position M97 is M97I, M97L, or M97V.

123. An Fc-based chimeric protein complex according to any one of claims 111 to 122, comprising one or more of the following mutations: Q1D, Q5V, A14P, A62S, A74S, M77T, M78V, K86R, and Q109L, and optionally all of Q1D, Q5V, A14P, D33H, A62S, A74S, M77T, M78V, K86R, and M97V.

124. The targeting portion is a full-length antibody, a single-domain antibody, a recombinant heavy chain-only antibody (VHH), a single-chain antibody, a shark heavy chain-only antibody (VNAR), a microprotein, darpin, anticarin, adonectin, an aptamer, Fv, Fab, Fab', F(ab'). ２ The Fc-based chimeric protein complex according to any one of claims 111 to 123, wherein the complex is a peptide-mimicking molecule, a natural ligand for a receptor, or a synthetic molecule.

125. The targeting portion is a variable domain heavy chain antibody (V Ｈ H) or humanized V Ｈ The Fc-based chimeric protein complex according to claim 124, comprising H.

126. The Fc-based chimeric protein complex according to any one of claims 111 to 125, wherein the targeting portion recognizes PD-L1, binds to it, and substantially functionally modulates its activity, or does not substantially functionally modulate its activity.

127. The Fc-based chimeric protein complex according to any one of claims 111 to 126, wherein the targeting portion recognizes and / or binds to the target without substantially neutralizing the activity of the target, or the targeting portion recognizes and / or binds to the target and substantially neutralizes the activity of the target.

128. An Fc-based chimeric protein complex according to any one of claims 111 to 127, further comprising one or more additional targeting moieties.

129. The Fc-based chimeric protein complex according to claim 128, wherein the one or more additional targeting portions bind to CD8, CD13, CD20, NKp46, Clec9A, Clec4c, PD-1, PD-L1, PD-L2, SIRP1α, FAP, XCR1, tenascin CA1, Flt3, or ECM protein.

130. The Fc-based chimeric protein complex according to any one of claims 111 to 129, wherein the recognition domain recognizes a tumor antigen and optionally modulates it functionally.

131. The Fc-based chimeric protein complex according to any one of claims 111 to 129, wherein the targeting portion recognizes an antigen on an immune cell and optionally modulates it functionally.

132. The Fc-based chimeric protein complex according to claim 131, wherein the immune cells are selected from T cells, B cells, dendritic cells, macrophages, neutrophils, NK cells, and NKT cells.

133. The Fc-based chimeric protein complex according to any one of claims 111 to 132, wherein the targeting portion recruits cytotoxic T cells to tumor cells or the tumor environment.

134. The Fc-based chimeric protein complex according to claim 133, further comprising one or more linkers.

135. The Fc-based chimeric protein complex according to claim 111, wherein the Fc domain is selected from IgG, IgA, IgD, IgM, or IgE.

136. The Fc-based chimeric protein complex according to claim 135, wherein the IgG is selected from IgG1, IgG2, IgG3, or IgG4.

137. The Fc-based chimeric protein complex according to claim 111, wherein the Fc domain is selected from human IgG, IgA, IgD, IgM, or IgE.

138. The Fc-based chimeric protein complex according to claim 137, wherein human IgG is selected from human IgG1, IgG2, IgG3, or IgG4.

139. The Fc-based chimeric protein complex according to any one of claims 108 to 135, wherein the signaling molecule is a modified signaling molecule and has reduced affinity or activity for the receptor of the signaling molecule compared to a wild-type signaling molecule.

140. The Fc-based chimeric protein complex according to claim 139, wherein the signaling substance is a modified signaling substance, and the targeting portion restores the affinity or activity of the modified signaling substance to the receptor of the signaling substance.

141. The Fc-based chimeric protein complex according to any one of claims 111 to 140, wherein the Fc chain pair formation is promoted by ion pair formation and / or knob-in-hole pair formation.

142. The Fc-based chimeric protein complex according to any one of claims 111 to 141, wherein the one or more mutations in the Fc domain result in ion pairing between the Fc chains in the Fc domain.

143. The Fc-based chimeric protein complex according to any one of claims 111 to 142, wherein the one or more mutations in the Fc domain result in the formation of a knob-in-hole pair in the Fc domain.

144. The Fc-based chimeric protein complex according to any one of claims 111 to 143, wherein the one or more mutations in the Fc domain result in a reduction or removal of the effector function of the Fc domain.

145. The Fc-based chimeric protein complex according to any one of claims 111 to 144, wherein the complex is a homodimer or a heterodimer.

146. The Fc-based chimeric protein complex according to any one of claims 111 to 145, wherein the Fc-based chimeric protein complex has the structure and / or orientation shown in any one of Figures 9A to F, 10A to H, 11A to H, 12A to D, 13A to F, 14A to J, 15A to D, 16A to F, 17A to J, 18A to F, 19A to L, 20A to L, 21A to F, 22A to L, 23A to L, 24A to J, 25A to J, 26A to F, and 27A to F.

147. The Fc-based chimeric protein complex according to claim 146, wherein the Fc-based chimeric protein complex has the structure and / or orientation shown in Figure 15B.

148. The Fc-based chimeric protein complex according to any one of claims 111 to 147, wherein the Fc-based chimeric protein complex has a trans orientation / structure with respect to any targeting moieties and signaling molecules to each other and / or any targeting moieties and / or any signaling molecules to each other.

149. The Fc-based chimeric protein complex according to any one of claims 111 to 147, wherein the Fc-based chimeric protein complex has a cis orientation / structure with respect to any targeting moieties and signaling molecules to each other and / or any targeting moieties and / or any signaling molecules to each other.

150. The Fc-based chimeric protein complex according to any one of claims 111 to 149, wherein the Fc comprises L234A, L235A, and one additional mutation selected from the substitutions K322A, K322Q, D265A, P329G, and P331S in human IgG1, and the numbering is in accordance with EU regulations.

151. The Fc-based chimeric protein complex according to any one of claims 111 to 150, wherein the Fc contains an S228P substitution in human IgG4, and the numbering is in accordance with EU regulations.

152. The Fc-based chimeric protein complex according to any one of claims 111 to 151, wherein the signaling molecule has reduced affinity or activity for the receptor of the signaling molecule compared to a wild-type signaling molecule.

153. The Fc-based chimeric protein complex according to any one of claims 111 to 153, wherein the targeting portion restores the affinity or activity of the modified signaling molecule to the receptor of the signaling molecule.

154. The Fc-based chimeric protein complex according to claim 153, wherein the modification in the modified signaling molecule enables a reduction in activity.

155. The Fc-based chimeric protein complex according to claim 153, wherein the agonist or antagonist activity of the modified signaling molecule is attenuated.

156. The Fc-based chimeric protein complex according to claims 111 to 155, wherein the signaling molecule is selected from one or more of interferon, interleukin, and tumor necrosis factor, and any of these is optionally modified or mutated.

157. The Fc-based chimeric protein complex according to claim 156, wherein the signaling molecule is selected from human: IFNα2, IFNα1, IFNβ, IFNγ, consensus interferon, TNF, TNFR, TGF-α, TGF-β, VEGF, EGF, PDGF, FGF, TRAIL, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-33, IGF-1, or EPO.

158. Human IFNα2, R33A, T106X ３ , R120E, R144X １ A145X ２ Selected from M148A, R149A, and L153A, and containing one or more mutations with respect to the amino acid sequence of SEQ ID NO: 81 or 82, X １ However, it is selected from A, S, T, Y, L, and I, X ２ However, selected from G, H, Y, K, and D, X ３ The Fc-based chimeric protein complex according to claim 156, selected from A and E.

159. Human IFNα1, A146G, C86X １ , and M149X ２ Selected from, and containing one or more mutations with respect to the amino acid sequence of SEQ ID NO: 83, X １ However, selected from A, Y, and S, X ２ The Fc-based chimeric protein complex according to claim 156, selected from V and A.

160. The Fc-based chimeric protein complex according to claim 156, wherein the human IFNβ comprises one or more mutations selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, and R152G with respect to the amino acid sequence of SEQ ID NO:

84.

161. The aforementioned human IL-1β has the following amino acid sequence with respect to SEQ ID NO: 100: A117G / P118G, R120G, R120A, L122A, T125G / L126G, R127G, Q130A, Q130W, Q131G, K132A, S137G / Q138Y, L145G, H146A, H146G, H146E, H146N, H146R, L145A / L147A, Q148E, Q148G, Q148L, Q148G / Q150G, Q150G / D151A, The Fc-based chimeric protein complex according to claim 156, comprising one or more mutations selected from M152G, F162A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209A, K209D, K209A / K210A, K219S, K219Q, E221S, E221K, E221S / N224A, N224S / K225S, E244K, and N245Q.

162. The Fc-based chimeric protein complex according to claim 156, wherein the human IL-2 comprises one or more mutations selected from R38A, F42A, Y45A, E62A, N88R, N88I, N88G, D20H, Q126L, Q126F, D109, and C125 with respect to the amino acid sequence of SEQ ID NO:

101.

163. The Fc-based chimeric protein complex according to claim 156, wherein the human TNFα comprises one or more mutations selected from R32G, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, Y870, Y87L, Y87A, Y87F, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145T, and Y87Q / I97A with respect to the amino acid sequence of SEQ ID NO:

97.

164. The Fc-based chimeric protein complex according to claim 156, wherein the signaling molecule is a modified IFNα2 having the R149A mutation with respect to the amino acid sequence of SEQ ID NO: 1 or 2, at any selection.

165. A recombinant nucleic acid composition encoding the PD-L1 targeting moiety according to any one of claims 1 to 28 and 75 to 110.

166. A host cell containing the nucleic acid described in claim 165.

167. The PD-L1 targeting portion according to any one of claims 1 to 28 and 75 to 110, wherein the targeting portion is suitable for use in patients having one or more of cancer, infectious diseases, immune disorders, and / or autoimmune diseases.

168. A method for treating or preventing cancer, which provides an effective amount to patients who need it. (a) The targeting portion according to any one of claims 1 to 28 and 75 to 110, or (b) The method comprising administering an Fc-based chimeric protein complex according to any one of claims 29 to 74 and 111 to 164.

169. The aforementioned cancers include basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatoma, neoplasm in situ, kidney cancer or renal cancer. Lymphomas including cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancers, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, abdominal cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancers, vulvar cancer, Hodgkin lymphoma and non-Hodgkin lymphoma, and lymphomas including B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse lymphoma) The method according to claim 168, comprising one or more selected from: sexual NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-incisional nuclear cell NHL, giant mass lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis associated with nevus, edema (e.g., associated with brain tumors), and Meigs syndrome.

170. A method for treating or preventing autoimmune diseases and / or neurodegenerative diseases, which provides an effective amount to patients in need. (a) The targeting portion according to any one of claims 1 to 28 and 75 to 110, or (b) The method comprising administering an Fc-based chimeric protein complex according to any one of claims 29 to 74 and 111 to 164.

171. The method according to claim 170, wherein the autoimmune disease and / or neurodegenerative disease is selected from multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barré syndrome, scleroderma, Goodpasture syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, primary sclerosing cholangitis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Meniere's syndrome, transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, lupus erythematosus, myasthenia gravis, Reiter's syndrome, and Graves' disease.

172. The method according to claim 171, wherein the autoimmune disease and / or neurodegenerative disease is multiple sclerosis.

173. Use of the targeting portion according to any one of claims 1 to 28 and 75 to 110 for the treatment or prevention of autoimmune diseases, neurodegenerative diseases, metabolic diseases, and / or cardiovascular diseases.

174. Use of the targeting portion according to any one of claims 1 to 28 and 75 to 110 for the preparation of a drug for the treatment of the prevention of autoimmune diseases, neurodegenerative diseases, metabolic diseases, and / or cardiovascular diseases.

175. A PD-L1 targeting moiety comprising an amino acid sequence having at least 90% sequence identity with one of the amino acid sequences selected from SEQ ID NOs: 1, 8-26, and 33-74.

176. A method for treating or preventing cancer, comprising administering an effective amount of the PD-L1 targeting portion described in claim 175 to a patient in need.

177. A method for treating or preventing an autoimmune disease, a neurodegenerative disease, a metabolic disease, and / or a cardiovascular disease, comprising administering an effective amount of the PD-L1 targeting portion described in claim 175 to a patient in need.

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