Immunoantigen-specific IL-18 immunocytokine and uses thereof

Abstract

The present disclosure relates to modified immunocytokine compositions comprising an antibody or antigen-binding fragment specific for an immune cell antigen and an IL-18 polypeptide. Methods of treatment with the immunocytokine compositions and methods of making the immunocytokine compositions are also provided herein.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 313,222, filed February 23, 2022, and U.S. Provisional Application No. 63 / 479,529, filed January 11, 2023, which applications are incorporated by reference herein in their entireties. [Background technology]

[0002] In 2022, approximately 1.8 million new cases of cancer will be diagnosed in the United States, and more than 600,000 people will die from cancer. Immunotherapies harness a subject's immune system to help treat the disease. Depending on the nature of the disease being treated, immunotherapies can be designed to activate or suppress the immune system. The goal of various immunotherapies for the treatment of cancer is to stimulate the immune system so that it recognizes and destroys tumors or other cancerous tissues.

[0003] Immune cells involved in the response to various cancers express certain proteins involved in regulating the immune response. These proteins, e.g., programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), etc., can downregulate the immune system and promote self-tolerance by suppressing T cell inflammatory activity. In light of these mechanisms, antibodies or antigen-binding fragments targeting these proteins have been identified as potential therapeutics. However, in some cases, single-mechanism therapies targeting only these proteins are insufficient for the treatment of cancer. Therefore, improved tools for cancer therapy are needed. Summary of the Invention

[0004] Described herein is an immunocytokine composition comprising an immune cell-associated antigen-specific antibody or antigen-binding fragment thereof and an interleukin-18 (IL-18) polypeptide.

[0005] In one aspect, provided herein is an immunocytokine composition comprising an IL-18 polypeptide, and an antibody or antigen-binding fragment thereof specific for an immune cell-associated antigen, and a linker, wherein the linker comprises a first point of attachment to the IL-18 polypeptide and a second point of attachment to the antibody or antigen-binding fragment thereof.

[0006] In some embodiments, the linker comprises a polymer. In some embodiments, the polymer comprises a water-soluble polymer. In some embodiments, the water-soluble polymer comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof. In some embodiments, the polymer has a weight average molecular weight of at least about 0.1 kDa, at least about 0.5 kDa, at least about 1 kDa, at least about 5 kDa, at least about 10 kDa, at least about 20 kDa, at least about 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, at least 120 kDa, at least 130 kDa, at least 140 kDa, at least 150 kDa, or more.

[0007] In some embodiments, the first point of attachment is at a residue that is neither N-terminal nor C-terminal of the IL-18 polypeptide. In some embodiments, the first point of attachment is to a residue within the region of residues 2-156 of the IL-18 polypeptide, where the numbering of residue positions is based on SEQ ID NO:1. In some embodiments, the first point of attachment is to a residue within the region of residues 38-144 of the IL-18 polypeptide. In some embodiments, the first point of attachment is at residue 38, 68, 69, 70, 76, 78, 85, 86, 95, 98, 121, 127, or 144 of the IL-18 polypeptide. In some embodiments, the first point of attachment is at residue 68, 69, 70, 85, 86, 95, or 98 of the IL-18 polypeptide. In some embodiments, the first point of attachment is at residue 68 of the IL-18 polypeptide. In some embodiments, the IL-18 polypeptide comprises an amino acid substitution at the first point of attachment. In some embodiments, the amino acid substitution at the first attachment point is to a non-natural amino acid. In some embodiments, the amino acid substitution at the first attachment point is to a natural amino acid. In some embodiments, the amino acid substitution at the first attachment point is E69C, K70C, E85C, M86C, T95C, or D98C.

[0008] In some embodiments, the second point of attachment is at an amino acid residue in the Fc region of the antibody or antigen-binding fragment. In some embodiments, the Fc region comprises an amino acid sequence having 90% or greater identity to the amino acid sequence of SEQ ID NO:260. In some embodiments, the second point of attachment is (a) amino acid residues 16-18 of SEQ ID NO:260, (b) amino acid residues 58-60 of SEQ ID NO:260, or (c) amino acid residue 87 of SEQ ID NO:260. In some embodiments, the second point of attachment is to a lysine residue on the antibody or antigen-binding fragment. In some embodiments, the IL-18 polypeptide exhibits reduced binding to IL-18 binding protein (IL-18BP) compared to the wild-type IL-18 polypeptide of SEQ ID NO:1. In some embodiments, the IL-18 polypeptide contains one or more amino acid substitutions at residue positions selected from Y01, F02, E06, V11, C38, K53, D54, S55, T63, C76, and C127, where the numbering of residue positions of the IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the one or more amino acid substitutions in the IL-18 polypeptide are selected from Y01G, F02A, E06K, V11I, C38S, C38A, K53A, D54A, S55A, T63A, C68S, C68A, C76S, C76A, C127S, and C127A. In some embodiments, the IL-18 polypeptide contains E06K and K53A amino acid substitutions. In some embodiments, the IL-18 polypeptide contains a T63A amino acid substitution. In some embodiments, the IL-18 polypeptide is synthetic.In some embodiments, the IL-18 polypeptide contains (a) a homoserine residue located at any one of residues 26-36; (b) a homoserine residue located at any one of residues 45-67; ​​(c) a homoserine residue located at any one of residues 70-80; (d) a homoserine residue located at any one of residues 110-130; (e) a norleucine or O-methyl-homoserine residue located at any one of residues 28-38; (f) a norleucine or O-methyl-homoserine residue located at any one of residues 46-56; (g) a norleucine or O-methyl-homoserine residue located at any one of residues 50-56; (h) a norleucine or O-methyl-homoserine residue located at any one of residues 80 to 90; (i) a norleucine or O-methyl-homoserine residue located at any one of residues 108 to 118; and (j) a norleucine or O-methyl-homoserine residue located at any one of residues 145 to 155, wherein the numbering of the residue positions of the IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the IL-18 polypeptide comprises one or more amino acid substitutions selected from homoserine (Hse)31, norleucine (Nle)33, O-methyl-homoserine (Omh)33, Hse50, Nle51, Omh51, Hse57, Nle60, Omh60, Hse63, Hse67, Hse75, Nle86, Omh86, Hse106, Nle113, Omh113, Hse116, Hse121, Nle150, and Omh150.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody, a humanized antibody, a grafted antibody, a chimeric antibody, a human antibody, a deimmunized antibody, or a bispecific antibody. In some embodiments, the antibody or antigen-binding fragment thereof, the antigen-binding fragment thereof, is a Fab, Fab', F(ab')2, bispecific F(ab')2, trispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), dsFv, bispecific scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, AVIMER®, minibody, diabody, bispecific diabody, triabody, tetrabody ... The antibody or antigen-binding fragment thereof may be a body, maxibody, camelid, VHH, minibody, intrabody, fusion protein comprising an antibody portion (domain antibody), single chain binding polypeptide, scFv-Fc, Fab-Fc, bispecific T cell engager (BiTE), tetravalent tandem diabody (TandAb), dual affinity retargeting antibody (DART), bispecific antibody (bscAb), single domain antibody (sdAb), fusion protein, or bispecific disulfide stabilized Fv antibody fragment (dsFv-dsFv'). In some embodiments, the antibody or antigen-binding fragment thereof comprises or is derived from IgG, IgM, IgE, IgA, IgD. In some embodiments, the antibody or antigen-binding fragment thereof comprises the IgG, and the IgG is or is derived from IgG1, IgG4. In some embodiments, the immune cell associated antigen is 4-IBB, B7-H3, B7-H4, BTLA, CD3, CCR8, CD8A, CD8B, CD16A, CD27, CD28, CD33, CD38, CD39, CD40, CD47, CD70, CD80, CD86, CD96, CD163, CLEC-1, CLEVER-1, CTLA-4, D40, GITR, ICOS, ILT2 / 3 / 4, LAG-3, MHCI, MHCII, NKG2A, NKG2D, NKp30, NKp44, NKp46, OX40, PD-1, PD-L1, PD-L2, PSGL-1, SIGLEC-9, SIGLEC-15, SIRP-alpha, TCR, TIGIT, TIM-3, VISTA, or VSIG4.

[0010] In some embodiments, the immune cell associated antigen is PD-1. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of tislelizumab, baizean, 0KVO411B3N, BGB-A317, hu317-1 / IgG4mt2, sintilimab, tyvyt, IBI-308, toripalimab, TeRuiPuLi, terepyril, tuoyi, JS-001, TAB-001, tamrelizumab, HR-301210, INCSHR-01210, SHR-1210, temiprimab, cemiplimab-rwlc, 6QVL057INT, H4H7798N, REGN-2810, SAR-43 9684, lambrolizumab, pembrolizumab, MK-3475, SCH-900475, h409A11, nivolumab, nivolumabBMS, BMS-936558, MDX-1106, ONO-4538, prorugolimab, forteca, BCD-100, penprimab, AK-105, zimberelimab, AB-122, GLS-010, WBP-3055, balstilimab, 1Q2QT5M7EO, AGEN-2034, AGEN-2034w, genolimuzumab, geptanolimab, APL-501, CBT-501, GB-226, doxorubicin, stallimab, ANB-011, GSK-4057190A, P0GVQ9A4S5, TSR-042, WBP-285, serplulimab, HLX-10, CS-1003, retifanlimab, 2Y3T5IF01Z, INCMGA-00012, INCMGA-0012, MGA-012, sasanlimab, LZZ0IC2EWP, PF-06801591, RN-888, spartalizumab, PDR-001, QOG25L6Z8Z, leratolimab / nivolumab, BMS-986213, cetrelimab, JNJ-3283, JNJ-63 723283, LYK98WP91F, tevotelimab, MGD-013, BCD-217, BAT-1306, HX-008, MEDI-5752, JTX-4014, kadonilimab, AK-104, BI-754091, pidilizumab, CT-011, MDV-9300, YBL-006, AMG-256, RG-6279, RO-7284755, BH-2950, ​​IBI-315, RG-6139, RO-7247669, ONO-4685, AK-112, 609-A, LY-3434172, T-3011, AMG-404,IBI-318, MGD-019, ONCR-177, LY-3462817, RG-7769, RO-7121661, F-520, XmAb-23104, Pd-1-pik, SG-001, S-95016, Sym-021, LZM-009, budigalimab, 6VDO4TY3OO, ABBV-181, PR-1648817, CC-90006, XmAb-2071 7, 2661380, AMP-224, B7-DCIg, EMB-02, ANB-030, PRS-332, [89Zr]deferoxamide-pembrolizumab, 89Zr-Df-pembrolizumab, [89Zr]Df-pembrolizumab, STI-1110, STI-A1110, CX-188, mPD-1, MCLA-134, 244C8, ENUM224C8, ENUM C8, 388D4, ENUM 388D4, ENUM D4, MEDI0680, NVP-LZV-184, or AMP-514, or modified forms thereof. In some embodiments, the antibody or antigen-binding fragment thereof comprises nivolumab, pembrolizumab, LZM-009, or cemiplimab, or modified forms thereof. ,

[0011] In some embodiments, the immune cell associated antigen is PD-L1. In some embodiments, the antibody is avelumab (Bavencio, 451238, KXG2PJ551I, MSB-0010682, MSB-0010718C, PF-06834635, CAS1537032-82-8), durvalumab (Imfinzi, 28X28X9OKV (UNII code), MEDI-4736, CAS1428935-60-7), atezolizumab (Tecentriq, 52CMI0WC3Y, MPDL-3280A, RG-7446, RO-5541267, CAS1380723-4 4-3), sugemalimab (CS-1001, WBP-3155), KN-046 (CAS2256084-03-2), APL-502 (CBT-502, TQB-2450), embafolimab (3D-025, ASC-22, KN-035, hu56V1-Fc-m1, CAS2102192-68-5), vintrafusp alfa (M-7824, MSB-0011359C, NW9K8C1JN3, CAS1918149-01-5), STI-1014 (STI-A1014, ZKAB-001), PD-L1 t-haNK, A-167 (HBM-9167, KL-A167), IMC-001 (STI-3031, STI-A-1015, STI-A1015, s), HTI-1088 (SHR-1316), IO-103, CX-072 (CytomX Therapeutics), AUPM-170 (CA-170), GS-4224, ND-021 (NM21-1480, PRO-1480), BNT-311 (DuoBody-PD-L1x4-1BB, GEN-1046), BGB-A333, IBI-322, NM-01, LY-3434172, LDP, CDX-527, IBI-318, 89Zr-DFO-REGN3504, ALPN-202 (CD80 vIgD-Fc), INCB-086550, LY-3415244, SHR-1701, JS-003 (JS003-30, JS003-SD), HLX-20 (PL2#3), E S-101 (INBRX-105, INBRX-105-1), MSB-2311, PD-1-Fc-OX40L (SL-279252, TAK-252), FS-118, FS118 mAb2, LAG-3 / PD-L1mAb2), FAZ-053 (LAE-005), lodapolimab (LY-3300054, NR4MAD6PPB, CAS2118349-31-6), MCLA-145, BMS-189, cosibelimab (CK-301, TG-1501, CAS2216751-26-5), IL-15Ralpha-SD / IL-15 (KD-033), WP-1066 (CAS85 7064-38-1), BMS-936559 (MDX-1105), BMS-986192, RC-98, CD-200AR-L (CD200AR-L), ATA-3271, IBC -Ab002, BMX-101, AVA-04-VbP, ACE-1708, KY-1043, ACE-05(YBL-013), ONC-0055(ONC0055, PRS-344 S-095012), TLJ-1-CK, GR-1405, PD1ACR-T, N-809 (N-IL15 / PDL1), CB-201, MEDI-1109, AVA-004 (AVA-04), CA-327, ALN-PDL, KY-1003, CD22 (aPD-L1) CAR-T cells (SL-22P), ATA-2271 (M28z1XXPD1DNR CAR T cells), or modified versions thereof. In some embodiments, the antibody is durvalumab, atezolizumab, or avelumab, or modified versions thereof.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence in Table 1.

[0013] In some embodiments, the IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-18 polypeptide of the immunocytokine provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 2-67. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence of SEQ ID NOs: 68-72.

[0014] In some embodiments, the immunocytokine composition has a binding affinity (K) for at least one Fc receptor that is within 10-fold of the binding affinity of an antibody that is not bound to an IL-18 polypeptide. D In some embodiments, the immunocytokine composition exhibits a binding affinity (K) for the antibody's antigen that is within 5-fold of the binding affinity of the antibody that is not bound to an IL-18 polypeptide. D In some embodiments, the immunocytokine composition exhibits a half maximal effective concentration (EC) that is within about 100-fold of the corresponding IL-18 polypeptide not included in the immunocytokine composition. 50 ) and the immune cytokine composition exhibited a lower EC 50 In some embodiments, the immunocytokine composition exhibits improved anti-tumor growth inhibition compared to the antibody alone, in some embodiments, the immunocytokine composition exhibits enhanced anti-tumor growth inhibition compared to the antibody and IL-18 polypeptide administered in combination.

[0015] In some embodiments, the immunocytokine compositions provided herein further comprise a second linker, the second linker comprising a third point of attachment to the antibody or antigen-binding fragment thereof and a fourth point of attachment to an additional cytokine. In some embodiments, the additional cytokine is selected from a second IL-18 polypeptide, an IL-7 polypeptide, or an IL-2 polypeptide. In some embodiments, the additional cytokine is a second IL-18 polypeptide. In some embodiments, the second IL-18 polypeptide is substantially identical to the IL-18 polypeptide. In some embodiments, the third point of attachment is to a different heavy chain than the second point of attachment of the antibody or antigen-binding fragment thereof.

[0016] In one aspect, provided herein is a pharmaceutical composition comprising an immunocytokine composition provided herein and one or more pharma- ceutically acceptable carriers or excipients. In some embodiments, the pharmaceutical composition is formulated for parenteral or enteral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous or subcutaneous administration. In some embodiments, the pharmaceutical composition is in lyophilized form. In some embodiments, the one or more pharma- ceutically acceptable carriers or excipients comprise one or more of each of carbohydrates, inorganic salts, antioxidants, surfactants, buffers, or any combination thereof. In some embodiments, the pharmaceutical composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more excipients.

[0017] In one aspect, the present disclosure provides a method for treating cancer in a subject in need of such treatment, comprising administering to the subject an effective amount of the immunocytokine composition provided herein or the pharmaceutical composition provided herein.In some embodiments, the cancer is melanoma, lung cancer, bladder cancer (BC), microsatellite instability high (MSI-H) / mismatch repair deficient (dMMR) solid tumor, high tumor mutation burden (TMB-H) solid tumor, triple negative breast cancer (TNBC), gastric cancer (GC), cervical cancer (CC), pleural mesothelioma (PM), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), or a combination thereof. In some embodiments, the cancer is a solid tumor consisting of bladder and urethral cancer, bone cancer, brain and spinal cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, eye cancer, gallbladder or bile duct cancer, germ cell tumors, head and neck cancer, kidney cancer, liver cancer, lung cancer, metastatic brain tumors, ovarian cancer, pancreatic cancer, childhood cancer, peripheral nerve sheath tumors, pituitary cancer, prostate cancer, skin cancer, stomach or gastric cancer, soft tissue cancer, testicular cancer, thyroid cancer, uterine cancer. In some embodiments, the cancer is a carcinoma, which includes cutaneous squamous cell carcinoma (CSCC), urothelial carcinoma (UC), renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma (ESCC), esophagogastric junction (GEJ) cancer, endometrial carcinoma (EC), Merkel cell carcinoma (MCC), or a combination thereof. In some embodiments, the cancer is leukemia, lymphoma, myeloma, or a combination thereof. In some embodiments, the cancer is leukemia, including acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, small lymphocytic lymphoma, or chronic myelogenous leukemia.In some embodiments, the cancer is a lymphoma, including Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma, peripheral T-cell lymphoma, Waldenstrom's macroglobulinemia, lymphedema lymphoma, marginal zone lymphoma, B-cell cutaneous lymphoma, extranodal natural killer T-cell lymphoma, T-cell lymphoblastic lymphoma, peripheral T-cell lymphoma, or T-cell cutaneous lymphoma.

[0018] In some embodiments, a method of producing an immunocytokine composition includes: a) covalently attaching a reactive group to a specific residue of an antibody or antigen-binding fragment thereof; b) contacting the reactive group with a complementary reactive group attached to the IL-18 polypeptide; and c) forming the composition.

[0019] In one aspect, provided herein is a method of producing a composition comprising an IL-18 polypeptide and an antibody or antigen-binding fragment thereof specific for an immune cell antigen, and a linker, the linker comprising a first point of attachment to the IL-18 polypeptide and a second point of attachment to the antibody or antigen-binding fragment thereof, the method comprising: a) providing the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprising at least one acceptor amino acid residue that reacts with the linker in the presence of a coupling enzyme, and b) reacting the antibody or antigen-binding fragment with the linker comprising a primary amine in the presence of an enzyme capable of causing formation of a covalent bond between the at least one acceptor amino acid residue and the linker, wherein the linker comprises a reactive group (R), the covalent bond is not at the R moiety, and the method is performed under conditions sufficient to cause the at least one acceptor amino acid residue to form a covalent bond via the linker to the reactive group, the covalent bond comprising the second point of attachment of the linker. In some embodiments, the enzyme comprises a transaminase. In some embodiments, the enzyme comprises transglutaminase.

[0020] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of the present disclosure have been shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0021] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the specification is intended to supersede and / or override any such conflicting material. [Brief description of the drawings]

[0022] [Figure 1A] 13 illustrates a non-limiting mechanism of action of the anti-PD-1 antibody / IL-18 immunocytokine provided herein, in which activated T cells exhibit increased activation through simultaneous blockade of PD-1 and stimulation with IL-18. [Figure 1B] 13 illustrates a non-limiting mechanism of action of the anti-PD-L1 antibody / IL-18 immunocytokine provided herein, in which the immunocytokine disrupts PD-L1 / PD-1 interaction and achieves IL-18R signaling. [Figure 1C] 1 shows a non-limiting mechanism of action of anti-PD-L1 antibodies / IL-18 immunocytokines acting on immune cells expressing both IL-18R and PD-L1. [Figure 1D]Shown is an immunocytokine composition having two different protein payloads (e.g., IL-18 and IL-2) linked to an antibody (e.g., PD-L1 antibody) that acts on immune cells to co-activate two different receptors (e.g., IL-18R and IL-2R). [Diagram 2] A representative immunocytokine composition with a drug-antibody ratio of 1 (DAR1) is shown. [Diagram 3] FIG. 1 shows a diagram of site-selective introduction of conjugation handles into the antibodies provided herein. [Figure 4] 1 shows the mechanism of action of IL-18 on IFNγ and IL-18BP production, and IL-18BP on IL-18 inhibitory activity. [Diagram 5] FIG. 1 shows a schematic diagram of a synthetic route that can be used to synthesize an IL-18 polypeptide for conjugation to an antibody. [Figure 6] FIG. 1 shows a schematic diagram of the coupling of a bifunctional probe (also referred to herein as a bifunctional linking reagent) to an IL-18 polypeptide provided herein. [Figure 7] 1 shows an exemplary reaction scheme that can be used to prepare antibody / IL-18 immunocytokine compositions provided herein with a drug-antibody ratio of 1 (top) or 2 (bottom). [Figure 8] 1 shows a reverse-phase HPLC chromatogram of a purified IL-18 / anti-PD-1 antibody provided herein (LZM009) to which DAR1 is attached at residue K248 (EU numbering) in the Fc region of the antibody. [Figure 9] 1 shows a size-exclusion HPLC chromatogram of a purified IL-18 / anti-PD-1 antibody provided herein (LZM009) to which DAR1 is attached at residue K248 (EU numbering) in the Fc region of the antibody. [Figure 10] 1 shows a mass spectrometry trace (Q-TOF) of a purified IL-18 / anti-PD-1 antibody provided herein (LZM009) to which DAR1 is bound at residue K248 (EU numbering) in the Fc region of the antibody. [Figure 11]RP-HPLC chromatogram of purified Composition A (detection: 280 nm). [Figure 12] Analytical SEC-HPLC chromatogram of purified Composition A (detection: 280 nm). [Figure 13] 1 shows a Q-TOF mass spectrometry deconvolution chromatogram of purified Composition A. [Figure 14A] Figure 14A and Figure 14B show plots measuring the ability of unmodified and conjugated anti-PD1 antibodies to bind to human PD1 / CD279 ligand, with the figures showing ELISA signal on the y-axis and the dose of biotinylated PD-1 protein on the x-axis. Figure 14A shows the unconjugated reference antibody. Tested in this figure are pembrolizumab, LZM-009, nivolumab, atezolizumab, durvalumab, and avelumab. [Figure 14B] Figure 14A and Figure 14B show plots measuring the ability of unmodified and conjugated anti-PD1 antibodies to bind to the human PD1 / CD279 ligand, with the figures showing the ELISA signal on the y-axis and the dose of biotinylated PD-1 protein on the x-axis. Figure 14B shows the conjugated antibody. Tested in this figure are Composition A and Composition B. [Figure 15A] Figure 15A and Figure 15B show plots measuring the ability of unmodified and conjugated antibodies to bind to human PD-L1 / B7-H1 ligand, with the figures showing ELISA signal on the y-axis and dose of biotinylated PD-L1 protein on the x-axis. Figure 15A shows the unconjugated reference antibody. Tested in this figure are pembrolizumab, LZM-009, nivolumab, atezolizumab, durvalumab, and avelumab. [Figure 15B] Figure 15A and Figure 15B show plots measuring the ability of unmodified and conjugated antibodies to bind to human PD-L1 / B7-H1 ligand, with the figures showing ELISA signal on the y-axis and dose of biotinylated PD-L1 protein on the x-axis. Figure 15B shows the conjugated antibody. Tested in this figure are Composition A and Composition B. [Figure 16] Figure 1 shows plots measuring the ability of unmodified and conjugated antibodies to bind to human PD-L1 / B7-H1 ligand, with the figure showing net BioLayer interferometry shift in nanometers on the y-axis and incubation dose of biotinylated PD-L1 protein on the x-axis. The figure shows average ELISA signal on the y-axis and dose of human Fc gamma receptor on the x-axis. The unconjugated reference antibodies are pembrolizumab and LZM-009. The conjugated antibody tested is Composition A. [Figure 17A] Figures 17A and 17B show plots measuring the ability of unmodified and conjugated anti-PD1 antibodies to interfere with the PD1 / PDL1 pathway, with the figures showing the normalized emission intensity of the effector cell NFAT-Lucia reporter on the y-axis and the dose of unmodified and conjugated anti-PD1 antibodies on the x-axis. Figure 17A shows that the unconjugated reference antibodies are pembrolizumab, LZM-009, nivolumab, atezolizumab, durvalumab, and avelumab. [Figure 17B] Figures 17A and 17B show plots measuring the ability of unmodified and conjugated anti-PD1 antibodies to interfere with the PD1 / PDL1 pathway, with the figures showing the normalized luminescence intensity of the effector cell NFAT-Lucia reporter on the y-axis and the dose of unmodified and conjugated anti-PD1 antibodies on the x-axis. Figure 17B shows the conjugated antibodies that were tested. Tested in this figure are Composition A and Composition B. [Figure 18A] A plot measuring the ability of unmodified and conjugated antibodies to bind to human Fc gamma receptor I (CD64) is shown. The figure shows the average ELISA signal on the y-axis and the dose of human Fc gamma receptor on the x-axis. The unconjugated reference antibodies are LZM-009 and atezolizumab. The conjugated antibodies tested are compositions A and B. [Figure 18B]Figure 1 shows a plot measuring the ability of unmodified and conjugated antibodies to bind to human Fc gamma receptor IIIa (CD16). The figure shows the average ELISA signal on the y-axis and the dose of human Fc gamma receptor on the x-axis. The unconjugated reference antibodies are LZM-009 and atezolizumab. The conjugated antibodies tested are compositions A and B. [Figure 19] Figure 1 shows a plot measuring the ability of unmodified and conjugated antibodies to bind to human Fc neonatal receptor. The figure shows the average AlphaLISA signal on the y-axis and the dose of human Fc neonatal receptor (FcRn) on the x-axis. The unconjugated reference antibodies are LZM-009 and Atezolizumab. The conjugated antibodies tested are Compositions A and B. [Figure 20] 1 shows plots measuring PD-1 and PD-L1 surface expression levels on NK92 cells. [Figure 21] Figure 1 shows plots measuring the ability of unconjugated IL-18 variants and corresponding IL-18 immunocytokines to stimulate secretion of IFN gamma by NK92 cells. The figure shows the average IFNg alphaLISA signal on the y-axis and the dose of unconjugated IL-18 variants and corresponding IL-18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL-18 wild type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. The corresponding IL-18 immunocytokines tested are compositions A, B, C, and D. [Figure 22] 1 shows plots measuring PD-1 and PD-L1 surface expression levels on KG-1 cells. [Figure 23]Figure 1 shows plots measuring the ability of unconjugated IL-18 variants and corresponding IL-18 immunocytokines to stimulate secretion of IFNgamma by parental PD-1 negative and engineered PD-1 positive KG-1 cells. The figure shows the average IFNg legendplex signal on the y-axis and the dose of unconjugated IL-18 variants and corresponding IL-18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL-18 wild type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. The corresponding IL-18 immunocytokines tested are compositions A, B, C, and D. [Figure 24] Figure 1 shows a plot measuring the ability of unconjugated IL-18 variants and corresponding IL-18 immunocytokines to bind to human IL-18 binding protein (IL-18BP). The figure shows the average free IL-18BP AlphaLISA signal on the y-axis and the dose of unconjugated IL-18 variants and corresponding IL-18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL-18 wild type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. The corresponding IL-18 immunocytokines tested are compositions A, B, C, and D. [Diagram 25] Figure 1 shows a plot measuring the ability of human IL-18 binding proteins to inhibit the secretion of IFN gamma by NK92 cells stimulated with 2 nM of unconjugated IL-18 variants and the corresponding IL-18 immunocytokine. The figure shows the average IFNg alphaLISA signal on the y-axis and the dose of human IL-18 binding protein on the x-axis. The unconjugated IL-18 variants are native IL-18 wild type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. The corresponding IL-18 immunocytokine tested are compositions A, B, C, and D. [Figure 26A]hPD1 Figure 1 shows plots illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on the growth of MC38 syngeneic colon carcinoma tumors in C57BL / 6 mice. The figure shows the mean tumor volume on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A, tested as a single agent at 0.3 and 1 mg / kg as an intravenous injection twice weekly (n=9, mean±SEM). [Figure 26B] hPD1 Figure 1 shows plots illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-L1 antibody on the growth of MC38 syngeneic colon carcinoma tumors in C57BL / 6 mice. The figure shows the mean tumor volume on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition B, tested as a single agent at 1 and 3 mg / kg as an intravenous injection twice weekly (n=9, mean±SEM). [Figure 27A] 1 shows a plot illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on body weight in hPD1 C57BL / 6 mice bearing MC38 syngeneic colon carcinoma tumors. The figure shows the mean body weight change on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A, tested as a single agent as an intravenous injection twice weekly at 0.3 and 1 mg / kg (n=9, mean±SEM). [Figure 27B] Figure 1 shows a plot illustrating the effect of unmodified PD-L1 antibody and IL-18 polypeptide-conjugated PD-L1 antibody on body weight in hPD1 C57BL / 6 mice bearing MC38 syngeneic colon carcinoma tumors. The figure shows the mean body weight change on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition B, tested as a single agent as an intravenous injection twice weekly at 1 and 3 mg / kg (n=9, mean ± SEM). [Figure 28A]hPD1 Figure 1 shows plots illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on the growth of MC38 syngeneic colon carcinoma tumors in C57BL / 6 mice. The figure shows the mean tumor volume on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as a twice weekly intravenous injection. As a control, Her2-targeted immunocytokine Composition C was applied as a single agent at 0.5 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 1 mg / kg (n=9, mean±SEM). [Figure 28B] hPD1 A plot illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on the growth of MC38 syngeneic colon cancer tumors in C57BL / 6 mice. The figure shows the mean tumor volume 17 days after the start of treatment on the y-axis. The immunocytokine tested in this figure is Composition A, tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as a twice weekly intravenous injection. As a control, Her2-targeting immunocytokine Composition C was applied as a single agent at 3 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9, One-way Anova test ***P value<0.001, **P value<0.01, *P value<0.1, ns not significant, TGI: tumor growth inhibition). [Figure 28C] hPD1 A plot illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on the growth of MC38 syngeneic colon cancer tumors in C57BL / 6 mice. The figure shows the tumor volume of each individual animal on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as two intravenous injections. As a control, the Her2-targeting immunocytokine Composition C was applied as a single agent at 0.5 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 1 mg / kg (n=9, CR: complete response). [Figure 29]Figure 1 shows plots illustrating the effect of unmodified PD-L1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on body weight in hPD1 C57BL / 6 mice bearing MC38 syngeneic colon cancer tumors. The figure shows the average body weight change on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as a twice weekly intravenous injection. As a control, Her2-targeted immunocytokine Composition C was applied as a single agent at 0.5 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 1 mg / kg (n=9, mean ± SEM). [Diagram 30] 1 shows plots illustrating the effect of unmodified PD-L1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on survival of hPD1 C57BL / 6 mice bearing MC38 syngeneic colon cancer tumors. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 0.1, 0.25, and 0.5 mg / kg as twice weekly intravenous injections. As a control, Her2-targeted immunocytokine Composition C was applied as a single agent at 0.5 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 1 mg / kg (n=9, mean±SEM; CR: complete response). [Figure 31A] FIG. 1 shows plots illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on the growth of B16F10 syngeneic melanoma tumors in hPD1 C57BL / 6 mice. The figure shows the mean tumor volume on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 0.3, 1, and 3 mg / kg as a twice weekly intravenous injection. As a control, the Her2-targeted immunocytokine Composition C was applied as a single agent at 3 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9, mean±SEM). [Figure 31B]hPD1 A plot illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on the growth of B16F10 syngeneic melanoma tumors in C57BL / 6 mice. The figure shows the mean tumor volume 10 days after the start of treatment on the y-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 0.3, 1, and 3 mg / kg as a twice-weekly intravenous injection. As a control, the Her2-targeting immunocytokine Composition C was applied as a single agent at 3 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9, TGI; tumor growth inhibition). [Figure 31C] hPD1 Figure 1 shows plots illustrating the effect of unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on the growth of B16F10 syngeneic melanoma tumors in C57BL / 6 mice. The figure shows the tumor volume of each individual animal on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 0.3, 1, and 3 mg / kg as an intravenous injection twice weekly. As a control, the Her2-targeted immunocytokine Composition C was applied as a single agent at 3 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9, CR: complete response). [Diagram 32] Figure 1 shows plots illustrating the effect of unmodified PD-L1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on body weight in hPD1 C57BL / 6 mice bearing B16F10 syngeneic melanoma tumors. The figure shows the average body weight change on the y-axis and time on the x-axis. The immunocytokine tested in this figure is Composition A, which was tested as a single agent at 0.3, 1, and 3 mg / kg as intravenous injections twice weekly. As a control, Her2-targeted immunocytokine Composition C was applied as a single agent at 3 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9, mean ± SEM). [Diagram 33]Figure 1 shows plots illustrating the effect of unmodified PD-L1 antibody and IL-18 polypeptide-conjugated PD-1 antibody on survival of hPD1 C57BL / 6 mice bearing B16F10 syngeneic melanoma tumors. The immunocytokine tested in this figure is Composition A, which was tested at 0.3, 1, and 3 mg / kg as a single agent as a twice weekly intravenous injection. As a control, Her2-targeted immunocytokine Composition C was applied as a single agent at 3 mg / kg and in combination with LZM-009 anti-PD-1 antibody at 10 mg / kg (n=9, mean ± SEM, CR: complete response). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description Disclosed herein is an antibody or antigen-binding fragment specific for an immune cell-associated antigen linked to an IL-18 polypeptide in an immunocytokine composition. In some cases, the immunocytokine compositions provided herein are useful as potent stimulators of one or more immune cell types (e.g., T cells, macrophages, etc.). In some embodiments, the immunocytokine compositions may act by one or more mechanisms of action.

[0024] In some embodiments, the antibodies of the immunocytokine composition allow for targeting of the immunocytokine composition to immune cells. In some embodiments, the immunocytokine composition can inhibit the activity of an immune cell-associated antigen by binding to the immune cell-associated antigen (e.g., inhibiting a checkpoint interaction such as PD-1 / PD-L1 interaction). In some embodiments, the immunocytokine composition induces IFNγ production in immune cells (e.g., T cells or NK cells). The antibody or antigen-binding fragment IL-18 immunocytokine composition of the present disclosure can have synergistic efficacy and improved tolerability by subjects. In some embodiments, the antibody or antigen-binding fragment IL-18 immunocytokine composition can significantly reduce the therapeutic dosage of an antibody or antigen-binding fragment, an IL-18 polypeptide, or both, to a subject with a disease such as cancer, compared to treatment with either or both individually or in combination. In some embodiments, the immunocytokine compositions provided herein are associated with fewer side effects than treatment with either or both individually or in combination, potentially due to the targeting nature of the antibody to immune cells.

[0025] An exemplary, non-limiting mechanism of action of the immunocytokines provided herein is shown in Figure 1A. In an exemplary embodiment, the immunocytokine composition comprises an anti-PD-1 antibody as the antibody or antigen-binding fragment of the immunocytokine composition. In this embodiment, the anti-PD-1 antibody portion of the immunocytokine binds to activated T cells (e.g., CD8 + The IL-18R selectively binds to PD-1 present on the surface of T cells (T cells). This binding prevents the checkpoint interaction of PD-1 with PD-L1 / 2, thus preventing the attenuation of T cell activity. At the same time, the IL-18 portion of the immune cytokine, which is effective at high local concentrations near the T cells due to ligation, further activates the T cells via IL-18R signaling. Although the exemplary embodiment illustrates the mechanism of action of an anti-PD-1 antibody, antibodies or antigen-binding fragments specific for other immune antigens provided herein may function according to a similar mechanism.

[0026] Another exemplary, non-limiting mechanism of action of the anti-PD-L1 antibody / IL-18 immunocytokine is shown in Figure 1B. In the exemplary embodiment, the anti-PD-L1 antibody portion of the immunocytokine binds to PD-L1 expressed on the surface of tumor cells. When T cells contact tumor cells, the interaction between PD-1 on the T cells and PD-L1 on the cells is blocked, preventing the attenuation of T cell activity. Furthermore, the IL-18 portion of the immunocytokine is free to signal through IL-18R on immune cells, thereby inducing the production of IFNγ and further activation of immune cells. Although this exemplary embodiment is demonstrated for PD-L1, other immune antigens provided herein in the immunocytokine may exhibit similar mechanisms of action.

[0027] Also disclosed herein is a method for producing the immunocytokine composition provided herein. In some embodiments, the immunocytokine composition is prepared using a chemical linker that allows the two parts of the immunocytokine composition to be linked together with high fidelity at preselected sites on each part. In some embodiments, the method provided herein can be used with a wide variety of antibodies or antigen-binding fragments to rapidly and easily generate a wide variety of immunogen-specific antibodies or antigen-binding fragments linked to an IL-18 polypeptide as an immunocytokine. In some embodiments, the method can be used with readily commercially available antibodies to allow for rapid linkage with an IL-18 polypeptide provided herein. A non-limiting diagram of one of the immunocytokines provided herein is shown in FIG. 2. This illustrates an IL-18 polypeptide linked to an antibody provided herein, with the attachment points of the linker to the IL-18 polypeptide at the side chains of residues and to the side chains of residues within the Fc region.

[0028] The following description and examples are intended to illustrate the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that there are numerous variations and modifications to the present disclosure that are encompassed within its scope.

[0029] Although various features of the present disclosure are described in the context of a single embodiment, those features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0030] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0031] Immunoantigen-specific antibodies linked to IL-18 polypeptides as immunocytokines Provided herein are antibodies and antigen-binding fragments that bind immune cell-associated antigens linked to IL-18 polypeptides as immunocytokine compositions. The immunocytokine compositions provided herein are effective for simultaneously delivering IL-18 polypeptides and antibodies or antigen-binding fragments to target cells, such as immune cells. This simultaneous delivery of both agents to the same cells has many advantages, including improved selectivity of IL-18 polypeptides, improved therapeutic potential of IL-18 polypeptides, and minimizing the risk of side effects from administering IL-18 therapy. In some embodiments, the immunocytokine compositions act through multiple mechanisms of action, including, but not limited to, interfering with the activity of immune cell-associated antigens (e.g., immune checkpoint evasion) and / or enhancing activation of immune cells in or around the tumor microenvironment.

[0032] The immunocytokine compositions provided herein utilize linkers to attach the antibodies or antigen-binding fragments to the IL-18 polypeptide. In some embodiments, the linkers are attached to each moiety (i.e., the antibodies or antigen-binding fragments and the IL-18 polypeptide) at a specific residue or a specific subset of residues. In some embodiments, the linkers are attached to each moiety in a site-selective manner so that the population of the immunocytokine composition is substantially homogenous. This is accomplished in a variety of ways, including site-selectively adding reagents for the conjugation reaction to the moiety to be conjugated, synthesizing or otherwise preparing the moiety to be conjugated with the desired reagents for the conjugation reaction, or a combination of these two approaches. Using these approaches, the attachment site (such as a specific amino acid residue) of the linker to each moiety can be precisely selected.

[0033] Furthermore, these approaches allow for a variety of linkers to be used in the compositions that are not limited to the amino acid residues required for the fusion protein. The combination of linker selection and correct attachment to the moieties also allows, in some embodiments, the linker to function to modulate the activity of one of the moieties when attached to an IL-18 polypeptide at a position that interacts with a protein that binds to the IL-18 polypeptide (e.g., an IL-18 binding protein).

[0034] In one aspect, provided herein is an immunocytokine composition comprising an IL-18 polypeptide and an antibody or antigen-binding fragment thereof specific for an immune cell-associated antigen. In some embodiments, the immunocytokine composition comprises a linker. In some embodiments, the linker comprises a first point of attachment to the IL-18 polypeptide. In some embodiments, the linker comprises a second point of attachment to the antibody or antigen-binding fragment thereof.

[0035] Immune cell specific antibodies In some embodiments, the antibody or antigen-binding fragment of the present disclosure specifically binds to an immune cell-associated antigen. The immune cell-associated antigen provided herein is an antigen that is expressed at a higher level in immune cells than in other cells. In some embodiments, the immune cell-associated antigen is expressed at a level at least 25% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, or at least 100% higher in immune cells than in other cells. In some embodiments, the immune cell-associated antigen is expressed at a level at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, or at least 10-fold higher in immune cells than in other cells.

[0036] An antibody selectively or preferentially binds to a target if it binds with higher affinity, avidity, more readily, and / or with longer duration than it binds to other substances. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to specific binding means that the affinity of the antibody, or antigen-binding fragment thereof, is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 1000-fold higher than the affinity of the antibody for an unrelated amino acid sequence. In some embodiments, an antibody or antigen-binding fragment of the present disclosure may inhibit the action / activity of a substance to which it binds. In some embodiments, an antibody or antigen-binding fragment of the disclosure can stimulate the action / activity of a substance to which it binds (e.g., an immune cell agonist antibody or antigen-binding fragment, e.g., one specific for CD16A, NKG2D, NKp30, or other targets).

[0037] As used herein, the term "antibody" refers to an immunoglobulin (Ig), polypeptide, or protein having a binding domain that is or is homologous to an antigen-binding domain. The term further includes "antigen-binding fragments" and other interchangeable terms for similar binding fragments described below. Native antibodies and native immunoglobulins (Igs) are usually heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain ("V") at one end and a variable domain ("V") at the other end. H "), followed by several constant domains ("C H Each light chain has at one end a variable domain (V L ) at its other end, and a constant domain ("C L "), in which the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0038] In some examples, the antibody or antigen-binding fragment includes an isolated antibody or antigen-binding fragment, a purified antibody or antigen-binding fragment, a recombinant antibody or antigen-binding fragment, a modified antibody or antigen-binding fragment, or a synthetic antibody or antigen-binding fragment.

[0039] The antibodies and antigen-binding fragments herein may be partially or wholly synthetically produced. The antibodies or antigen-binding fragments may be polypeptides or proteins having a binding domain that may be or may be homologous to an antigen-binding domain. In one example, the antibodies or antigen-binding fragments may be produced in a suitable in vivo animal model and then isolated and / or purified.

[0040] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins (Ig) can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. An Ig or a part thereof can, in some cases, be a human Ig. In some instances, C H The three domains may be derived from immunoglobulins. In some cases, the chains or portions of the antibody or antigen-binding fragment, modified antibody or antigen-binding fragment, or binder may be derived from Ig. In such cases, the Ig may be or be derived from IgG, IgA, IgD, IgE, or IgM. If the Ig is an IgG, it may be a subtype of IgG, which may include IgG1, IgG2a, IgG2b, IgG3, or IgG4. In some cases, the C H The three domains may be derived from or from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgG. In some instances, the antibodies or antigen-binding fragments comprise or are derived from IgG1. In some embodiments, the antibodies or antigen-binding fragments comprise or are derived from IgG4. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgG2. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgM or are monomeric forms of IgM. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgE. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgD. In some embodiments, the antibodies or antigen-binding fragments described herein comprise or are derived from IgA.

[0041] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ" or "K") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0042] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two CDR determination techniques: (1) based on interspecies sequence variation (Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda Md., p647-669, hereafter referred to as "Kabat"), and (2) based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). As used herein, CDRs can refer to CDRs defined by either approach, or by a combination of both approaches.

[0043] With respect to antibodies, the term "variable domain" refers to the variable domain of an antibody that is used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions (also known as CDRs) in both the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called "framework regions" or "FRs". Unmodified heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4) and mainly adopt a β-sheet configuration incorporating the three CDRs, which form loops connecting and sometimes part of the β-sheet structure. The CDRs within each chain are held together in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat).

[0044] As used herein, the terms "hypervariable region" and "CDR" refer to the amino acid residues of an antibody that are responsible for antigen binding. CDRs contain amino acid residues from three sequence regions that bind to an antigen in a complementary manner, and H Chain and V LFor each chain, they are known as CDR1, CDR2, and CDR3. In the light chain variable domain, the CDRs usually correspond to about residues 24-34 (CDRL1), 50-56 (CDRL2), and 89-97 (CDRL3), and in the heavy chain variable domain, the CDRs usually correspond to about residues 31-35 (CDRH1), 50-65 (CDRH2), and 95-102 (CDRH3), according to Kabat et al., supra. It is understood that the CDRs of different antibodies may contain insertions, and therefore the numbering of the amino acids may differ. The Kabat numbering system accounts for such insertions in a numbering scheme that utilizes letters attached to specific residues (e.g., 27A, 27B, 27C, 27D, 27E, and 27F of CDRL1 in the light chain) to reflect any insertions in the numbering between different antibodies. Alternatively, according to Chothia and Lesk (J. Mol. Biol., 196:901-917 (1987)), in a light chain variable domain, the CDRs usually correspond to about residues 26-32 (CDRL1), 50-52 (CDRL2), and 91-96 (CDRL3), and in a heavy chain variable domain, the CDRs usually correspond to about residues 26-32 (CDRH1), 53-55 (CDRH2), and 96-101 (CDRH3).

[0045] As used herein, "framework region," "FW" or "FR" refers to framework amino acid residues that form part of an antigen binding pocket or groove. In some embodiments, the framework residues form loops that are part of the antigen binding pocket or groove, and the amino acid residues within the loops may or may not contact the antigen. Framework regions generally include the regions between the CDRs. In accordance with Kabat et al., supra, in the light chain variable domain, the FRs typically correspond to about residues 0-23 (FRL1), 35-49 (FRL2), 57-88 (FRL3), and 98-109, and in the heavy chain variable domain, the FRs typically correspond to about residues 0-30 (FRH1), 36-49 (FRH2), 66-94 (FRH3), and 103-133. As discussed above in the Kabat numbering for the light chain, the heavy chain accounts for insertions as well (e.g., 35A, 35B of CDRH1 in the heavy chain). Alternatively, according to Chothia and Lesk, supra, in light chain variable domains, the FRs usually correspond to approximately residues 0-25 (FRL1), 33-49 (FRL2), 53-90 (FRL3), and 97-109 (FRL4), and in heavy chain variable domains, the FRs usually correspond to approximately residues 0-25 (FRH1), 33-52 (FRH2), 56-95 (FRH3), and 102-113 (FRH4). The loop amino acids of the FRs can be assessed and determined by examining the three-dimensional structure of the antibody heavy chain and / or antibody light chain. The three-dimensional structure can be analyzed for solvent-accessible amino acid positions, since such positions are more likely to form loops and / or provide antigen contacts in the antibody variable domain. Some of the solvent-accessible positions can tolerate amino acid sequence diversity, while others (e.g., structural positions) are generally less diversified. The three-dimensional structure of the antibody variable domain can be derived from a crystal structure or protein modeling.

[0046] In this disclosure, the following abbreviations (in parentheses) are used according to convention and where appropriate: heavy chain (H chain), light chain (L chain), heavy chain variable region (VH), light chain variable region (VL), complementarity determining region (CDR), first complementarity determining region (CDR1), second complementarity determining region (CDR2), third complementarity determining region (CDR3), heavy chain first complementarity determining region (VH CDR1), heavy chain second complementarity determining region (VH CDR2), heavy chain third complementarity determining region (VH CDR3), light chain first complementarity determining region (VL CDR1), light chain second complementarity determining region (VL CDR2), and light chain third complementarity determining region (VL CDR3).

[0047] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The numbering of residues in the Fc region is the same as in the EU index numbering in Kabat. The Fc region of an immunoglobulin generally consists of two constant domains, the C and C domains. H 2 and C H Includes 3.

[0048] An "antibody" useful in the present disclosure includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a bispecific antibody, an grafted antibody, a multispecific antibody, a heteroconjugate antibody, a humanized antibody, a human antibody, a deimmunized antibody, a variant thereof, a fusion thereof, an immunoconjugate thereof, an antigen-binding fragment thereof, and / or any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylation variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. In certain embodiments of the methods and conjugates provided herein, the antibody requires an Fc region to allow attachment of a linker between the antibody and the protein (e.g., attachment of a linker using an affinity peptide such as in AJICAP™ technology).

[0049] In some cases, the antibody is a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may exist in small amounts. In contrast to polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen (epitope). The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0050] In some cases, the antibody is a humanized antibody. As used herein, "humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are certain chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and biological activity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody, nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody comprises substantially all or at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to the variable domains of a non-human immunoglobulin, and all or substantially all of the FR regions being the variable domains of a human immunoglobulin consensus sequence. The humanized antibody will most desirably also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have a modified Fc region, for example as described in WO99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0051] Optionally, the antibodies or antigen-binding fragments described herein can be evaluated for immunogenicity and deimmunized (i.e., the antibody is rendered less immunoreactive by modifying one or more T cell epitopes) as needed. As used herein, a "deimmunized antibody" means that one or more T cell epitopes in the antibody sequence have been modified to reduce the T cell response following administration of the antibody to a subject compared to an antibody that has not been deimmunized. Analysis of the immunogenicity and T cell epitopes present in the antibodies and antigen-binding fragments described herein can be performed using software and specific databases. Exemplary software and databases include iTope™, developed by Antitope, Cambridge, UK. iTope™ is an in silico technology for analyzing peptide binding to human MHC class II alleles. The iTope™ software predicts peptide binding to human MHC class II alleles, thereby providing an initial screening for the location of such "potential T cell epitopes." iTope™ software predicts favorable interactions between peptide amino acid side chains and specific binding pockets within the binding grooves of 34 human MHC class II alleles. Location of key binding residues is achieved by in silico generation of 9-mer peptides that span the test antibody variable region sequence, overlapping by one amino acid. Each 9-mer peptide can be tested against each of the 34 MHC class II allotypes and scored based on their potential "fit" and interaction with the MHC class II binding groove. Peptides that yield high average binding scores (>0.55 in the iTope™ scoring function) for more than 50% of the MHC class II alleles are considered potential T-cell epitopes. In such regions, the core 9 amino acid sequences for peptide binding within the MHC class II groove are analyzed to determine the MHC class II pocket residues (P1, P4, P6, P7, and P9) and potential T-cell receptor (TCR) contact residues (P-1, P2, P3, P5, P8).After identification of any T cell epitope, amino acid residue changes, substitutions, additions, and / or deletions can be introduced to eliminate the identified T cell epitope. Such changes can be made to maintain the structure and function of the antibody while still eliminating the identified epitope. Exemplary changes can include, but are not limited to, conservative amino acid changes.

[0052] The antibody may be a human antibody. As used herein, "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human and / or that is made using any suitable technique for making the human antibody. This definition of a human antibody includes antibodies that comprise at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody that comprises a mouse light chain and a human heavy chain polypeptide. In one embodiment, the human antibody is selected from a phage library that expresses a human antibody. Human antibodies can also be made by introducing human immunoglobulin loci into a transgenic animal, e.g., a mouse in which the endogenous immunoglobulin genes have been partially or completely inactivated. Alternatively, human antibodies may be prepared by immortalizing human B lymphocytes that produce antibodies directed against a target antigen (such B lymphocytes may be harvested from an individual or immunized in vitro).

[0053] Any of the antibodies herein may be bispecific. Bispecific antibodies are antibodies that have binding specificities for at least two different antigens and may be prepared using the antibodies disclosed herein. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities. Bispecific antibodies may be composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. In this asymmetric structure, an immunoglobulin light chain is present in only one half of the bispecific molecule, facilitating the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations.

[0054] In one approach to making bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion is with an immunoglobulin heavy chain constant domain, including at least part of the hinge, CH2 and CH3 regions. The first heavy chain constant region (CH1), containing the site necessary for light chain binding, may be present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows for great flexibility in adjusting the mutual ratios of the three polypeptide fragments in embodiments where optimal yields are obtained when the ratios of the three polypeptide chains used in the construction are not equal. However, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector when high yields are obtained when at least two polypeptide chains are expressed in equal ratios, or when the ratios are not particularly important.

[0055] In some cases, the antibody herein is a chimeric antibody. "Chimeric" forms of non-human (e.g., mouse) antibodies include chimeric antibodies that contain minimal sequences derived from non-human Ig. In most cases, chimeric antibodies are mouse antibodies in which at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, is inserted in place of the mouse Fc. Chimeric or hybrid antibodies can also be prepared in vitro using suitable methods in synthetic protein chemistry, including those involving cross-linking agents.

[0056] Provided herein are antibodies and antigen-binding fragments thereof, modified antibodies and antigen-binding fragments thereof, and binding agents that specifically bind to one or more epitopes on one or more target antigens.In one case, the binding agent selectively binds to an epitope on a single antigen.In another example, the binding agent is bivalent and either selectively binds to two distinct epitopes on a single antigen or binds to two distinct epitopes on two distinct antigens.In another example, the binding agent is multivalent (i.e., trivalent, tetravalent, etc.), and the binding agent binds to three or more distinct epitopes on a single antigen or binds to three or more distinct epitopes on two or more (multiple) antigens.

[0057] Any antigen-binding fragment of the antibodies herein is also contemplated. The terms "antigen-binding portion of an antibody," "antigen-binding domain," "antibody fragment," or "functional fragment of an antibody" are used interchangeably herein and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, bispecific F(ab')2, trispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), dsFv, bispecific scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, AVIMER®, minibodies, diabodies, bispecific diabodies, triabodies, tetrabodies, minibodies, maxibodies, camelids, VHH, minibodies, intrabodies, fusion proteins comprising an antibody portion (e.g., domain antibodies), single chain binding polypeptides, scFv-Fc, Fab-Fc, bispecific T cell engagers (BiTEs; where two scFvs are produced as a single polypeptide chain, each scFv may comprise any of the amino acid sequences, CDR combinations, or VL / VL combinations described herein. (comprising an Fc region), tetravalent tandem diabodies (TandAbs; antibody fragments generated in a head-to-tail configuration as non-covalently linked homodimeric folds, e.g., TandAbs comprising scFvs (wherein the scFvs comprise an amino acid sequence, CDR combination, or VL / VL combination as described herein), dual affinity retargeting antibodies (DARTs; in which different scFvs are linked by stabilized interchain disulfide bonds), bispecific antibodies (bscAbs; two single chain Fv fragments linked via a glycine-serine linker), single domain antibodies (sdAbs), fusion proteins, bispecific disulfide stabilized Fv antibody fragments (dsFv-dsFv'; two different disulfide stabilized Fv antibody fragments connected by a flexible linker peptide). In certain embodiments of the invention, full length antibodies (e.g., antigen binding fragment and Fc region) are preferred.

[0058] Heteroconjugate polypeptides comprising two covalently linked antibodies or antigen-binding fragments of antibodies are also within the scope of the present disclosure. Appropriate linkers can be used to multimerize the binding agents. Non-limiting examples of linking peptides include (GS) n (SEQ ID NO: 224), (GGS) n (SEQ ID NO: 225), (GGGS) n (SEQ ID NO: 226), (GGSG) n (SEQ ID NO: 227), (GGSGG) n (SEQ ID NO: 228), (GGGGS) n (SEQ ID NO: 229), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, the linking peptide can be (GGGGS)3 (SEQ ID NO: 230) or (GGGGS)4 (SEQ ID NO: 231). In some embodiments, the linking peptide bridges the carboxy terminus of one variable region with the amino terminus of the other variable region by about 3.5 nm. Linkers of other sequences have been designed and used. However, the linker can be modified for additional functions, such as drug attachment or attachment to a solid support.

[0059] As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. Apparent affinity can be determined by methods such as enzyme-linked immunosorbent assay (ELISA) or any other suitable technique. Avidity can be determined by methods such as Scatchard analysis or any other suitable technique.

[0060] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents, K D The binding affinity (K D) is 500nM, 475nM, 450nM, 425nM, 400nM, 375nM, 350nM, 325nM, 300nM, 275nM, 250nM, 225nM, 200nM, 175nM, 150nM, 125nM, 100nM, 90nM, 80nM, 70nM, 50nM, 50nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 43nM, 42nM, 41nM, 40nM, 39nM, 38nM, 37nM, 36nM, 35nM, 34nM, 33nM, 32nM, 3 1nM, 30nM, 29nM, 28nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9n M, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 990pM, 980pM, 970pM, 960pM, 950pM, 940pM, 930pM, 920pM, 910pM, 900pM, 890pM, 880pM, 870pM , 860pM, 850pM, 840pM, 830pM, 820pM, 810pM, 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710pM, 700pM, 690pM, 6 80pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610pM, 600pM, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530pM, 520pM, 510pM, 500p M, 490pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420pM, 410pM, 400pM, 390pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM, 290pM, 280pM, 270pM, 260pM, 250pM, 240pM, 230pM, 220pM, 210pM, 200pM, 190pM, 180pM, 170pM, or any integer therebetween.Binding affinity can be determined using surface plasmon resonance (SPR), KINEXA® biosensor, scintillation proximity assay, enzyme-linked immunosorbent assay (ELISA), ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, yeast display, or any combination thereof. Binding affinity can also be screened using a suitable bioassay.

[0061] Also provided herein are affinity matured antibodies. The following methods can be used to tune the affinity of an antibody and to characterize the CDRs. One method of characterizing the CDRs of an antibody and / or changing (e.g. improving) the binding affinity of a polypeptide such as an antibody is called "library scanning mutagenesis". In general, library scanning mutagenesis works as follows: one or more amino acid positions in the CDR are substituted with two or more (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids. This generates a small library of clones (in some embodiments, one for each amino acid position analyzed), each with a complexity of two or more members (when two or more amino acids are substituted at each position). In general, the library also includes clones that contain natural (unsubstituted) amino acids. A small number of clones from each library, for example about 20-80 clones (depending on the complexity of the library), can be screened for binding specificity or affinity to the target polypeptide (or other binding target), and candidates with increased, the same, decreased, or no binding are identified. Binding affinity can be determined using Biacore surface plasmon resonance analysis to detect differences in binding affinity of about 2-fold or greater.

[0062] In some instances, the antibody or antigen-binding fragment is bispecific or multispecific and can specifically bind to multiple antigens. In some cases, such bispecific or multispecific antibodies or antigen-binding fragments can specifically bind to two or more different antigens. In some cases, the bispecific antibody or antigen-binding fragment can be a bivalent antibody or antigen-binding fragment. In some cases, the multispecific antibody or antigen-binding fragment can be a bivalent antibody or antigen-binding fragment, a trivalent antibody or antigen-binding fragment, or a tetravalent antibody or antigen-binding fragment.

[0063] The antibodies or antigen-binding fragments described herein can be isolated, purified, recombinant, or synthetic.

[0064] When an antibody name is used, it is intended to encompass generic or biosimilar versions of the antibody named herein that share the same amino acid sequence as the indicated antibody.

[0065] The antibodies described herein can be made by any suitable method. Antibodies can often be produced in large quantities, especially when high-level expression vectors are utilized.

[0066] In one embodiment, the antibody or antigen-binding fragment of the disclosure comprises a fusion protein or peptide immunotherapeutic.

[0067] Immune cell-associated antigen-specific antibodies In some embodiments, the antibody or antigen-binding fragment thereof is specific for an immune cell-associated antigen. In some embodiments, the immune cell-associated antigen is associated with a subtype of immune cell (e.g., lymphocyte, neutrophil, macrophage, etc.). In some embodiments, the immune cell-associated antigen is associated with a T cell, a monocyte, and / or a natural killer (NK) cell. In some embodiments, the immune cell-associated antigen is associated with a T cell. In some embodiments, the immune cell antigen is associated with an effector T cell, a cytotoxic T cell, a helper T cell, a regulatory T cell, and / or a memory T cell.

[0068] In some embodiments, the immune cell-associated antigen is an immune checkpoint molecule. In some embodiments, the immune cell-associated antigen is a costimulatory antigen. In some embodiments, the immune cell-associated antigen is a macrophage cell surface antigen. In some embodiments, the immune cell-associated antigen is a NK cell surface antigen. In some embodiments, the immune cell-associated antigen is a T cell surface antigen (e.g., CD8A, CD8B).

[0069] In some embodiments, the immune cell associated antigen is 4-IBB, B7-H3, B7-H4, BTLA, CD3, CCR8, CD8A, CD8B, CD16A, CD27, CD28, CD33, CD38, CD39, CD40, CD47, CD70, CD80, CD86, CD96, CD163, CLEC-1, CLEVER-1, CTLA-4, D40, GITR, ICOS, ILT2 / 3 / 4, LAG-3, MHCI, MHCII, NKG2A, NKG2D, NKp30, NKp44, NKp46, OX40, PD-1, PD-L1, PD-L2, PSGL-1, SIGLEC-9, SIGLEC-15, SIRP-alpha, TCR, TIGIT, TIM-3, VISTA, or VSIG4. In some embodiments, the immune cell-associated antigen is PD-1. In some embodiments, the immune cell-associated antigen is CCR8, CD8A, CD8B, CD16A, CD96, CD226, CTLA-4, ICOS, LAG-3, NKG2A, NKG2D, NKp30, NKp44, NKp46, PD-1, PD-L1, TIGIT, or TIM-3.

[0070] In some embodiments, the immune cell associated antigen is 4-1BB. In some embodiments, the immune cell associated antigen is B7-H3. In some embodiments, the immune cell associated antigen is B7-H4. In some embodiments, the immune cell associated antigen is BTLA. In some embodiments, the immune cell associated antigen is CD3. In some embodiments, the immune cell associated antigen is CCR8. In some embodiments, the immune cell associated antigen is CD8A. In some embodiments, the immune cell associated antigen is CD8B. In some embodiments, the immune cell associated antigen is CD16A. In some embodiments, the immune cell associated antigen is CD27. In some embodiments, the immune cell associated antigen is CD33. In some embodiments, the immune cell associated antigen is CD38. In some embodiments, the immune cell associated antigen is CD39. In some embodiments, the immune cell associated antigen is CD40. In some embodiments, the immune cell associated antigen is CD47. In some embodiments, the immune cell associated antigen is CD80. In some embodiments, the immune cell associated antigen is CD86. In some embodiments, the immune cell associated antigen is CD96. In some embodiments, the immune cell associated antigen is CD163. In some embodiments, the immune cell associated antigen is CLEC-1. In some embodiments, the immune cell associated antigen is CLEVER-1. In some embodiments, the immune cell associated antigen is CTLA4. In some embodiments, the immune cell associated antigen is D40. In some embodiments, the immune cell associated antigen is GITR. In some embodiments, the immune cell associated antigen is ICOS. In some embodiments, the immune cell associated antigen is ILT2 / 3 / 4. In some embodiments, the immune cell associated antigen is LAG-3. In some embodiments, the immune cell associated antigen is MHCI. In some embodiments, the immune cell associated antigen is MHCII.In some embodiments, the immune cell associated antigen is NKG2A. In some embodiments, the immune cell associated antigen is NKp30. In some embodiments, the immune cell associated antigen is NKp44. In some embodiments, the immune cell associated antigen is NKp46. In some embodiments, the immune cell associated antigen is OX40. In some embodiments, the immune cell associated antigen is PD-1. In some embodiments, the immune cell associated antigen is PD-L1. In some embodiments, the immune cell associated antigen is PD-L2. In some embodiments, the immune cell associated antigen is PSGL-1. In some embodiments, the immune cell associated antigen is SIGLEC-9. In some embodiments, the immune cell associated antigen is SIGLEC-15. In some embodiments, the immune cell associated antigen is SIRP-·. In some embodiments, the immune cell associated antigen is TCR. In some embodiments, the immune cell associated antigen is TIGIT. In some embodiments, the immune cell-associated antigen is TIM-3. In some embodiments, the immune cell-associated antigen is VISTA. In some embodiments, the immune cell-associated antigen is VSIG4.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment. Programmed cell death protein 1 (also known as PD-1 and CD279) is a cell surface receptor that plays a role in downregulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 is an immune cell inhibitory molecule expressed on activated B cells, T cells, and myeloid cells. PD-1 represents an immune checkpoint and prevents autoimmunity through a dual mechanism that promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis of regulatory T cells. PD-1 is a member of the CD28 / CTLA-4 / ICOS costimulatory receptor family that delivers negative signals that affect T cell and B cell immunity. PD-1 is a monomer both in solution and on the cell surface, in contrast to CTLA-4 and other family members, which are all disulfide-linked homodimers. Signaling through the PD-1 inhibitory receptor upon binding to its ligand, PD-L1, suppresses immune responses to self-antigens and tumors and plays a role in maintaining peripheral immune tolerance. The interaction between PD-1 and PD-L1 results in a reduction in tumor-infiltrating lymphocytes, a decrease in T cell receptor-mediated proliferation, and immune evasion by cancer cells. A non-limiting example of a human PD-1 amino acid sequence is MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 331).

[0072] In one embodiment, an anti-PD-1 antibody or antigen-binding fragment of the disclosure comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) described herein. In another embodiment, an anti-PD-1 antibody or antigen-binding fragment of the disclosure comprises a combination of complementarity determining regions (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) described herein. In one embodiment, the anti-PD-1 antibody or anti-PD-1 antigen-binding fragment of the disclosure is a modified tislelizumab, Baizean, 0KVO411B3N, BGB-A317, hu317-1 / IgG4mt2, sintilimab, Tyvyt, IBI-308, toripalimab, TeRuiPuLi, terepryl, Tuoyi, JS-001, TAB-001, camrelizumab, HR-301210, INCSHR-01210, SHR-1210, semi Primab, cemiplimab-rwlc, LIBTAYO®, 6QVL057INT, H4H7798N, REGN-2810, SAR-439684, avelumab, BAVENCIO®, 451238, KXG2PJ551I, MSB-0010682, MSB-0010718C, PF-06834635, durvalumab, IMFINZI®, 28X28X9OKV, MEDI-4736, lambrolizumab, pem Brolizumab, KEYTRUDA®, MK-3475, SCH-900475, h409A11, nivolumab, nivolumab BMS, OPDIVO®, BMS-936558, MDX-1106, ONO-4538, prorugolimab, Forteca, BCD-100, penprimab, AK-105, zimblerimab, AB-122, GLS-010, WBP-3055, balstilimab, 1Q2QT5M7EO, AGEN-203 4, AGEN-2034w, genolimuzumab, geptanolimab, APL-501, CBT-501, GB-226, dostallimab, ANB-011, GSK-4057190A, P0GVQ9A4S5, TSR-042, WBP-285, serplulimab, HLX-10, CS-1003, retifanlimab, 2Y3T5IF01Z, INCMGA-00012, INCMGA-0012, MGA-012, sasanlimab, LZZ0IC2EWP,PF-06801591, RN-888, spartalizumab, NVP-LZV-184, PDR-001, QOG25L6Z8Z, leratolimab / nivolumab, BMS-986213, cetrelimab, JNJ-3283, JNJ-63723283, LYK98WP91F, tebotelimab, MGD-013, BCD-217, BAT-1306, HX-008, MEDI-57 52, JTX-4014, cadnilimab, AK-104, BI-754091, pidilizumab, CT-011, MDV-9300, YBL-006, AMG-256, RG-6279, RO-7284755, BH-2950, ​​IBI-315, RG-6139, RO-7247669, ONO-4685, AK-112, 609-A, LY-3434172, T-3011 , MAX-10181, AMG-404, IBI-318, MGD-019, INCB-086550, ONCR-177, LY-3462817, RG-7769, RO-7121661, F-520, XmAb-23104, Pd-1-pik, SG-001, S-95016, Sym-021, LZM-009, budigalimab, 6VDO4TY3OO, ABBV-18 1, PR-1648817, CC-90006, XmAb-20717, 2661380, AMP-224, B7-DCIg, EMB-02, ANB-030, PRS-332, [89Zr]deferoxamide-pembrolizumab, 89Zr-Df-pembrolizumab, [89Zr]Df-pembrolizumab, STI-1110, STI-A1110, CX-188, mPD-1 Pb-Tx, MCLA-134, 244C8, ENUM224C8, ENUMC8, 388D4, ENUM388D4, ENUM D4, MEDI0680, or AMP-514.

[0073] In some embodiments, the anti-PD-1 antibodies or anti-PD-1 antigen-binding fragments of the disclosure comprise tislelizumab, sintilimab, toripalimab, telepryl, camrelizumab, cemiplimab, pembrolizumab, nivolumab, prorugolimab, penprimab, zimvelerimab, balstilimab, genolimuzumab, geptanolimab, dostallimab, serplulimab, retifanlimab, sasanlimab, spartalizumab, cetrelimab, tebotelimab, kadonilimab, Apidilimab, LZM-009, or budigalimab. In one embodiment, the anti-PD-1 antibody or anti-PD-1 antigen-binding fragment of the disclosure comprises a modified tislelizumab, sintilimab, toripalimab, telepryl, camrelizumab, cemiplimab, pembrolizumab nivolumab, prorugolimab, penprimab, zimvelerimab, balstilimab, genolimuzumab, geptanolimab, dostallimab, serplulimab, retifanlimab, sasanlimab, spartalizumab, cetrelimab, tebotelimab, kadonilimab, Apidilimab, LZM-009, or budigalimab.

[0074] In some embodiments, the anti-PD-1 polypeptide is nivolumab, pembrolizumab, LZM-009, dostallimab, sintilimab, spartalizumab, tislelizumab, or cemiplimab. In some embodiments, the anti-PD-1 polypeptide is dostallimab, sintilimab, spartalizumab, or tislelizumab. In some embodiments, the anti-PD-1 polypeptide is nivolumab, pembrolizumab, LZM-009, or cemiplimab.

[0075] In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the anti-PD1 antibody is a modified pembrolizumab.

[0076] In some embodiments, the anti-PD-1 antibody is a biosimilar of tislelizumab, sintilimab, toripalimab, telepryl, camrelizumab, cemiplimab, pembrolizumab nivolumab, prorugolimab, pempulimab, zimberelimab, balstilimab, genolimuzumab, geptanolimab, dostallimab, serplulimab, retifanlimab, sasanlimab, spartalizumab, cetrelimab, tebotelimab, kadonilimab, Apidilimab, LZM-009, or budigalimab. In some embodiments, the anti-PD-1 antibody is a biosimilar of any one of the antibodies provided herein.

[0077] Table 1 provides sequences of exemplary anti-PD-1 antibodies and anti-PD-1 antigen-binding fragments that can be modified to prepare anti-PD-1 immunoconjugates. Table 1 also provides combinations of CDRs that can be utilized in the modified anti-PD-1 immunoconjugates. References herein to anti-PD-1 antibodies may alternatively refer to anti-PD-1 antigen-binding fragments.

[0078] In some cases, the SEQ ID NOs listed in Table 1 contain the full-length heavy or light chain of the indicated antibody with the VH or VL, respectively, shown in bold. When reference is made herein to the VH or VL of a SEQ ID NO in Table 1 that contains a full-length heavy or light chain, it is intended to refer to the bolded portion of the sequence. For example, reference to "a VH having the amino acid sequence shown in SEQ ID NO: 332" refers to the bolded portion of SEQ ID NO: 332 in Table 1.

[0079] The anti-PD-1 antibody or antigen-binding fragment may comprise a VH having the amino acid sequence of any one of SEQ ID NOs: 332, 334, 336, 338, 340, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, and 378. The anti-PD-1 antibody or antigen-binding fragment may comprise a VL having the amino acid sequence of any one of SEQ ID NOs: 333, 335, 337, 339, 341, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, and 379.

[0080] The anti-PD-1 antibody or antigen-binding fragment may comprise a heavy chain or VH having the amino acid sequence of any one of SEQ ID NOs: 332, 334, 336, 338, 340, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, and 378, or a portion corresponding to the VH. The anti-PD-1 antibody or antigen-binding fragment may comprise a light chain or VL having the amino acid sequence of any one of SEQ ID NOs: 333, 335, 337, 339, 341, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, and 379, or a portion corresponding to the VL thereof.

[0081] In one example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:332, and a VL having the amino acid sequence set forth in SEQ ID NO:333. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:334, and a VL having the amino acid sequence set forth in SEQ ID NO:335. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:336, and a VL having the amino acid sequence set forth in SEQ ID NO:337. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:338, and a VL having the amino acid sequence set forth in SEQ ID NO:339. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:340, and a VL having the amino acid sequence set forth in SEQ ID NO:341. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:346, and a VL having the amino acid sequence set forth in SEQ ID NO:347. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:348, and a VL having the amino acid sequence set forth in SEQ ID NO:349. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:350, and a VL having the amino acid sequence set forth in SEQ ID NO:351. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:352, and a VL having the amino acid sequence set forth in SEQ ID NO:353. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:354, and a VL having the amino acid sequence set forth in SEQ ID NO:355. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:356, and a VL having the amino acid sequence set forth in SEQ ID NO:357.In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:358, and a VL having the amino acid sequence set forth in SEQ ID NO:359. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:360, and a VL having the amino acid sequence set forth in SEQ ID NO:361. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:362, and a VL having the amino acid sequence set forth in SEQ ID NO:363. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:364, and a VL having the amino acid sequence set forth in SEQ ID NO:365. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:366, and a VL having the amino acid sequence set forth in SEQ ID NO:367. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:368, and a VL having the amino acid sequence set forth in SEQ ID NO:369. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:370, and a VL having the amino acid sequence set forth in SEQ ID NO:371. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:372, and a VL having the amino acid sequence set forth in SEQ ID NO:373. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:374, and a VL having the amino acid sequence set forth in SEQ ID NO:375. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:376, and a VL having the amino acid sequence set forth in SEQ ID NO:377. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:378, and a VL having the amino acid sequence set forth in SEQ ID NO:379.

[0082] In one example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:332, and a VL having the amino acid sequence set forth in SEQ ID NO:333. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:334, and a VL having the amino acid sequence set forth in SEQ ID NO:335. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:336, and a VL having the amino acid sequence set forth in SEQ ID NO:337. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:338, and a VL having the amino acid sequence set forth in SEQ ID NO:339. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:340, and a VL having the amino acid sequence set forth in SEQ ID NO:341. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:346, and a VL having the amino acid sequence set forth in SEQ ID NO:347. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:348, and a VL having the amino acid sequence set forth in SEQ ID NO:349. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:350, and a VL having the amino acid sequence set forth in SEQ ID NO:351. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:352, and a VL having the amino acid sequence set forth in SEQ ID NO:353. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:354, and a VL having the amino acid sequence set forth in SEQ ID NO:355. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:356, and a VL having the amino acid sequence set forth in SEQ ID NO:357.In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:358, and a VL having the amino acid sequence set forth in SEQ ID NO:359. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:360, and a VL having the amino acid sequence set forth in SEQ ID NO:361. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:362, and a VL having the amino acid sequence set forth in SEQ ID NO:363. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:364, and a VL having the amino acid sequence set forth in SEQ ID NO:365. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:366, and a VL having the amino acid sequence set forth in SEQ ID NO:367. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:368, and a VL having the amino acid sequence set forth in SEQ ID NO:369. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:370, and a VL having the amino acid sequence set forth in SEQ ID NO:371. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:372, and a VL having the amino acid sequence set forth in SEQ ID NO:373. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:374, and a VL having the amino acid sequence set forth in SEQ ID NO:375. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:376, and a VL having the amino acid sequence set forth in SEQ ID NO:377. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:378, and a VL having the amino acid sequence set forth in SEQ ID NO:379.

[0083] In one example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 380, a VH CDR2 having the amino acid sequence of SEQ ID NO: 381, a VH CDR3 having the amino acid sequence of SEQ ID NO: 382, ​​a VL CDR1 having the amino acid sequence of SEQ ID NO: 383, a VL CDR2 having the amino acid sequence of SEQ ID NO: 384, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 385. In one example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 386, a VH CDR2 having the amino acid sequence of SEQ ID NO: 387, a VH CDR3 having the amino acid sequence of SEQ ID NO: 388, a VL CDR1 having the amino acid sequence of SEQ ID NO: 389, a VL CDR2 having the amino acid sequence of SEQ ID NO: 390, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 391. In one example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 392, a VH CDR2 having the amino acid sequence of SEQ ID NO: 393, a VH CDR3 having the amino acid sequence of SEQ ID NO: 394, a VL CDR1 having the amino acid sequence of SEQ ID NO: 395, a VL CDR2 having the amino acid sequence of SEQ ID NO: 396, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 397. In one example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 398, a VH CDR2 having the amino acid sequence of SEQ ID NO: 399, a VH CDR3 having the amino acid sequence of SEQ ID NO: 400, a VL CDR1 having the amino acid sequence of SEQ ID NO: 401, a VL CDR2 having the amino acid sequence of SEQ ID NO: 402, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 403.

[0084] In one example, the anti-PD-1 antibody comprises a fusion protein. Such a fusion protein can be, for example, a double-sided Fc fusion protein comprising the extracellular domain (ECD) of programmed cell death 1 (PD-1) and the ECD of tumor necrosis factor (ligand) superfamily member 4 (TNFSF4 or OX40L), fused via the hinge-CH2-CH3 Fc domain of human IgG4 expressed in CHO-K1 cells, the fusion protein having the exemplary amino acid sequence of SEQ ID NO: 104.

[0085] In some embodiments, the antibody or antigen-binding fragment thereof is an anti-PD-1 antibody or antigen-binding fragment. Programmed death-ligand 1 (PD-L1) is a ligand for the immunosuppressive receptor "Programmed Death Receptor 1 (PD-1)" that is primarily expressed on activated T cells and B cells and can negatively regulate antigen receptor signaling. The ligands for PD-1 (PD-L1 and PD-L2) can be constitutively expressed or induced on many cell types, including non-hematopoietic tissues and various tumor types. PD-L1 is expressed on B cells, T cells, myeloid cells, and dendritic cells (DCs), but also on peripheral cells, pseudovascular endothelial cells, and non-lymphoid organs such as the heart and lungs. A non-limiting example of a human PD-L1 amino acid sequence is MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 330).

[0086] In one embodiment, the anti-PD-L1 antibodies or antigen-binding fragments of the disclosure comprise a combination of heavy chain variable regions (VH) and light chain variable regions (VL) described herein. In another embodiment, the anti-PD-1L antibodies or antigen-binding fragments of the disclosure comprise a combination of complementarity determining regions (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) described herein. In one embodiment, the anti-PD-L1 antibody or anti-PD-L1 antigen-binding fragment of the disclosure is a modified avelumab (BAVENCIO, 451238, KXG2PJ551I, MSB-0010682, MSB-0010718C, PF-06834635, CAS1537032-82-8: EMD Serono, Merck & Co., Merck KGaA, Merck Serono, National Cancer Institute (NCI), Pfizer), durvalumab (IMFINZI, 28X28X9OKV (UNII code), MEDI-4736, CAS1428935-60-7: AstraZeneca, Celgene, Children's Hospital Los Angeles (CHLA), City of Hope National Medical Center, MedImmune, Memorial Sloan-Kettering Cancer Center, Mirati Therapeutics, National Cancer Institute (NCI), Pfizer), Institute (NCI), Samsung Medical Center (SMC), Washington University), atezolizumab (Tecentriq, 52CMI0WC3Y, MPDL-3280A, RG-7446, RO-5541267, CAS1380723-44-3: Academisch Medisch Centrum (AMC), Chugai Pharmaceutical, EORTC, Genentech, Immune Design (Merck & Co.), Memorial Sloan- Kettering Cancer Center, National Cancer Institute (NCI), Roche,Roche Center for Medical Genomics) (CS-1001, WBP-3155:CStone Pharmaceuticals, EQRx, Pfizer), KN-046(CAS2256084-03-2:Jiangsu Alphamab). Biopharmaceuticals, Sinovent, APL-502(CBT-502, TQB-2450:Apollomics, Jiangsu Chia Tai Tianqing Pharmaceutical) ダダ ダ (3D-025 ASC-22 KN-035 hu56V1-Fc-m1 CAS2102192-68-5:3D Medicines. Ascletis. Jiangsu Alphamab Biopharmaceuticals,Suzhou Alphamab,Tracon Pharmaceuticals, Inc.) Marketing Corporation (M-7824, MSB-0011359C, NW9K8C1JN3, CAS1918149-01-5:EMD Serono, GlaxoSmithKline, Merck KGaA, National Cancer Institute (NCI), STI-1014 (STI-A1014, ZKAB-001: Lee's Pharmaceutical, Sorrento Therapeutics), PD-L1 t-haNK(ImmunityBio,NantKwest),A-167(HBM-9167,KL-A167:Harbor BioMed,Sichuan Kelun-Biotech Biopharmaceutical),BMI-001(STI-3031,STI-A-1015,STI-A1015,ImmuneOnce Therapeutics,Sorrento Therapeutics, HTI-1088 (SHR-1316: Atridia, Jiangsu Hengrui), IO-103 (IO Biotech), CX-072 (CytomX). Therapeutics), AUPM-170(CA-170:Aurigene,Curis),GS-4224(Gilead),ND-021(NM21-1480,PRO-1480:CStone Pharmaceuticals,Numab Therapeutics),BNT-311(DuoBody-PD-L1x4-1BB,GEN-1046:BioNTech,Genmab),BGB-A333(BeiGene),IBI-322(Innovent Biologics),NM-01(Nanomab Technology,Shanghai First People’s Hospital),LY-3434172(Eli Lilly),LDP(Dragonboat Biopharmaceutical),CDX-527(Celldex Therapeutics),IBI-318(Innovent Biologics, Lilly),89Zr-DFO-REGN3504(Regeneron),ALPN-202(CD80 vIgD-Fc:Alpine Immune Sciences),INCB-086550(Incyte),LY-3415244(Eli Lilly),SHR-1701(Jiangsu Hengrui),JS-003(JS003-30,JS003-SD:Shanghai Junshi Biosciences),HLX-20(PL2#3:Henlix Biotech,Shanghai Henlius Biotech),ES-101(INBRX-105,INBRX-105-1:Elpiscience BioPharma,Inhibrx),MSB-2311(MabSpace Biosciences),PD-1-Fc-OX40L(SL-279252,TAK-252:Heat Biologics,Shattuck Labs,Takeda),FS-118,FS118 mAb2,LAG-3 / PD-L1 mAb2:F-star Therapeutics,Merck & Co., Merck KGaA),FAZ-053(LAE-005: Laekna Therapeutics,Novartis),Lodapolimab(LY-3300054,NR4MAD6PPB,CAS2118349-31-6:Eli Lilly), MCLA-145(Incyte,Merus),BMS-189 (BMS-986189,Bristol-Myers SquibbのPD-L1-Milla),コシベリマブ(CK-301,TG-1501,CAS2216751-26-5:Checkpoint Therapeutics,Dana-Farber Cancer Institute,Samsung Biologics,TG Therapeutics),IL-15Ralpha-SD / IL-15(KD-033:Kadmon),WP-1066(CAS857064-38-1:M.D.Anderson Cancer Center,Moleculin Biotech),BMS-936559(MDX-1105:Bristol-Myers Squibb,Medarex,National Institute Allergy Infect Dis.),BMS-986192(Bristol-Myers Squibb),RC-98(RemeGen),CD-200AR-L(CD200AR-L:OX2 Therapeutics,University of Minnesota),ATA-3271(Atara Biotherapeutics),IBC-Ab002(ImmunoBrain Checkpoint),BMX-101(Biomunex Pharmaceuticals),AVA-04-VbP(Avacta),ACE-1708(Acepodia Biotech),KY-1043(Kymab,Provenance Biopharmaceuticals),ACE-05(YBL-013:Y-Biologics),ONC-0055(ONC0055,PRS-344 S-095012:Pieris Pharmaceuticals,Servier),TLJ-1-CK(I-Mab Biopharma),GR-1405(Chinese Academy of Medical Sciences),PD-1ACR-T(Taipei Medical University),N-809(N-IL15 / PD-L1:ImmunityBio),CB-201(Crescendo Biologics),MEDI-1109(MedImmune),AVA-004(AVA-04:Avacta),CA-327(Aurigene,Curis),ALN-PDL(Alnylam Pharmaceuticals),KY-1003(Kymab),These include CD22(aPD-L1) CAR-T cells (SL-22P: Hebei Senlang Biotechnology), ATA-2271 (M28z1XXPD-1DNR CAR T cells: Atara Biotherapeutics) and Zeushield cytotoxic T lymphocytes (Second Xiangya Hosp Central South Univ.).

[0087] In some embodiments, the anti-PD-L1 antibody is avelumab, durvalumab, atezolizumab, sugemalimab, emvafolimab, lodapolimab, or cosibelimab, or modified versions thereof. In some embodiments, the anti-PD-L1 antibody is avelumab, durvalumab, atezolizumab, sugemalimab, emvafolimab, lodapolimab, or cosibelimab. In some embodiments, the antibody is a biosimilar of avelumab, durvalumab, atezolizumab, sugemalimab, emvafolimab, lodapolimab, or cosibelimab.

[0088] Table 1 provides exemplary sequences of anti-PD-L1 antibodies and anti-PD-L1 antigen-binding fragments that may be modified to prepare anti-PD-L1 immunoconjugates. Table 1 also provides exemplary combinations of CDRs that may be utilized in the modified anti-PD-L1 immunoconjugates. References herein to anti-PD-L1 antibodies may alternatively refer to anti-PD-L1 antigen-binding fragments.

[0089] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence of any one of SEQ ID NOs: 232, 234, 236, 238, 242, 244, or 248. The anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence of any one of SEQ ID NOs: 233, 235, 237, 239, 243, 245, or 249. In one example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence of SEQ ID NO: 232, and a light chain or VL having the amino acid sequence of SEQ ID NO: 233. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence of SEQ ID NO: 234, and a light chain or VL having the amino acid sequence of SEQ ID NO: 235. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence of SEQ ID NO:236, and a light chain or VL having the amino acid sequence of SEQ ID NO:237. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence of SEQ ID NO:238, and a light chain or VL having the amino acid sequence of SEQ ID NO:239. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:242, and a VL having the amino acid sequence of SEQ ID NO:243. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO:244, and a VL having the amino acid sequence of SEQ ID NO:245. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence of SEQ ID NO:248, and a light chain or VL having the amino acid sequence of SEQ ID NO:249.

[0090] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence of any one of SEQ ID NOs: 232, 234, 236, 238, 242, 244, or 248, or a portion corresponding to the VH thereof. The anti-PD-L1 antibody or antigen-binding fragment comprises a light chain or VL having the amino acid sequence of any one of SEQ ID NOs: 233, 235, 237, 239, 243, 245, or 249, or a portion corresponding to the VL thereof. In one example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence set forth in SEQ ID NO: 232, and a light chain or VL having the amino acid sequence set forth in SEQ ID NO: 233. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence set forth in SEQ ID NO: 234, and a light chain or VL having the amino acid sequence set forth in SEQ ID NO: 235. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence set forth in SEQ ID NO:236, and a light chain or VL having the amino acid sequence set forth in SEQ ID NO:237. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence set forth in SEQ ID NO:238, and a light chain or VL having the amino acid sequence set forth in SEQ ID NO:239. In another example, the anti-PD-1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:242, and a VL having the amino acid sequence set forth in SEQ ID NO:243. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a VH having the amino acid sequence set forth in SEQ ID NO:244, and a VL having the amino acid sequence set forth in SEQ ID NO:245. In another example, the anti-PD-L1 antibody or antigen-binding fragment comprises a heavy chain or VH having the amino acid sequence set forth in SEQ ID NO:248, and a light chain or VL having the amino acid sequence set forth in SEQ ID NO:249.

[0091] In one example, the anti-PD-L1 antibody or antigen-binding fragment comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 250, a VH CDR2 having the amino acid sequence of SEQ ID NO: 251, a VH CDR3 having the amino acid sequence of SEQ ID NO: 252, a VL CDR1 having the amino acid sequence of SEQ ID NO: 253, a VL CDR2 having the amino acid sequence of SEQ ID NO: 254, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 255.

[0092] In one example, the anti-PD-L1 antibody comprises a single domain binding antibody having the amino acid sequence of SEQ ID NO:256, a trispecific fusion single chain antibody construct having the amino acid sequence of SEQ ID NO:257, or a bispecific tetrameric antibody-like engager having the amino acid sequence of SEQ ID NO:258. [Table 1] TIFF2025508823000003.tif236159TIFF2025508823000004.tif231159TIFF2025508823000005.tif234159TIFF2025508823000006.tif23415 9TIFF2025508823000007.tif234159TIFF2025508823000008.tif237159TIFF2025508823000009.tif237159TIFF2025508823000010.tif23615 9TIFF2025508823000011.tif238159TIFF2025508823000012.tif238159TIFF2025508823000013.tif234159TIFF2025508823000014.tif23315 9TIFF2025508823000015.tif236159TIFF2025508823000016.tif234159TIFF2025508823000017.tif232159TIFF2025508823000018.tif58159

[0093] Modifications to the Fc region Disclosed herein is an antibody or antigen-binding fragment thereof comprising an Fc region, the Fc region comprising at least one covalently linked linker. In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is covalently linked to a tyrosine, aspartic acid, glutamic acid, arginine, histidine, or lysine residue. In some embodiments, the chemical linker is covalently linked to a lysine, cysteine, or tyrosine residue. In some embodiments, the chemical linker is covalently linked to a cysteine ​​residue. In some embodiments, the chemical linker is covalently linked to a lysine residue. In some embodiments, the chemical linker is covalently linked to a constant region of an antibody.

[0094] In some embodiments, the antibody comprises an Fc region. In some embodiments, the Fc region is an IgG Fc region, an IgA Fc region, an IgD Fc region, an IgM Fc region, or an IgE Fc region. In some embodiments, the Fc region is an IgG Fc region, an IgA Fc region, or an IgD Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, the Fc region is a humanized Fc region. In some embodiments, the Fc region is an IgG Fc. In some examples, the IgG Fc region is an IgG1 Fc region, an IgG2a Fc region, or an IgG4 Fc region.

[0095] One or more mutations can be introduced into the Fc region to reduce the Fc-mediated effector functions of the antibody or antigen-binding fragment, such as antibody-dependent cellular cytotoxicity (ADCC) and / or complement function. In some examples, the modified Fc comprises a humanized IgG4 kappa isotype containing an S229P Fc mutation. In some examples, the modified Fc comprises a human IgG1 kappa, in which the heavy chain CH2 domain is engineered using triple mutations, such as (a) L238P, L239E, and P335S, or (2) K248, K288; and K317.

[0096] Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Cys Pro Cys Ser Cys Asp Lys Thr His Thr Cys Pro Cys Pro 1 Glu Xa Proa Glyx Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Asp Val His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asp Ser Thr Tyr Val Val Leu Val Leu Ser Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Pro Lys Asn Gln Val Ser Leu Thr Cys Leu Val Tyr Ply Gly I Asp Val Ser Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Xaa Thr Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala His His His Asn Asn Leu Sern LeuGly, where Xaa can be any naturally occurring amino acid. In some embodiments, the Fc region comprises one or more mutations that render the Fc region susceptible to modification or conjugation at a particular residue, such as, for example, by incorporation of a cysteine ​​residue at a position of SEQ ID NO:260 that does not contain a cysteine. Alternatively, the Fc region may be modified to incorporate modified natural or non-natural amino acids that comprise a conjugation handle, such as one connected to the modified natural or non-natural amino acid via a linker. In some embodiments, the Fc region does not comprise any mutations that facilitate attachment of the linker to an additional cytokine (e.g., an IL-18 polypeptide). In some embodiments, the chemical linker is attached to a natural residue represented by SEQ ID NO:260. In some embodiments, the chemical linker is attached to a native lysine residue represented by SEQ ID NO:260.

[0097] In some embodiments, the chemical linker is attached to the Fc region at one of amino acid residues at positions 10 to 90 of SEQ ID NO: 260. In some embodiments, the chemical linker is attached to the Fc region at one of amino acid residues at positions 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 1 to 80, 10 to 90, 10 to 100, 10 to 110, 10 to 120, 10 to 130, 10 to 140, 10 to 150, 10 to 160, 10 to 170, 10 to 180, 10 to 190, or 10 to 200 of SEQ ID NO: 260. In some embodiments, the chemical linker is attached to the Fc region at one of amino acid residues at positions 10 to 30, 50 to 70, or 80 to 100 of SEQ ID NO: 260. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues 20-40, 65-85, or 90-110 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues 15-26, 55-65, or 85-90 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues 25-35, 70-80, or 95-105 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues 30, 32, 72, 74, or 101 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at any one of amino acid residues K30, K32, K72, K74, or K101 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 30 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 32 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 72 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 74 of SEQ ID NO:260. In some embodiments, the chemical linker is attached to the Fc region at amino acid residue 101 of SEQ ID NO:260.

[0098] The chemical linker may be covalently bound to one amino acid residue of the Fc region of the antibody. In some embodiments, the chemical linker is covalently bound to a non-terminal residue of the Fc region. In some embodiments, the non-terminal residue is in the CH1, CH2, or CH3 region of the antibody. In some embodiments, the non-terminal residue is in the CH2 region of the antibody.

[0099] In some embodiments, the chemical linker is covalently attached to an amino acid residue of the antibody or antigen-binding fragment that selectively binds to the immune associated antigen (e.g., an anti-PD-1 antibody) such that the function of the antibody or antigen-binding fragment is maintained (e.g., without denaturing the polypeptide). For example, when the antibody or antigen-binding fragment is a human IgG (e.g., human IgG1), exposed lysine and exposed tyrosine residues are present at the following positions (see the website www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html (EU numbering)). Exemplary exposed lysine residues: CH2 domain (positions 246, 248, 274, 288, 290, 317, 320, 322, and 338), CH3 domain (positions 360, 414, and 439). Exemplary exposed tyrosine residues: CH2 domain (positions 278, 296, and 300), CH3 domain (position 436).

[0100] Human IgG, such as human IgG1, may also be modified with lysine or tyrosine residues at any one of the positions listed above to provide ideal surface-exposed residues for subsequent modification.

[0101] In some embodiments, the chemical linker is covalently attached to an amino acid residue in the constant region of the antibody. In some embodiments, the chemical linker is covalently attached to an amino acid residue in the CH1, CH2, or CH3 region. In some embodiments, the chemical linker is covalently attached to an amino acid residue in the CH2 region. In some embodiments, the chemical linker may be covalently attached to one amino acid residue in the following residue groups according to EU numbering in human IgG Fc: amino acid residues 1-478, amino acid residues 2-478, amino acid residues 1-477, amino acid residues 2-477, amino acid residues 10-467, amino acid residues 30-447, amino acid residues 50-427, amino acid residues 100-377, amino acid residues 150-327, amino acid residues 200-327, amino acid residues 240-327, and amino acid residues 240-320.

[0102] In some embodiments, the chemical linker is covalently attached to one lysine residue of the human IgG Fc region. In some embodiments, the chemical linker is covalently attached to Lys246 of the Fc region of the antibody, and the amino acid residue position number is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Lys248 of the Fc region of the antibody, and the amino acid residue position number is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Lys288 of the Fc region of the antibody, and the amino acid residue position number is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Lys290 of the Fc region of the 0 antibody, and the amino acid residue position number is based on Eu numbering. In some embodiments, the chemical linker is covalently attached to Lys317 of the antibody, and the amino acid residue position number is based on Eu numbering.

[0103] The chemical linker may be covalently attached to an amino acid residue selected from a subset of amino acid residues. In some embodiments, the subset comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues of the Fc region of the antibody. The chemical linker may be covalently attached to one of two lysine residues of the Fc region of the antibody.

[0104] In some embodiments, the antibody will comprise two linkers covalently linked to the Fc region of the antibody. In some embodiments, each of the two linkers will be covalently linked to different heavy chains of the antibody. In some embodiments, each of the two linkers will be covalently linked to different heavy chains of the antibody at the same residue position. In some embodiments, each of the two linkers will be covalently linked to different heavy chains of the antibody at different residue positions. When the two linkers are covalently linked to different residue positions, any combination of residue positions provided herein can be used in combination.

[0105] In some embodiments, the first chemical linker is covalently linked to Lys248 of the first Fc region of the antibody, and the second chemical linker is covalently linked to Lys288 of the second Fc region of the antibody, where the amino acid residue position number is based on Eu numbering. In some embodiments, the first chemical linker is covalently linked to Lys248 of the first Fc region of the antibody, and the second chemical linker is covalently linked to Lys317 of the second Fc region of the antibody, where the amino acid residue position number is based on Eu numbering. In some embodiments, the first chemical linker is covalently linked to Lys288 of the first Fc region of the antibody, and the second chemical linker is covalently linked to Lys317 of the second Fc region of the antibody, where the amino acid residue position number is based on Eu numbering.

[0106] Methods for modifying the Fc region Also provided herein are methods for preparing modified Fc regions of antibodies or antigen-binding fragments, e.g., for attachment to the antibodies or antigen-binding fragments of linkers, conjugation handles, IL-18 polypeptides, or any combination thereof. A variety of methods for site-specific modification of the Fc region of an antibody are known in the art.

[0107] Modification of the linker with an affinity peptide configured for site-specific attachment to the antibody In some embodiments, the Fc region is modified to incorporate a linker, a conjugation handle, or a combination thereof. In some embodiments, the modification is performed by contacting the Fc region with an affinity peptide having a payload configured to attach a linker or other group to the Fc region, such as at a specific residue of the Fc region. In some embodiments, the linker is attached using a reactive group that forms a bond with a residue of the Fc region. In some embodiments, the affinity peptide comprises a cleavable linker. The cleavable linker is configured on the affinity peptide such that after the linker or other group is attached to the Fc region, the affinity peptide can be removed, leaving only the desired linker or other group attached to the Fc region. The linker or other group can then be further used to additionally attach additional groups, such as a cytokine or a linker attached to the cytokine, to the Fc region.

[0108] Non-limiting examples of such affinity peptides can be found at least in PCT Publication Nos. WO2018199337A1, WO2019240288A1, WO2019240287A1, and WO2020090979A1, each of which is incorporated by reference in its entirety as if set forth herein. In some embodiments, the affinity peptide is a peptide modified to deliver a linker / conjugation handle payload to one or more specific residues of the Fc region of an antibody. In some embodiments, the affinity peptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a peptide selected from the following: (1) QETNPTENLYFQQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 261), (2) QTADNQKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDCSQSANLLAEAQQLNDAQAPQA (SEQ ID NO: 262), (3) QETKNMQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 263), (4) QETFNKQCQRRFYEALHDPNLNEEQRNARIRSIRDDDC (SEQ ID NO: 264), (5) QE TFNMQCQRRFYEALHDPNLNKEQRNARIRSIRDDDC (SEQ ID NO: 265), (6) QETFNMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 266), (7) QETMQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 267), (8) QETQCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 268), (9) QETCQRRFYEALHDPNLNEEQRNARIRSIKDDC (SEQ ID NO: 269), (10) QETRGNCAYHKGQLVWCTYH (SEQ ID NO: 270), and (11) QETRGNCAYHKGQIIWCTYH (SEQ ID NO: 271), or the corresponding peptides truncated by 1, 2, 3, 4, or 5 residues at the N-terminus.

[0109] Exemplary affinity peptides having a cleavable linker and conjugation handle payload capable of attaching a payload to residue K248 of the antibodies provided herein are shown below (reported in Matsuda et al., “Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody-Drug Conjugates,” Mol. Pharmaceutics 2021, 18, 11, 4058-4066). [ka]

[0110] Alternate affinity peptides targeting alternative residues in the Fc region are described in the references cited above for AJICAP™ technology, and such affinity peptides can be used to attach desired functional groups to alternative residues in the Fc region (e.g., K246, K288, etc.). For example, the disulfide groups of the affinity peptides described above may instead be replaced with thioesters to provide sulfhydryl protecting groups as cleavable portions of the linking group (e.g., the relevant portion of the affinity peptide is [ka] or another cleavable linker as discussed below). Such alternative affinity peptides include, for example, those described in "AJICAP Second Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody-Drug Conjugation Technology for Antibody-Drug Conjugate Production" (Working Paper, Fujii et al., DOI: 10.26434 / chemrxiv-2023-9p5p7, chemrxiv.org / engage / chemrxiv / article-details / 63d5f7131125965a9e7df8a5 (accessed February 20, 2023, Version 1 published January 30, 2023)). Exemplary affinity peptides provided above include those shown below, where the structure on the left targets K248 in the Fc region and the structure on the right targets K288 in the Fc region (EU numbering). [ka]

[0111] The affinity peptide of the present disclosure may include a cleavable linker. In some embodiments, the cleavable linker of the affinity peptide connects the affinity peptide to a group that is attached to the Fc region and is configured so that the peptide can be cleaved after the group that includes the linker or conjugation handle is attached. In some embodiments, the cleavable linker is a divalent group. In some embodiments, the cleavable linker is a thioester group, an ester group, a sulfane group, a methanimine group, an oxyvinyl group, a thiopropanoate group, an ethane-1,2-diol group, an (imidazol-1-yl)methan-1-one group, a selenoether group, a silyl ether group, a dioxysilane group, an ether group, a dioxymethane group, a tetraoxospiro[5.5]undecane group, an acetamidoethyl phosphoramidite group, a bis(methylthio)-pyrazolopylidene ... The cleavable linker may include a cyclodextrin-dione group, a 2-oxo-2-phenylethyl formate group, a 4-oxybenzyl carbamate group, a 2-(4-hydroxy-oxyphenyl)diazinyl)benzoate group, a 4-amino-2-(2-amino-2-oxoethyl)-4-oxobut-2-enoate group, a 2-(2-methylenehydrazinyl)pyridine group, an N'-methyleneformohydrazide group, or an isopropyl carbamate group, any of which may be unsubstituted or substituted. Compositions and attachment points of the cleavable linker to the affinity peptide, as well as related methods of use, are described at least in PCT Publication No. WO2018199337A1, PCT Publication No. WO2019240288A1, PCT Publication No. WO2019240287A1, and PCT Publication No. WO2020090979A1.

[0112] In some embodiments, the cleavable linker is: [ka] During the ceremony, - one of A or B is the point of attachment of the linker and the other of A or B is the point of attachment to the affinity peptide; -Each R 2a is independently H or optionally substituted alkyl; -Each R 2b is independently H or optionally substituted alkyl; -R 2c is H or optionally substituted alkyl, -J is a methyl, N, S, Si or O atom, and -r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0113] The affinity peptide comprises a reactive group configured to allow the covalent attachment of a linker / conjugation handle to the Fc region. In some embodiments, the reactive group is selective for a specific amino acid residue functional group, such as a lysine residue, a tyrosine residue, a serine residue, a cysteine ​​residue, or a functional group of a non-natural amino acid residue of the Fc region that is incorporated to facilitate the attachment of a linker. The reactive group may be any suitable functional group, such as an activated ester (e.g., an N-hydroxysuccinimide ester or derivative thereof, a pentafluorophenyl ester, etc.) for reaction with lysine, or a sulfhydryl reactive group (e.g., an alpha-beta unsaturated carbonyl or a Michael acceptor such as a maleimide) for reaction with cysteine. In some embodiments, the reactive group is: [ka] During the ceremony, -Each R 5a , R 5b , and R 5c is independently H, halogen, or optionally substituted alkyl; - each j is 1, 2, 3, 4, or 5; and - each k is 1, 2, 3, 4, or 5.

[0114] In some embodiments, an affinity peptide is used to deliver a reactive moiety to a desired amino acid residue, so that the reactive moiety is exposed upon cleavage of the cleavable linker. As a non-limiting example, the reactive group forms a covalent bond with a desired residue of the Fc region of an antibody or antigen-binding fragment due to the interaction between the affinity peptide and the Fc region. Following the formation of this covalent bond, the cleavable linker is cleaved under appropriate conditions to expose the reactive moiety (e.g., if the cleavable linker comprises a thioester, a free sulfhydryl group is attached to the Fc region after cleavage of the cleavable linker). This new reactive moiety can then be used to subsequently add additional moieties, such as conjugation handles, by a reagent that comprises a conjugation handle tethered to a sulfhydryl reactive group (e.g., an alpha-halogenated carbonyl group, an alpha-beta unsaturated carbonyl group, a maleimide group, etc.).

[0115] In some embodiments, affinity peptides are used to deliver free sulfhydryl groups to the lysines of the Fc region. In some embodiments, the free sulfhydryl groups are then reacted with bifunctional linking reagents to attach new conjugation handles to the Fc region. In some embodiments, the new conjugation handles are then used to form linkers for the bound cytokines. In some embodiments, the new conjugation handles are alkyne functional groups. In some embodiments, the new conjugation handles are DBCO functional groups.

[0116] Exemplary bifunctional linking reagents useful for this purpose are of the formula ABC, where A is a sulfhydryl-reactive conjugation handle (e.g., maleimide, α,β-unsaturated carbonyl, a-carbonyl halide), B is a linking group, and C is a novel conjugation handle (e.g., an alkyne such as DBCO). Specific non-limiting examples of bifunctional linking reagents include: [ka] and related molecules (eg, isomers) wherein each n is independently an integer from 1 to 6 and each m is independently an integer from 1 to 30.

[0117] Alternatively, the affinity peptide can be configured such that the conjugation handle is attached to the Fc region (such as by a linker group) immediately following formation of the covalent bond between the reactive group and a residue of the Fc region. In such a case, the affinity peptide is cleaved and immediately thereafter the conjugation handle is ready for subsequent conjugation to an IL-18 polypeptide.

[0118] Alternative methods for modifying the Fc region While the affinity peptide-mediated modification of the Fc region of the antibodies described above has many advantages over other methods that can be used to site-specifically modify Fc regions (e.g., ease of use, ability to rapidly generate many different antibody conjugates, ability to use many "off the shelf" commercially available antibodies without the need for time-consuming protein engineering, etc.), other methods of making such modifications are also contemplated as being within the scope of this disclosure.

[0119] In some embodiments, the present disclosure generally relates to transglutaminase-mediated site-specific antibody drug conjugates (ADCs) that include: 1) a glutamine-containing tag, an endogenous glutamine (e.g., a naturally occurring glutamine that is not engineered, such as a glutamine in a variable domain, CDR, etc.), and / or an endogenous glutamine that has been made reactive by antibody engineering or engineered transglutaminase; and 2) an amine donor agent that includes an amine donor unit, a linker, and a drug moiety. Non-limiting examples of such transglutaminase-mediated site-specific modifications are described at least in PCT Publication No. WO2020188061, U.S. Patent Publication No. US2019194641, U.S. Patent Publication No. US2021128743, U.S. Patent No. US9764038, and U.S. Patent No. US10434180, which are incorporated by reference in their entireties as if set forth herein in their entireties.

[0120] In another aspect, the disclosure provides an engineered Fc-containing polypeptide complex comprising the formula: (Fc-containing polypeptide-TA), where T is an engineered acyl donor glutamine-containing tag at a specific site, and A is an amine donor agent, where the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at the carboxyl terminus, amino terminus, or another site of the Fc-containing polypeptide, and the acyl donor glutamine-containing tag comprises the amino acid sequence XXQX, where X is any amino acid (e.g., X can be the same or different amino acids), and the engineered Fc-containing polypeptide complex comprises an amino acid substitution at position 295 for glutamine to asparagine (Q295N; EU numbering scheme).

[0121] In some embodiments, the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys (such as the ability to form a covalent bond as an amine donor in the presence of an acyl donor and transglutaminase) in the polypeptide or Fc-containing polypeptide. In some embodiments, the polypeptide or Fc-containing polypeptide comprises an amino acid modification at the last amino acid position within the carboxyl terminus compared to the wild-type polypeptide at the same position. The amino acid modification may be an amino acid deletion, insertion, substitution, mutation, or any combination thereof.

[0122] In some embodiments, the immunocytokine composition comprises a full-length antibody heavy chain and an antibody light chain, and an acyl donor glutamine-containing tag is located at the carboxyl-terminus of the heavy chain, the light chain, or both the heavy and light chains.

[0123] In some embodiments, the immunocytokine composition comprises an antibody, which is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment. In some embodiments, the antibody is an IgG.

[0124] In another aspect, provided herein is a method for preparing an engineered Fc-containing polypeptide complex comprising the formula (Fc-containing polypeptide-TA), wherein T is an engineered acyl donor glutamine-containing tag at a specific site, A is an amine donor agent, which is site-specifically conjugated to the acyl donor glutamine-containing tag at the carboxyl terminus, amino terminus, or another site of the Fc-containing polypeptide, the acyl donor glutamine-containing tag comprising the amino acid sequence XXQX, where X is any amino acid (e.g., X can be the same or different amino acids), and the engineered Fc-containing polypeptide complex comprises an acyl donor glutamine-containing tag that is conjugated to the carboxyl terminus, amino terminus, or another site of the Fc-containing polypeptide, the acyl donor glutamine-containing tag comprising the amino acid sequence XXQX, where X is any amino acid (e.g., X can be the same or different amino acids). The Fc-containing polypeptide complex comprises an amino acid substitution at position 295 for glutamine to asparagine (Q295N, EU numbering scheme), and the method comprises the steps of a) providing an engineered (Fc-containing polypeptide)-T molecule comprising the Fc-containing polypeptide and the acyl donor glutamine-containing tag, b) contacting the amine donor agent with the engineered (Fc-containing polypeptide)-T molecule in the presence of transglutaminase, and c) allowing the engineered (Fc-containing polypeptide)-T to covalently bind to the amine donor agent to form an engineered Fc-containing polypeptide complex.

[0125] In another aspect, provided herein is a method for preparing an engineered polypeptide complex comprising the formula: polypeptide-TA, where T is an engineered acyl donor glutamine-containing tag at a specific site, and A is an amine donor agent, where the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at the carboxyl terminus, amino terminus, or another site of the polypeptide, the acyl donor glutamine-containing tag comprising the amino acid sequence LLQGPX, where X is A or P (SEQ ID NO:272), or GGLLQGPP (SEQ ID NO:273), the method comprising the steps of: a) providing an engineered polypeptide-T molecule comprising a polypeptide and an acyl donor glutamine-containing tag, b) contacting the amine donor agent with the engineered polypeptide-T molecule in the presence of a transglutaminase, and c) allowing the engineered polypeptide-T to covalently bind to the amine donor agent to form the engineered Fc-containing polypeptide complex.

[0126] In some embodiments, an engineered polypeptide complex described herein (e.g., the engineered Fc-containing polypeptide complex, the engineered Fab-containing polypeptide complex, or the engineered antibody complex) has a conjugation efficiency of at least about 51%. In another aspect, the invention provides a pharmaceutical composition comprising an engineered polypeptide complex as described herein (e.g., the engineered Fc-containing polypeptide complex, the engineered Fab-containing polypeptide complex, or the engineered antibody complex) and a pharma- ceutically acceptable excipient.

[0127] In some embodiments, provided herein is a method for conjugating a moiety of interest (Z) to an antibody, comprising: (a) providing an antibody having at least one acceptor amino acid residue (e.g., a naturally occurring amino acid) that is reactive with a linking reagent (linker) in the presence of a coupling enzyme, such as a transamidase (e.g., in the primary sequence of the constant region); and (b) reacting the antibody with a linking reagent (e.g., a linker comprising a primary amine) that comprises a reactive group (R), optionally a protected reactive group, or optionally an unprotected reactive group, in the presence of an enzyme capable of causing the formation of a covalent bond between the acceptor amino acid residue and the linking reagent (other than the R moiety) under conditions sufficient to obtain an antibody that comprises an acceptor amino acid residue that is (covalently) linked to the reactive group (R) via a linking agent. Optionally, the acceptor residue of the antibody or antibody fragment is adjacent to a non-aspartic acid residue at the +2 position. Optionally, the residue at the +2 position is a non-aspartic acid residue. In one embodiment, the residue at position +2 is a non-aspartic acid, non-glutamine residue. In one embodiment, the residue at position +2 is a non-aspartic acid, non-asparagine residue. In one embodiment, the residue at position +2 is a non-negatively charged amino acid (an amino acid other than aspartic acid or glutamic acid). Optionally, the acceptor glutamine is in the Fc domain of the antibody heavy chain, and optionally further in the CH2 domain. Optionally, the antibody does not contain heavy chain N297-linked glycosylation. Optionally, the acceptor glutamine is at position 295 and the residue at position +2 is the residue at position 297 (EU index numbering) of the antibody heavy chain.

[0128] In one aspect, a method for conjugating a moiety of interest (Z) to an antibody is provided herein, the method comprising the steps of: (a) providing an antibody having at least one acceptor glutamine residue; and (b) reacting the antibody with a linker (lysine-based linker) comprising a reactive group (R), preferably a primary amine comprising a protected reactive group, in the presence of transglutaminase (TGase) under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked (covalently) to the reactive group (R) via the linker. Optionally, the acceptor glutamine residue of the antibody or antibody fragment is adjacent to a non-aspartic acid residue at the +2 position. Optionally, the residue at the +2 position is a non-aspartic acid residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, non-glutamine residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, non-asparagine residue. In one embodiment, the residue at position +2 is a non-negatively charged amino acid (an amino acid other than aspartic acid or glutamic acid). Optionally, the acceptor glutamine is in the Fc domain of the antibody heavy chain, and optionally further in the CH2 domain. Optionally, the antibody does not contain heavy chain N297-linked glycosylation. Optionally, the acceptor glutamine is at position 295, and the residue at position +2 is the residue at position 297 (EU index numbering) of the antibody heavy chain. The antibody containing the acceptor residue or acceptor glutamine residue linked to the reactive group (R) via a linker containing a primary amine (lysine-based linker) is then reacted with a reaction partner containing a moiety of interest (Z) to generate an antibody containing the acceptor residue or acceptor glutamine residue linked to the moiety of interest (Z) via the linker.Thus, in one embodiment, the method further comprises the step (c); (c) (i) reacting the antibody of step b) comprising an acceptor glutamine linked to a reactive group (R) via a linker comprising a primary amine (lysine-based linker), optionally immobilized on a solid support, with (ii) a compound comprising a moiety of interest (Z) and a reactive group (R') capable of reacting with the reactive group R, under conditions sufficient to obtain an antibody comprising an acceptor glutamine linked to a moiety of interest (Z) via a linker comprising a primary amine (lysine-based linker). Preferably, the compound comprising a moiety of interest (Z) and a reactive group (R') capable of reacting with the reactive group R is provided in less than 80-fold, 40-fold, 20-fold, 10-fold, 5-fold or 4 molar equivalents relative to the antibody. In one embodiment, the antibody comprises two acceptor glutamines, and the compound comprising a moiety of interest (Z) and a reactive group (R') is provided in less than 10 molar equivalents relative to the antibody. In one embodiment, the antibody comprises two acceptor glutamines and the compound comprising the moiety of interest (Z) and the reactive group (R') is provided in 5 or less molar equivalents relative to the antibody. In one embodiment, the antibody comprises four acceptor glutamines and the compound comprising the moiety of interest (Z) and the reactive group (R') is provided in 20 or less molar equivalents relative to the antibody. In one embodiment, the antibody comprises four acceptor glutamines and the compound comprising the moiety of interest (Z) and the reactive group (R') is provided in 10 or less molar equivalents relative to the antibody. In one embodiment, step (b) and / or step (c) are performed in aqueous conditions. Optionally, step (c) comprises: immobilizing a sample of an antibody comprising a functionalized acceptor glutamine residue of formula II on a solid support to provide a sample comprising immobilized antibody, reacting the sample comprising immobilized antibody, optionally recovering any unreacted compounds, reintroducing such recovered compounds to the solid support for reaction with the immobilized antibody, and eluting the antibody complex to provide an antibody composition comprising a Z moiety.

[0129] In alternative embodiments, the amino acid residues comprising the conjugation handle can be incorporated into the Fc region of the antibody (e.g., during expression of the antibody) at a desired position (e.g., any of the positions provided herein). In some embodiments, the amino acid residues comprising the conjugation handle are non-naturally occurring amino acids.

[0130] Conjugation Handle Chemistry In some embodiments, an appropriately modified Fc region of the antibody or antigen-binding fragment will comprise a conjugation handle that is used to conjugate the antibody or antigen-binding fragment to an IL-18 polypeptide to produce the immunocytokine compositions provided herein.

[0131] Any suitable reactive group capable of reacting with a complementary reactive group attached to an IL-18 polypeptide may be used as a conjugation handle. In some embodiments, the conjugation handle comprises reagents for metal-mediated processes such as Cu(I)-catalyzed or "copper-free" alkyne-azide triazole formation reactions (e.g., strain-promoted cycloadditions), Staudinger ligation, inverse electron demand Diels-Alder (IEDDA) reactions, "photoclick" chemistry, tetrazine cycloadditions with trans-cyclooctene, potassium acyltrifluoroborate (KAT) ligation, or olefin metathesis and Suzuki-Miyaura or Sonogashira cross-couplings.

[0132] In some embodiments, the conjugation handle comprises a reagent for a "copper-free" alkyne azide triazole forming reaction. Non-limiting examples of the alkyne for the azide triazole forming reaction include cyclooctyne reagents (e.g., (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol-containing reagents, dibenzocyclooctyne-amine reagents, difluorocyclooctyne, or derivatives thereof). In some embodiments, the alkyne functional group is attached to an Fc region. In some embodiments, the azide functional group is attached to an Fc region.

[0133] In some embodiments, the conjugation handle comprises a reactive group selected from azides, alkynes, tetrazines, halides, sulfhydryls, disulfides, maleimides, activated esters, alkenes, aldehydes, ketones, imines, hydrazines, potassium acyltrifluoroborates, hydroxylamines (e.g., O-substituted hydroxylamines), and hydrazides. In some embodiments, the IL-18 polypeptide comprises a reactive group complementary to the conjugation handle of the Fc region. In some embodiments, the conjugation handle and the complementary conjugation handle comprise a "CLICK" chemical reagent. Exemplary groups of click chemistry moieties are provided in Hein et al., "Click Chemistry, A Powerful Tool for Pharmaceutical Sciences," Pharmaceutical Research, volume 25, pages 2216-2230 (2008); Thirumurugan et al., "Click Chemistry for Drug Development and Diverse Chemical-Biology Applications," Chem. Rev. 2013, 113, 7, 4905-4979; US20160107999A1; US10266502B2; and US20190204330A1, each of which is incorporated by reference in its entirety.

[0134] Linker Structure In some embodiments, the linker used to link the antibody or antigen-binding fragment to the IL-18 polypeptide includes a point of attachment at both moieties. The point of attachment can be any of the residues provided herein to facilitate attachment. The linker structure can be any suitable structure to create a spatial link between the two moieties. In some embodiments, the linker provides a covalent linkage of both moieties. In some embodiments, the linker is a chemical linker (e.g., not an expressed polypeptide, such as in a fusion protein). In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-peptide linker (e.g., not consisting of amino acid residues).

[0135] Chemical Linkers In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker comprises at least one moiety that is not composed of an amino acid residue. In some embodiments, the linker comprises a polymer. In some embodiments, the linker comprises a water-soluble polymer. In some embodiments, the linker comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof. In some embodiments, the linker comprises a poly(alkylene oxide). In some embodiments, the poly(alkylene oxide) is polyethylene glycol or polypropylene glycol, or a combination thereof. In some embodiments, the poly(alkylene oxide) is polyethylene glycol.

[0136] In some embodiments, the linker is a bifunctional linker. In some embodiments, the bifunctional linker comprises an amide group, an ester group, an ether group, a thioether group, or a carbonyl group. In some embodiments, the linker comprises a non-polymeric linker. In some embodiments, the linker is a non-polymeric bifunctional linker. In some embodiments, the non-polymeric bifunctional linker comprises 4-(N-maleimidomethyl)cyclohexane-1-carboxylate succinimidyl, maleimidocaproyl; valine-citrulline; allyl(4-methoxyphenyl)dimethylsilane, 6-(allyloxycarbonylamino)-1-hexanol, 4-aminobutyraldehyde diethylacetal, or (E)-N-(2-aminoethyl)-4-{2-[4-(3-azidopropoxy)phenyl]diazenyl}benzamide hydrochloride.

[0137] The linker may be branched or linear. In some embodiments, the linker is linear. In some embodiments, the linker is branched. In some embodiments, the linker comprises a linear portion of the chain (e.g., between the first attachment point and the second attachment point) of at least 10, 20, 50, 100, 500, 1000, 2000, 3000, or 5000 atoms. In some embodiments, the linker comprises a linear portion of the chain of at least 10, 20, 30, 40, or 50 atoms. In some embodiments, the linker comprises a linear portion of at least 10 atoms. In some embodiments, the linker is branched and comprises a linear portion of the chain of at least 10, 20, 50, 100, 500, 1000, 2000, 3000, or 5000 atoms. In some embodiments, the linker may have 1 to 1000 atoms, 1 to 900 atoms, 1 to 800 atoms, 1 to 500 atoms, 1 to 400 atoms, 1 to 300 atoms, 1 to 200 atoms, 1 to 100 atoms, 1 to 50 atoms, 10 to 1000 atoms, 10 to 900 atoms, 10 to 800 atoms, 10 to 500 atoms, 10 to 400 atoms, 10 to 300 atoms, 10 to 200 atoms, 10 to 100 atoms, 10 to The linker comprises a linear portion of 50 atoms, 25-1000 atoms, 25-900 atoms, 25-800 atoms, 25-500 atoms, 25-400 atoms, 25-300 atoms, 25-200 atoms, 25-100 atoms, 25-50 atoms, 50-1000 atoms, 50-900 atoms, 50-800 atoms, 50-500 atoms, 50-400 atoms, 50-300 atoms, 50-200 atoms, or 50-100 atoms. In some embodiments, the linker has a linear length of about 10 angstroms to about 200 angstroms. In some embodiments, the linker has a linear chain length of about 10-500, 10-200, 10-150, 10-125, 10-100, 10-75, 10-50, 25-200, 25-150, 25-125, 25-100, 25-75, 25-50, 50-200, 50-150, 50-100, or 50-75 angstroms.

[0138] In some embodiments, the linker has a molecular weight of about 200 daltons to about 2000 daltons. In some embodiments, the linker has a molecular weight of about 200 daltons to about 5000 daltons. In some embodiments, the linker has a molecular weight of 200 daltons to about 100,000 daltons. In some embodiments, the linker has a molecular weight of at least about 500 daltons, at least about 1,000 daltons, at least about 5,000 daltons, at least about 10,000 daltons, at least about 15,000 daltons, at least about 20,000 daltons, at least about 25,000 daltons, or at least about 30,000 daltons. In some embodiments, the linker has a molecular weight of up to about 100,000 daltons, up to about 50,000 daltons, up to about 40,000 daltons, up to about 30,000 daltons, up to about 25,000 daltons, up to about 20,000 daltons, up to about 15,000 daltons, up to about 10,000 daltons, or up to about 5,000 daltons.

[0139] In some embodiments, the linker comprises a reaction product of one or more pairs of a conjugation handle and its complementary conjugation handle. In some embodiments, the reaction product comprises a triazole, a hydrazone, a pyridazine, a sulfide, a disulfide, an amide, an ester, an ether, an oxime, an alkene, or any combination thereof. In some embodiments, the reaction product comprises a triazole. The reaction product can be separated from the first and second attachment points by any portion of the linker. In some embodiments, the reaction product is substantially at the center of the linker. In some embodiments, the reaction product has one attachment point substantially closer than the other attachment point.

[0140] In some embodiments, the linker has the formula (X): [ka] The structure includes In the formula, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , L 8 , and L 9 each independently represents -O-, -NR L -, -(C1-C6 alkylene)NR L -, -NR L (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R L )2 + -, -N(R L )2 + -(C1-C6 alkylene)-, -OP(=O)(OR L )O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -C(=O)NR L (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR L -, -NR L C(=O)-, -(C1-C6 alkylene)NR L C(=O)-, -NR L C(=O)(C1-C6 alkylene)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -,-C(=O)NR L S(=O)2-, -S(=O)2NR LC(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd -, the reaction product of a conjugation handle and a complementary conjugation handle, or the absence thereof; R L are each independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; each [ka] is a point of attachment to said antibody or antigen-binding fragment or said IL-18 polypeptide.

[0141] In some embodiments, the linker is comprised of multiple structures of formula (X) that form a linkage between the antibody or antigen-binding fragment and the IL-18 polypeptide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more structures of formula (X) attached end-to-end, with the terminal [ka] only shows the point of attachment to the antibody or antigen-binding fragment or IL-18 polypeptide).

[0142] In some embodiments, the polymer has the formula (X'): [ka] and a linker comprising the structure In the formula, each L' is independently -O-, -NR L -, -(C1-C6 alkylene)NR L -, -NR L (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R L )2 + -, -N(R L )2 + -(C1-C6 alkylene)-, -OP(=O)(OR L )O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -C(=O)NR L (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR L -, -NR L C(=O)-, -(C1-C6 alkylene)NR L C(=O)-, -NR L C(=O)(C1-C6 alkylene)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR LC(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd -, the reaction product of a conjugation handle with a complementary conjugation handle, or absent; (C1-C6 alkylene), R L each is independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; g is an integer from 1 to 100; each [ka] is the point of attachment to the modified IL-18 polypeptide or the antibody or antigen-binding fragment.

[0143] In some embodiments, the linker of formula (X) or formula (X′) has the following structure: [ka] (In the formula, [ka] is a first point of attachment to a lysine residue on the antibody or antigen-binding fragment, L is a linking group, and [ka] is the point of attachment to the linking group that is connected to the first point of attachment. or positional isomers thereof.

[0144] In some embodiments, L is the structure [ka] wherein each n is independently an integer from 1 to 6 and each m is an integer from 1 to 30. In some embodiments, each m is independently 2 or 3. In some embodiments, each m is an integer from 1 to 24, 1 to 18, 1 to 12, or 1 to 6.

[0145] In some embodiments, the compound of formula (X) or (X a ) or the linker of formula (X') is of the structure [ka] (In the formula, [ka] is a first point of attachment to a lysine residue of the polypeptide that selectively binds PD-1, L is a linking group, and [ka] is the point of attachment to the linking group that is connected to the first point of attachment. or positional isomers thereof.

[0146] In some embodiments, L″ is the structure [ka] wherein each n is independently an integer from 1 to 6 and each m is an integer from 1 to 30. In some embodiments, each m is independently 2 or 3. In some embodiments, each m is an integer from 1 to 24, 1 to 18, 1 to 12, or 1 to 6.

[0147] In some embodiments, L or L″ is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more. [ka] where each n is independently an integer from 1 to 30. In some embodiments, each n is independently an integer from 1 to 6. In some embodiments, L or L″ comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits.

[0148] In some embodiments, L or L″ is a group represented by the formula (X″) [ka] The structure is In the formula, L 1a , L 2a , L 3a , L 4a , L 5a each independently represents -O-, -NR La -, -(C1-C6 alkylene)NR La -, -NR La (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R La )2 + (C1-C6 alkylene)-, -N(R L )2 + -,-OP(=O)(OR La)O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR La -, -C(=O)NR La (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR La -, -NR La C(=O)-, -(C1-C6 alkylene)NR La C(=O)-, -NR La C(=O)(C1-C6 alkylene)-, -OC(=O)NR La -, -NR La C(=O)O-, -NR La C(=O)NR La -, -NR La C(=S)NR La -, -CR La =N-, -N=CR La , -NR La S(=O)2-, -S(=O)2NR La -, -C(=O)NR La S(=O)2-, -S(=O)2NR La C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C2-C 20 Heteroarylene, -(CH2-CH2-O) qe -, -(O-CH2-CH2) qf -, -(CH2-CH(CH3)-O) qg -, -(O-CH(CH3)-CH2) qh -, the reaction product of a conjugation handle with a complementary conjugation handle, or absent; (C1-C6 alkylene) Each R Laare independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qe, qf, qg, and qh independently represents an integer of 1 to 100.

[0149] In some embodiments, L or L″ comprises a linear chain of 2 to 10, 2 to 15, 2 to 20, 2 to 25, or 2 to 30 atoms. In some embodiments, the linear chain comprises one or more alkyl groups (e.g., lower alkyl (C1-C4)), one or more aromatic groups (e.g., phenyl), one or more amide groups, one or more ether groups, one or more ester groups, or any combination thereof.

[0150] In some embodiments, the linking group attached to the first point of attachment (e.g., the point of attachment to the IL-18 polypeptide) comprises poly(ethylene glycol). In some embodiments, the linking group comprises from about 2 to about 30 poly(ethylene glycol) units. In some embodiments, the linking group attached to the first point of attachment (e.g., the point of attachment to the IL-18 polypeptide) is a functional group that binds to a cytokine provided herein that comprises an azide (e.g., a triazole is the reaction product of an azide).

[0151] In some embodiments, the reaction product of the conjugation handle and the complementary conjugation handle independently comprises a triazole, a hydrazone, a pyridazine, a sulfide, a disulfide, an amide, an ester, an ether, an oxime, or an alkene. In some embodiments, the reaction product of the conjugation handle and the complementary conjugation handle comprises a triazole. In some embodiments, the reaction product of the conjugation handle and the complementary conjugation handle comprises: [ka] or the structure of a positional isomer or derivative thereof.

[0152] Peptide Linker In some embodiments, the antibody or antigen-binding fragment is linked to the IL-18 polypeptide via a peptide linker. In some embodiments, the antibody or antigen-binding fragment is linked to the IL-18 polypeptide as a fusion protein. In such cases, the linker comprises one or more peptide bonds between the antibody or antigen-binding fragment and the IL-18 polypeptide. In some embodiments, the linker between the antibody or antigen-binding fragment fusion protein and the IL-18 polypeptide is a bond. In some embodiments, the linker between the antibody or antigen-binding fragment fusion protein and the IL-18 polypeptide is a linkage peptide. Non-limiting examples of linkage peptides include (GS) n (SEQ ID NO: 224), (GGS) n (SEQ ID NO: 225), (GGGS) n (SEQ ID NO: 226), (GGSG) n (SEQ ID NO: 227), or (GGSGG) n (SEQ ID NO: 228), (GGGGS) n (SEQ ID NO: 229), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, the connecting peptide can be (GGGGS)3 (SEQ ID NO: 230) or (GGGGS)4 (SEQ ID NO: 231). In some embodiments, the IL-18 polypeptide is fused (optionally via a connecting peptide) to the C-terminus of the antibody or antigen-binding fragment. In some embodiments, the IL-18 polypeptide is fused (optionally via a connecting peptide) to the N-terminus of the antibody or antigen-binding fragment.

[0153] Cleavable Linkers In some embodiments, the linker (e.g., a chemical linker or a peptide linker provided herein) is a cleavable linker. In some embodiments, the cleavable linker is cleaved at, near, or in the tumor microenvironment. In some embodiments, the tumor is mechanically or physically cleaved at, near, or in the tumor microenvironment. In some embodiments, the tumor is chemically cleaved at, near, or in the tumor microenvironment. In some embodiments, the cleavable linker is a reduction-sensitive linker. In some embodiments, the cleavable linker is an oxidation-sensitive linker. In some embodiments, the cleavable linker is cleaved as a result of pH at, near, or in the tumor microenvironment. In some embodiments, the cleavable linker is cleaved by tumor metabolites at, near, or in the tumor microenvironment. In some embodiments, the cleavable linker is cleaved by proteases at, near, or in the tumor microenvironment.

[0154] IL-18 Polypeptides The present disclosure describes antibodies or antigen-binding fragments linked to interleukin-18 (IL-18) polypeptides as immunocytokine compositions and their use as therapeutic agents. IL-18 is a pro-inflammatory cytokine that induces biological activities that initiate or promote host defense and inflammation following infection or injury. IL-18 has been implicated in autoimmune diseases, myocardial function, emphysema, metabolic syndrome, psoriasis, inflammatory bowel disease, hemophagocytic syndrome, macrophage activation syndrome, sepsis, and acute kidney injury. In some disease models, IL-18 plays a protective role.

[0155] IL-18 also plays a major role in the production of IFNγ from T cells and natural killer cells. IFNγ is a T helper type 1 cytokine produced primarily by T cells, NK cells, and macrophages and is essential for innate and adaptive immunity against viral, some bacterial, and protozoal infections. IFNγ is also an important activator of macrophages and an inducer of class II major histocompatibility complex (MHC) molecule expression.

[0156] IL-18 forms a signaling complex by binding to the IL-18 alpha chain (IL-18Rα), the ligand-binding chain of mature IL-18. However, the binding affinity of IL-18 to IL-18Rα is low. In cells expressing the co-receptor IL-18 receptor beta chain (IL-18Rβ), a high-affinity heterodimeric complex is formed, which then activates cell signaling.

[0157] The activity of IL-18 is balanced by the presence of a high affinity naturally occurring IL-18 binding protein (IL-18BP). IL-18BP binds to IL-18 and neutralizes the biological activity of IL-18. Cell surface IL-18Rα competes with IL-18BP for IL-18 binding. Increased disease severity may be associated with an imbalance of IL-18 to IL-18BP, resulting in elevated levels of free IL-18 in the circulation. Figure 4 illustrates the mechanism of action of IL-18, IFNγ production, IL-18BP production, and inhibition of IL-18 activity by IL-18BP. IL-18 induces IFNγ production, which in turn induces IL-18BP production. IL-18BP then competes with IL-18Rα to inhibit IL-18 activity.

[0158] In some embodiments, the IL-18 polypeptides of the immunocytokines provided herein exhibit reduced binding to IL-18BP and retain binding to IL-18 receptor. The IL-18 polypeptides provided herein with this property can retain IL-18 receptor signaling activity (including induction of IFNγ production) even in the presence of IL-18BP. This allows the immunocytokines provided herein to retain IL-18 signaling activity well beyond a short period after administration or after repeated administration. In some embodiments, the modified IL-18 polypeptides with this property include modifications (e.g., substitutions, polymer attachments, or deletions) at one or more amino acid residues that convey this property to the IL-18 polypeptide. Examples of IL-18 polypeptides with this property are provided herein as well as those otherwise known, such as those described in Patent Cooperation Treaty Publication No. WO2019051015A1, which is incorporated herein by reference as if set forth in its entirety. In addition to the IL-18 polypeptides provided herein, these otherwise known IL-18 polypeptides or analogs thereof can be similarly modified at the point of attachment to the linkers provided herein.

[0159] Attachment points of chemical linkers to IL-18 polypeptides The immunocytokines provided herein include a linker having a point of attachment to an IL-18 polypeptide. In some embodiments, the linker is a chemical linker. As described above, the linker has a separate point of attachment to an antibody or antigen-binding fragment at any residue provided herein. The point of attachment to the IL-18 polypeptide is to a residue provided herein.

[0160] In some embodiments, the linker is attached to an amino acid residue of the IL-18 polypeptide. In some embodiments, the linker is attached to any amino acid residue of the IL-18 polypeptide (e.g., at a position corresponding to any one of positions 1-157 of SEQ ID NO:1). In some embodiments, the linker is attached at a non-terminal residue of the IL-18 polypeptide (e.g., a residue at a position corresponding to any one of positions 2-156 of SEQ ID NO:1). In some embodiments, the linker is attached at a non-terminal residue of the IL-18 polypeptide, and the IL-18 polypeptide is extended or truncated by one or more amino acids relative to SEQ ID NO:1.

[0161] In some embodiments, the linker is attached to the IL-18 polypeptide at a residue within the region including residues 2-156, where the numbering of residue positions is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue within the region including residues 30-150. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue within the region including residues 33-43, residues 60-100, residues 65-75, residues 80-90, residues 85-100, residues 90-110, residues 115-130, residues 120-130, or residues 140-150. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue selected from residues 38, 68, 69, 70, 76, 78, 85, 86, 95, 98, 121, 127, and 144. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue selected from 68, 69, 70, 85, 86, and 98. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 68, 69, or 70. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 85, 86, 95, or 98. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 68. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 69. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 70. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 85. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 86. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 95. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 98.

[0162] In some embodiments, the linker is attached to the IL-18 polypeptide at a residue known in the art to be compatible with attachment of a polymer to the IL-18 polypeptide without significantly affecting the biological activity of the IL-18 polypeptide. Examples of these residues include residues 38, 76, 78, 121, 127, and 144, as described in PCT Publication No. WO2004091517A2, which is incorporated herein by reference in its entirety.

[0163] In some embodiments, the residue to which the linker is attached is a natural amino acid residue. In some embodiments, the residue to which the linker is covalently attached is selected from cysteine, aspartic acid, asparagine, glutamic acid, glutamine, serine, threonine, lysine, and tyrosine. In some embodiments, the residue to which the linker is covalently attached is selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, lysine, and tyrosine. In some embodiments, the linker is covalently attached to a cysteine. In some embodiments, the linker is covalently attached to a lysine. In some embodiments, the linker is covalently attached to a glutamine. In some embodiments, the linker is covalently attached to an asparagine. In some embodiments, the residue to which the linker is attached is a tyrosine. In some embodiments, the residue to which the linker is attached is a natural amino acid at that position in SEQ ID NO:1.

[0164] In some embodiments, the linker is attached to a different natural amino acid that is substituted at the relevant position. The substitution can be for a naturally occurring amino acid that is more suitable for the attachment of additional functional groups (e.g., aspartic acid, cysteine, glutamic acid, lysine, serine, threonine, or tyrosine), a modified derivative of any natural amino acid, or any non-natural amino acid (e.g., an amino acid that contains a desired conjugation handle, such as an azide, an alkyne, or other click chemistry reagent). In some embodiments, the linker is site-specifically covalently attached to the natural amino acid.

[0165] In some embodiments, the linker is attached at a non-natural amino acid residue. In some embodiments, the non-natural amino acid residue comprises a conjugation handle. In some embodiments, the conjugation handle facilitates the addition of the linker to the modified IL-18 polypeptide. The conjugation handle can be any of the conjugation handles provided herein. In some embodiments, the linker is site-specifically covalently attached to the non-amino acid. Non-limiting examples of amino acid residues that comprise conjugation handles are disclosed, for example, in PCT Publication Nos. WO2015054658A1, WO2014036492A1, WO2021133839A1, WO2006069246A2, and WO2007079130A2, each of which is incorporated by reference as if set forth in its entirety.

[0166] In some embodiments, the linker is covalently attached at residue 68. In some embodiments, the linker is covalently attached at residue C68, C68E, C68D, C68Q, C68K, C68N, or C68Y. In some embodiments, the linker is covalently attached at residue C68. In some embodiments, the linker is covalently attached to the unnatural amino acid at residue 68.

[0167] In some embodiments, the linker is covalently attached at residue 69. In some embodiments, the linker is covalently attached at residue E69, E69C, E69D, E69Q, E69K, E69N, or E69Y. In some embodiments, the linker is covalently attached at residue E69. In some embodiments, the linker is covalently attached at residue E69C. In some embodiments, the linker is covalently attached to the non-natural amino acid at residue 69.

[0168] In some embodiments, the linker is covalently attached at residue 70. In some embodiments, the linker is covalently attached at residue K70, K70C, K70D, K70Q, K70E, K70N, or K70Y. In some embodiments, the linker is covalently attached at residue K70. In some embodiments, the linker is covalently attached at residue K70C. In some embodiments, the linker is covalently attached to the unnatural amino acid at residue 70.

[0169] In some embodiments, the linker is covalently attached at residue 85. In some embodiments, the linker is covalently attached at residue E85, E85C, E85D, E85Q, E85K, E85N, or E85Y. In some embodiments, the linker is covalently attached at residue E85. In some embodiments, the linker is covalently attached at residue E85C. In some embodiments, the linker is covalently attached to the unnatural amino acid at residue 85.

[0170] In some embodiments, the linker is covalently attached at residue 86. In some embodiments, the linker is covalently attached at residue M86C, M86D, M86Q, M86K, M86N, M86E, or M86Y. In some embodiments, the linker is covalently attached at residue M86C. In some embodiments, the linker is covalently attached to the unnatural amino acid at residue 86.

[0171] In some embodiments, the polymer is covalently attached at residue 95. In some embodiments, the polymer is covalently attached at residue T95, T95C, T95D, T95Q, T95K, T95N, T95E, or T95Y. In some embodiments, the polymer is covalently attached at residue T95C, T95D, T95Q, T95K, T95N, T95E, or T95Y. In some embodiments, the polymer is covalently attached at residue T95C. In some embodiments, the polymer is covalently attached to the unnatural amino acid at residue 95.

[0172] In some embodiments, the linker is covalently attached at residue 98. In some embodiments, the linker is covalently attached at residue D98, D98C, D98Q, D98K, D98N, D98E, or D98Y. In some embodiments, the linker is covalently attached at residue D98C. In some embodiments, the linker is covalently attached to the unnatural amino acid at residue 98.

[0173] In some embodiments, the linker is covalently attached via a modified natural amino acid. In some embodiments, the modified natural amino acid comprises a conjugation handle. In some embodiments, the linker is covalently attached via a modified natural amino acid α. In some embodiments, the modified amino acid α is an amino acid-PEG-azide group. In some embodiments, the modified amino acid α is a glutamic acid, aspartic acid, lysine, cysteine, or tyrosine modified to incorporate an azide group linked to the amino acid via a PEG spacer. In some embodiments, the modified amino acid α has a structure selected from the following: [ka] Each n is independently an integer from 1 to 30. In some embodiments, n is an integer from 1 to 20, 1 to 10, 2 to 30, 2 to 20, 2 to 10, 5 to 30, 5 to 20, or 5 to 10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 12. The modified amino acid α may be incorporated at any point of attachment of the IL-18 polypeptides provided herein. In some embodiments, the modified amino acid alpha is located at a position on the modified IL-18 polypeptide selected from residue 68, residue 69, residue 70, residue 85, residue 86, residue 95, or residue 98.

[0174] When an IL-18 polypeptide contains unnatural amino acids or modified natural amino acids (e.g., those provided herein for conjugation purposes), these amino acids can be incorporated into the IL-18 polypeptide using a number of techniques known in the art for introducing such modifications. For example, recombinant proteins with unnatural amino acids can be produced using the methods described in Patent Cooperation Treaty Publication Nos. WO2016115168, WO2002085923, WO2005019415, and WO2005003294. Alternatively or in combination, unnatural or modified natural amino acids can be incorporated into chemically synthesized proteins during synthesis.

[0175] Modifications to IL-18 Polypeptides In some embodiments, the IL-18 polypeptide of the immunocytokine comprises one or more modifications relative to the polypeptide of SEQ ID NO: 1. The modifications provided herein are in addition to any modifications at the attachment points described above. In some embodiments, the numbering of the residue positions of the modified IL-18 polypeptide is based on SEQ ID NO: 1 as the reference sequence.

[0176] Modifications of IL-18 polypeptides described herein include mutations, addition of various functional groups, deletion of amino acids, addition of amino acids, or any other changes to the wild-type version of the protein or protein fragment. Functional groups that can be added to the polypeptide include polymers, linkers, alkyl groups, detectable molecules such as chromophores or fluorophores, reactive functional groups, or any combination thereof. In some embodiments, functional groups are added to individual amino acids of the polypeptide. In some embodiments, functional groups are added site-specifically to the polypeptide.

[0177] In some embodiments, the IL-18 polypeptide of the immunocytokine contains one or more modifications (e.g., amino acid substitutions at residues to which the linker is not attached) in addition to the modifications necessary to attach the linker to the relevant residues of the IL-18 polypeptide. 37~193 (SEQ ID NO:1), which reflects the biologically active form of IL-18. Endogenously, IL-18 is initially expressed with an additional 36 amino acid segment at the N-terminus, which is cleaved by caspases to mediate biological activity.

[0178] In some embodiments, the IL-18 polypeptide of the immunocytokine described herein contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more modified amino acid residues.

[0179] In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:1.

[0180] In some embodiments, the IL-18 polypeptide of the immunocytokine provided herein comprises the amino acid sequence of any one of SEQ ID NOs:2-203 provided herein. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence that is at least about 85% identical to an amino acid sequence of SEQ ID NOs:2-203. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO:30. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO:30. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO:59. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence that is at least about 85% identical to SEQ ID NO:59.

[0181] In some embodiments, an IL-18 polypeptide of an immunocytokine described herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 amino acid substitutions, wherein the amino acid substitutions are compared to SEQ ID NO: 1. In some embodiments, the IL-18 polypeptide comprises between 1 and 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 1 or 2 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 6 amino acid substitutions, 1 to 7 amino acid substitutions, 1 to 8 amino acid substitutions, 2 to 3 amino acid substitutions, 2 to 4 amino acid substitutions, 2 to 5 amino acid substitutions, 2 to 6 amino acid substitutions, 2 to 7 amino acid substitutions, 2 to 8 amino acid substitutions, 2 to 9 amino acid substitutions, 3 or 4 amino acid substitutions, 3 to 5 amino acid substitutions, 3 to 6 amino acid substitutions, 3 to 7 amino acid substitutions, 3 to 9 amino acid substitutions, 4 or 5 amino acid substitutions, 4 to 6 amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 amino acid substitutions, 5 or 6 amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 amino acid substitutions, 6 or 7 amino acid substitutions, 6 to 9 amino acid substitutions, or 7 to 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises a maximum of 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions.

[0182] In some embodiments, an IL-18 polypeptide comprising an immunocytokine described herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 amino acid substitutions, where the amino acid substitutions are compared to SEQ ID NO: 1. In some embodiments, the IL-18 polypeptide comprises 1-9 naturally occurring amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 1 or 2 naturally occurring amino acid substitutions, 1-3 naturally occurring amino acid substitutions, 1-4 naturally occurring amino acid substitutions, 1-5 naturally occurring amino acid substitutions, 1-6 naturally occurring amino acid substitutions, 1-7 naturally occurring amino acid substitutions, 1-8 naturally occurring amino acid substitutions, 2-3 naturally occurring amino acid substitutions, 2-4 naturally occurring amino acid substitutions, 2-5 naturally occurring amino acid substitutions, 2-6 naturally occurring amino acid substitutions, 2-7 naturally occurring amino acid substitutions, 2-8 naturally occurring amino acid substitutions, 2-9 naturally occurring amino acid substitutions, 3 naturally occurring amino acid substitutions, 4 naturally occurring amino acid substitutions, 5 naturally occurring amino acid substitutions, 6 naturally occurring amino acid substitutions, 7 naturally occurring amino acid substitutions, 8 naturally occurring amino acid substitutions, 9 naturally occurring amino acid substitutions, 10 naturally occurring amino acid substitutions, 11 naturally occurring amino acid substitutions, 12 naturally occurring amino acid substitutions, 13 naturally occurring amino acid substitutions, 14 naturally occurring amino acid substitutions, 15 naturally occurring amino acid substitutions, 16 naturally occurring amino acid substitutions, 17 naturally occurring amino acid substitutions, 18 naturally occurring amino acid substitutions, 19 naturally occurring amino acid substitutions, 20 naturally occurring amino acid substitutions, 21 naturally occurring amino acid substitutions, 22 naturally occurring amino acid substitutions, 23 naturally occurring amino acid substitutions, 24 naturally occurring amino acid substitutions, 25 naturally occurring amino acid substitutions, 26 naturally occurring amino acid substitutions, 27 naturally occurring In some embodiments, the IL-18 polypeptide comprises 3 or 4 naturally occurring amino acid substitutions, 3 to 5 naturally occurring amino acid substitutions, 3 to 6 naturally occurring amino acid substitutions, 3 to 7 naturally occurring amino acid substitutions, 3 to 9 naturally occurring amino acid substitutions, 4 or 5 naturally occurring amino acid substitutions, 4 to 6 naturally occurring amino acid substitutions, 4 to 7 naturally occurring amino acid substitutions, 4 to 9 naturally occurring amino acid substitutions, 5 or 6 naturally occurring amino acid substitutions, 5 to 7 naturally occurring amino acid substitutions, 5 to 9 naturally occurring amino acid substitutions, 6 or 7 naturally occurring amino acid substitutions, 6 to 9 naturally occurring amino acid substitutions, or 7 to 9 naturally occurring amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 3 naturally occurring amino acid substitutions, 4 naturally occurring amino acid substitutions, 5 naturally occurring amino acid substitutions, 6 naturally occurring amino acid substitutions, 7 naturally occurring amino acid substitutions, or 9 naturally occurring amino acid substitutions. In some embodiments, the IL-18 polypeptide further comprises up to 4 naturally occurring amino acid substitutions, 5 naturally occurring amino acid substitutions, 6 naturally occurring amino acid substitutions, 7 naturally occurring amino acid substitutions, or 9 naturally occurring amino acid substitutions. In some embodiments, the IL-18 polypeptide further comprises up to 10 non-standard amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-natural amino acid substitutions.In some embodiments, the IL-18 polypeptide further comprises unnatural amino acid substitutions at residues M33, M51, N60, M86, M113, and / or M150. In some embodiments, the unnatural amino acid residues substituted for methionine are each independently norleucine or O-methyl-homoserine. In some embodiments, the IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 75, and 116. In some embodiments, the IL-18 polypeptide further comprises homoserine (Hse)31, Hse75, and Hse116. In some embodiments, the IL-18 polypeptide further comprises homoserine (Hse)31, Hse63, and Hse116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 63, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse63, Hse75, and Hse116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 67, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse67, Hse75, and Hse116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 57, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse57, Hse75, and Hse116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 50, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse50, Hse75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises non-natural amino acid substitutions at residues 31, 50, 75, and 121.In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse)31, Hse50, Hse75, and Hse121.

[0183] In some embodiments, the IL-18 polypeptide of the immunocytokine described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least nine amino acid substitutions relative to any one of SEQ ID NOs: 68, 92, 116, 140, or 170. In some embodiments, the modified IL-18 polypeptide described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least nine additional amino acid substitutions relative to any one of SEQ ID NOs: 68, 92, 116, 140, or 170. In some embodiments, the IL-18 polypeptide comprises between 1 and 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 1 or 2 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 6 amino acid substitutions, 1 to 7 amino acid substitutions, 1 to 8 amino acid substitutions, 2 to 3 amino acid substitutions, 2 to 4 amino acid substitutions, 2 to 5 amino acid substitutions, 2 to 6 amino acid substitutions, 2 to 7 amino acid substitutions, 2 to 8 amino acid substitutions, 2 to 9 amino acid substitutions, 3 or 4 amino acid substitutions, 3 to 5 amino acid substitutions, 3 to 6 amino acid substitutions, 3 to 7 amino acid substitutions, 3 to 9 amino acid substitutions, 4 or 5 amino acid substitutions, 4 to 6 amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 amino acid substitutions, 5 or 6 amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 amino acid substitutions, 6 or 7 amino acid substitutions, 6 to 9 amino acid substitutions, or 7 to 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises a maximum of 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions.

[0184] In some embodiments, one modification is at amino acid residue 6. In some embodiments, one modification is within amino acid residues 53-63. In some embodiments, one modification is at amino acid residue 53. In some embodiments, one modification is at amino acid residue 63.

[0185] In some embodiments, the IL-18 polypeptide comprises at least one modification to the amino acid sequence of SEQ ID NO:1 selected from Y01X, F02X, E06X, S10X, V11X, D17X, C38X, M51X, K53X, D54X, S55X, T63X, C68X, C76X, and C127X, where each X is independently a natural or unnatural amino acid. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at the attachment point of the linker, such as at residue 69, residue 70, residue 85, residue 86, residue 95, or residue 98. In some embodiments, the IL-18 polypeptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 1 selected from Y01G, F02A, E06K, S10T, V11I, D17N, C38S, C38A, C38Q, M51G, K53A, D54A, S55A, T63A, C68S, C68A, C76S, C76A, C127A, and C127S. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at the attachment point of the linker, such as E69C, K70C, E85C, M86C, T95C, or D98C.

[0186] In one aspect, described herein is a modified interleukin-18 (IL-18) polypeptide comprising E06K and K53A, wherein the numbering of residue positions of the IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the IL-18 polypeptide further comprises V11I. In some embodiments, the IL-18 polypeptide further comprises T63A. In some embodiments, the IL-18 polypeptide further comprises at least one of Y01X, S55X, F02X, D54X, C38X, C68X, E69X, K70X, C76X, or C127X, wherein each X is independently an amino acid or amino acid derivative. In some embodiments, the IL-18 polypeptide further comprises at least one of Y01G, S55A, F02A, D54A, C38S, C38A, C38Q, C68S, C68A, E69C, K70C, C76S, C76A, C127S, or C127A. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at the attachment point of the linker, such as at residue 69, residue 70, residue 85, residue 86, residue 95, or residue 98.

[0187] In some embodiments, the IL-18 peptide comprises at least one modification relative to the amino acid sequence of SEQ ID NO: 1, the modification being E06X, V11X, K53X, S55X, or T63X, where X is a natural or unnatural amino acid. In some embodiments, the IL-18 peptide comprises at least two modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications being either E06X and K53X, E06X and S55X, K53X and S55X, E06X and T63X, or K53X and T63X, where X is a natural or unnatural amino acid. In some embodiments, the IL-18 peptide comprises at least three modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications being either E06X, K53X and S55X, or E06X, K53X and T63X, where X is a natural or unnatural amino acid. In some embodiments, the IL-18 peptide comprises at least four modifications relative to the amino acid sequence of SEQ ID NO:1, the modifications including E06X, K53X, S55X, and T63X; E06X, K53X, S55X, and Y01X; E06X, K53X, S55X, and F02X; E06X, K53X, S55X, and D54X; E06X, K53X, S55X, and M51X; or C38X, C68X, C76X, and C127X, where X is a natural or unnatural amino acid. In each embodiment in which multiple amino acid residues are replaced with a natural or unnatural amino acid X, each X is independently the same or a different amino acid.

[0188] In some embodiments, the IL-18 peptide comprises at least one modification relative to the amino acid sequence of SEQ ID NO: 1, the modification being E06K, V11I, K53A, S55A, or T63A. In some embodiments, the IL-18 peptide comprises at least two modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications comprising E06K and K53A, E06K and S55A, K53A and S55A, E06K and T63A, or K53A and T63A. In some embodiments, the IL-18 peptide comprises at least three modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications comprising E06K, K53A, and S55A; E06K, V11I, and K53A; E06K, C38A, and K53A; or E06K, K53A, and T63A. In some embodiments, the IL-18 peptide comprises at least four modifications relative to the amino acid sequence of SEQ ID NO:1, the modifications including E06K, K53A, S55A, and T63A; E06K, K53A, S55A, and Y01G; E06K, K53A, S55A, and F02A; E06K, K53A, S55A, and D54A; E06K, K53A, S55A, and M51G; or C38S, C68S, C76S, and C127S. In some embodiments, the IL-18 peptide comprises at least six modifications relative to the amino acid sequence of SEQ ID NO:1, the modifications comprising E06K, K53A, C38S, C68S, C76S, and C127S; or K53A, T63A, C38S, C68S, C76S, and C127S. In some embodiments, the modified IL-18 polypeptide comprises at least seven modifications relative to the sequence of SEQ ID NO:1, the seven modifications comprising E6K, V11I, C38A, K53A, T63A, C76A, C127A. In some embodiments, the IL-18 peptide comprises at least eight modifications relative to the amino acid sequence of SEQ ID NO:1, the modifications comprising Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A. In some embodiments, the IL-18 peptide comprises at least eight modifications relative to the amino acid sequence of SEQ ID NO:1, the modifications including Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A.

[0189] In one aspect, provided herein is a modified IL-18 polypeptide provided herein, further comprising E06K and K53A, wherein the numbering of residue positions of the IL-18 polypeptide is based on SEQ ID NO:1 as a reference sequence. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO:30. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO:59. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at one or more cysteine ​​residues. In some embodiments, the IL-18 polypeptide comprises one or more cysteines substituted with either serine or alanine. In some embodiments, the IL-18 polypeptide comprises an amino acid substitution at each cysteine ​​residue of SEQ ID NO:1. In some embodiments, each cysteine ​​residue is substituted with serine or alanine. In some embodiments, the IL-18 polypeptide comprises amino acid substitutions at 1, 2, 3, 4, 5, or 6 methionine residues. In some embodiments, each substitution at a methionine residue is for an O-methyl-L-homoserine residue or a norleucine residue. In some embodiments, each methionine residue is substituted with an O-methyl-L-homoserine residue. In some embodiments, the IL-18 polypeptide comprises a homoserine residue at one of positions 31, 116, and 63 and 75. In some embodiments, the IL-18 polypeptide comprises a homoserine residue at one of positions 31, 116, 75, and 50, 57, 63, and 67. In some embodiments, the IL-18 polypeptide comprises a homoserine residue at one of positions 31, 121, 75, and 50, 57, 63, and 67.

[0190] In some embodiments, the IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NOs: 2-12. In some embodiments, the IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NOs: 13-23. In some embodiments, the modified IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 24-33. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO: 30 or SEQ ID NO: 59. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO: 30. In some embodiments, the polypeptide sequence is at least about 90% identical to SEQ ID NO: 30. In some embodiments, the polypeptide sequence is at least about 95% identical to SEQ ID NO: 30. In some embodiments, the polypeptide sequence is identical to SEQ ID NO: 30. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO: 59. In some embodiments, the polypeptide is at least about 90% identical to SEQ ID NO: 59. In some embodiments, the polypeptide sequence is at least about 95% identical to SEQ ID NO: 59. In some embodiments, the IL-18 polypeptide is recombinant. In some embodiments, the IL-18 polypeptide is one presented in Table 2. In some embodiments, the IL-18 polypeptide is one set forth in Table 3.

[0191] biological activity In some embodiments, the immunocytokine composition exhibits one or more activities associated with the antibody or antigen-binding fragment and / or the IL-18 polypeptide.

[0192] In some embodiments, the immunocytokine composition exhibits an ability to bind to the IL-18 receptor. In some embodiments, the immunocytokine composition exhibits an ability to bind to the IL-18 receptor equivalent to WT IL-18. In some embodiments, the immunocytokine composition exhibits an ability to bind to the IL-18 receptor (IL-18Rαβ) that is reduced by up to 2-fold, up to 5-fold, up to 10-fold, up to 20-fold, up to 50-fold, up to 100-fold, up to 200-fold, up to 300-fold, up to 400-fold, or up to 1000-fold compared to WT IL-18. In some embodiments, the immunocytokine composition exhibits an increased ability to bind to IL-18Rαβ. In some embodiments, the immunocytokine composition exhibits an ability to bind to IL-18Rαβ that is increased by at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold compared to WT IL-18.

[0193] In some embodiments, the immunocytokine composition exhibits the ability to stimulate the production of IFNγ upon contact with a cell (e.g., an immune cell such as a NK cell). In some embodiments, the ability of the immunocytokine composition to stimulate IFNγ production is somewhat reduced compared to WT IL-18. In some embodiments, the half-maximal effective concentration (EC 50 ) is up to 100-fold higher, up to 50-fold higher, up to 20-fold higher, up to 10-fold higher, up to 5-fold higher, or up to 2-fold higher than WT IL-18. In some embodiments, the ability of the immunocytokine composition to stimulate IFNγ production is increased compared to WT IL-18. In some embodiments, the half-maximal effective concentration (EC 50) is at least 5-fold lower, at least 10-fold lower, at least 20-fold lower, at least 50-fold lower, at least 75-fold lower, or at least 100-fold higher than WT IL-18.

[0194] In some embodiments, the immunocytokine composition exhibits the ability to stimulate the production of IFNγ upon contact with a cell (e.g., an immune cell such as a NK cell), which is only somewhat reduced compared to an IL-18 polypeptide not included in the immunocytokine composition (e.g., an unconjugated IL-18 polypeptide). In some embodiments, the EC of IFNγ stimulation 50 is up to 5-fold higher, up to 10-fold higher, up to 50-fold higher, or up to 100-fold higher than an IL-18 polypeptide not included in the immunocytokine composition. In some embodiments, the immunocytokine composition has a half maximal effective concentration (EC) that is within about 100-fold of the corresponding IL-18 polypeptide not included in the immunocytokine composition. 50 In some embodiments, the immune cytokine composition has a lower EC50 value than WT IL-18. 50 In some embodiments, the immune cytokine composition has an EC that is at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, or 100-fold lower than WT IL-18. 50 Shows.

[0195] In some embodiments, the immunocytokine composition exhibits a reduced ability to bind to IL-18 binding protein (IL-18BP). In some embodiments, the ability of the immunocytokine composition to bind to IL-18BP is reduced by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, or at least 100-fold compared to WT IL-18. In some embodiments, the immunocytokine composition exhibits no substantial ability to bind to IL-18BP.

[0196] In some embodiments, the immunocytokine composition exhibits a reduced ability to have its IFNγ production stimulatory activity inhibited by IL-18BP. In some embodiments, the ability of the immunocytokine composition to be inhibited by IL-18BP is measured by measuring the half-maximal inhibitory concentration (IC 50 In some embodiments, the immune cytokine composition is measured as an IC of inhibition of WT IL-18 by IL-18BP. 50 IC by IL-18BP is at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, at least 15-fold higher, at least 20-fold higher, at least 25-fold higher, at least 30-fold higher, at least 40-fold higher, or at least 50-fold higher than 50 In some embodiments, the immunocytokine composition exhibits an IC of inhibition of WT IL-18 by IL-18BP. 50 IC by IL-18BP was at least 100-fold higher than that by 50 In some embodiments, the immunocytokine composition exhibits an IC of inhibition of WT IL-18 by IL-18BP. 50 IC by IL-18BP was at least 200-fold higher than that by 50 In some embodiments, the immunocytokine composition exhibits an IC of inhibition of WT IL-18 by IL-18BP. 50 IC by IL-18BP was at least 500-fold higher than that by 50 In some embodiments, the immunocytokine composition exhibits an IC of inhibition of WT IL-18 by IL-18BP. 50 IC by IL-18BP was at least 1000-fold higher than that by 50 Shows.

[0197] In some embodiments, the immunocytokine composition retains the binding associated with the antibody or antigen-binding fragment. In some embodiments, the immunocytokine composition retains the binding of the antibody or antigen-binding fragment to its antigen. In some embodiments, the immunocytokine composition retains the binding affinity (K) of the antibody to its antigen that is within 5-fold of the binding affinity of the antibody that is not bound to the IL-18 polypeptide. D In some embodiments, the immunocytokine composition exhibits a binding affinity (K) for the antibody's antigen that is within 2.5-fold of the binding affinity of the antibody that is not bound to the IL-18 polypeptide. D ). In some embodiments, the binding is determined by ELISA. In some embodiments, the binding is determined by BLI.

[0198] In some embodiments, the immunocytokine composition retains binding to one or more Fc receptors associated with the antibody or antigen-binding fragment. In some embodiments, the Fc receptors are selected from FcRn, CD64, CD32a, CD16, and CD32b, or any combination thereof. In some embodiments, the immunocytokine composition retains binding to at least one Fc receptor with a binding affinity (K ) within 10-fold of the binding affinity of the antibody that is not bound to an IL-18 polypeptide. D In some embodiments, the immunocytokine composition exhibits a binding affinity (K) for at least one Fc receptor that is less than 10-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than the binding affinity of an antibody that is not bound to an IL-18 polypeptide. D In some embodiments, the immunocytokine composition exhibits a binding affinity (K) for each of FcRn, CD64, CD32a, CD16, and CD32B that is less than 10-fold greater, less than 5-fold greater, less than 4-fold greater, less than 3-fold greater, less than 2-fold greater, or greater than the binding affinity of an antibody that is not bound to an IL-18 polypeptide.D In some embodiments, the immunocytokine composition exhibits a binding affinity (K) for each of FcRn, CD64, CD32a, CD16, and CD32B that is within 10-fold of the binding affinity of an antibody that is not bound to an IL-18 polypeptide. D In some embodiments, the immunocytokine composition exhibits a binding affinity (K) for each of FcRn, CD64, CD32a, CD16, and CD32B that is within 20-fold of the binding affinity of an antibody that is not bound to an IL-18 polypeptide. D In some embodiments, the immunocytokine composition exhibits a binding affinity (K) for each of FcRn, CD64, CD32a, CD16, and CD32B that is within 50-fold of the binding affinity of an antibody that is not bound to an IL-18 polypeptide. D In some embodiments, the immunocytokine composition exhibits a binding affinity (K) for each of FcRn, CD64, CD32a, CD16, and CD32B that is within 100-fold of the binding affinity of an antibody that is not bound to an IL-18 polypeptide. D ) is shown.

[0199] In some embodiments, the immunocytokine composition exhibits synergistic efficacy due to the presence of both molecules in one molecule. In some embodiments, the immunocytokine composition exhibits improved activity compared to either molecule alone. In some embodiments, the immunocytokine composition exhibits improved anti-tumor growth inhibition compared to an antibody alone. In some embodiments, the immunocytokine composition exhibits improved anti-tumor growth inhibition compared to an antibody and an IL-18 polypeptide administered in combination. In some embodiments, the IL-18 polypeptide is administered in a half-life extended form (e.g., PEGylated, conjugated to an Fc region (e.g., an Fc fusion), or conjugated to a negative control antibody). In some embodiments, the immunocytokine composition exhibits similar or enhanced anti-tumor activity at the same concentration as an antibody administered alone. In some embodiments, the immunocytokine composition exhibits similar or enhanced anti-tumor activity when administered at a dose that is less than 0.5, 0.25, or 0.1 times the dose of the antibody alone.

[0200] Orthogonal Payloads In one non-limiting example, the antibody / IL-18 immunocytokine of the present disclosure may comprise a dual orthogonal payload. The antibody / IL-18 immunocytokine may comprise an antibody, an IL-18 polypeptide, and a payload linked to the antibody by a chemically orthogonal linking group. The orthogonal payload may be an amino acid, an amino acid derivative, a peptide, a protein, a cytokine, an alkyl group, an aryl or heteroaryl group, a therapeutic small molecule drug, a polyethylene glycol (PEG) moiety, a lipid, a sugar, biotin, a biotin derivative, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a peptide nucleic acid (PNA), either substituted, unsubstituted, modified, or unmodified. In some embodiments, the orthogonal payload is a therapeutic small molecule. In some embodiments, the orthogonal payload is a PEG moiety. In some embodiments, the orthogonal payload is an additional cytokine, such as, for example, IL-7 or IL-2. In one exemplary example, human IL-7 is DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 210), or is a modified human IL-7 (e.g., an IL-7 having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to human IL-7). In one typical example, the human IL-2 has an amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 211), or is a modified human IL-2 (e.g., an IL-2 having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to human IL-2).

[0201] composition In one aspect, provided herein is a pharmaceutical composition comprising an antibody linked to a modified IL-18 polypeptide as described herein and a pharma- ceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises one or more excipients, including but not limited to carbohydrates, inorganic salts, antioxidants, surfactants, buffers, or any combination thereof. In some embodiments, the pharmaceutical composition further comprises one, two, three, four, five, six, seven, eight, nine, ten, or more excipients, including but not limited to carbohydrates, inorganic salts, antioxidants, surfactants, buffers, or any combination thereof.

[0202] In some embodiments, the pharmaceutical composition further comprises a carbohydrate. In certain embodiments, the carbohydrate is selected from the group consisting of fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrin, dextran, starch, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, cyclodextrin, and combinations thereof.

[0203] Alternatively, or in addition, the pharmaceutical composition further comprises an inorganic salt, hi certain embodiments, the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium sulfate, or a combination thereof.

[0204] Alternatively, or in addition, the pharmaceutical composition further comprises an antioxidant, hi certain embodiments, the antioxidant is selected from the group consisting of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, potassium metabisulfite, propyl gallate, sodium metabisulfite, sodium thiosulfate, vitamin E, 3,4-dihydroxybenzoic acid, and combinations thereof.

[0205] Alternatively, or in addition, the pharmaceutical composition further comprises a surfactant, hi certain embodiments, the surfactant is selected from the group consisting of polysorbates, sorbitan esters, lipids, phospholipids, phosphatidylethanolamines, fatty acids, fatty acid esters, steroids, EDTA, zinc, and combinations thereof.

[0206] Alternatively, or in addition, the pharmaceutical composition further comprises a buffering agent, hi certain embodiments, the buffering agent is selected from the group consisting of citric acid, sodium phosphate, potassium phosphate, acetic acid, ethanolamine, histidine, an amino acid, tartaric acid, succinic acid, fumaric acid, lactic acid, Tris, HEPES, or a combination thereof.

[0207] In some embodiments, the pharmaceutical composition is formulated for parenteral or enteral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous (IV) or subcutaneous (SQ) administration. In some embodiments, the pharmaceutical composition is in lyophilized form.

[0208] In one aspect, described herein is a liquid or lyophilized composition comprising a described antibody or antigen-binding fragment linked to a modified IL-18 polypeptide. In some embodiments, the IL-18 polypeptide, the antibody or antigen-binding fragment linked to a modified IL-18 polypeptide is a lyophilized powder. In some embodiments, the lyophilized powder is resuspended in a buffer solution. In some embodiments, the buffer solution comprises a buffering agent, a sugar, a salt, a surfactant, or any combination thereof. In some embodiments, the buffer solution comprises a phosphate salt. In some embodiments, the phosphate salt is sodium Na2HPO4. In some embodiments, the salt is sodium chloride. In some embodiments, the buffer solution comprises phosphate buffered saline. In some embodiments, the buffer solution comprises mannitol. In some embodiments, the lyophilized powder is suspended in a solution comprising about 10 mM Na2HPO4 buffer, about 0.022% SDS, and about 50 mg / mL mannitol, and has a pH of about 7.5.

[0209] Dosage form The immunocytokine compositions described herein may be in various dosage forms. In some embodiments, the immunocytokine compositions are administered as lyophilized powders. In some embodiments, the immunocytokine compositions are administered as suspensions. In some embodiments, the immunocytokine compositions are administered as solutions. In some embodiments, the immunocytokine compositions are administered as injection solutions. In some embodiments, the immunocytokine compositions are administered as IV solutions.

[0210] Treatment Method In one aspect, the present disclosure provides a method for treating cancer in a subject in need of such treatment, comprising administering to the subject an effective amount of an immunocytokine composition or pharmaceutical composition provided herein. In some embodiments, the cancer is a solid cancer. The cancer or tumor can be, for example, a primary cancer or tumor or a metastatic cancer or tumor. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cancer, gastric cancer, testicular cancer, thymus cancer, thyroid cancer, ureter cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0211] In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, AIDS-related lymphoma, multiple myeloma, plasma cell tumor, post-transplant lymphoproliferative disorder, or Waldenstrom's macroglobulinemia.

[0212] Combination therapy with one or more additional active agents is contemplated herein.

[0213] An effective response is achieved when a subject experiences a partial or total alleviation or reduction in signs or symptoms of the disease, specifically including, but not limited to, an extension of survival. Expected progression-free survival can be measured in months to years, depending on prognostic factors, including the number of recurrences, stage of the disease, and other factors. Extension of survival includes, but is not limited to, at least 1 month (mo), at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, etc. Overall survival or progression-free survival can also be measured in months to years. Alternatively, an effective response can be when the subject's symptoms or cancer burden remain static and do not worsen. Further treatment of the indications is described in more detail below. In some cases, the cancer or tumor is reduced by at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0214] In some embodiments, the immunocytokine composition is administered in a single dose of an effective amount of the immunocytokine composition, including the following further embodiments: (i) the immunocytokine composition is administered once daily, or (ii) the immunocytokine composition is administered once daily, or (ii) the immunocytokine composition is administered to a subject multiple times during the day. In some embodiments, the conjugate is administered daily, every other day, twice weekly, three times weekly, once weekly, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every twelve weeks, every third day, every fourth day, every fifth day, every sixth day, twice weekly, three times weekly, four times weekly, five times weekly, six times weekly, monthly, twice monthly, three times monthly, four times monthly, once every two months, once every three months, once every four months, once every five months, or once every six months. Administration includes, but is not limited to, injection by any suitable route (eg, parenteral, enteral, intravenous, subcutaneous, etc.).

[0215] Manufacturing method In one embodiment described herein, a method of making an immunocytokine composition includes providing an antibody or antigen-binding fragment thereof (e.g., an antibody or antigen-binding fragment provided herein), the antibody comprising a reactive group (e.g., a conjugation handle), contacting the reactive group with a complementary reactive group attached to an IL-18 polypeptide, and forming the immunocytokine composition. The resulting composition is any of the compositions provided herein.

[0216] In some embodiments, providing an antibody comprising a reactive group comprises attaching the reactive group to the antibody. In some embodiments, the reactive group is site-specifically added. In some embodiments, attaching the reactive group to the antibody comprises contacting the antibody with an affinity group comprising a reactive functional group that forms a bond with a specific residue of the antibody. In some embodiments, attaching the reactive group to the antibody comprises contacting the antibody with an enzyme. In some embodiments, the enzyme is configured to site-specifically attach the reactive group to a specific residue of the antibody. In some embodiments, the enzyme is a glycosylation enzyme or a transglutaminase enzyme.

[0217] In some embodiments, the method further comprises attaching a complementary reactive group to the cytokine.

[0218] definition All terms are intended to be understood as commonly understood by one of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0219] The following definitions supplement the definitions in the art and are directed to this application, and are not to be attributed to any related or unrelated cases, such as any commonly owned patent or application. Although methods and materials similar or equivalent to those described herein can be used in the practice of testing the present disclosure, preferred methods and materials are described herein. Therefore, the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be limiting.

[0220] The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. In this application, the use of the singular includes the plural unless expressly stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly indicates otherwise.

[0221] In this application, the use of "or" means "and / or" unless otherwise stated. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may convey that any combination is specifically intended. For illustrative purposes only, the following phrase "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" may be used conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0222] The term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending in part on the method of measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold of a value. Unless otherwise stated, when a particular value is described in this application and claims, the term "about" is deemed to mean within an acceptable error range for the particular value.

[0223] As used in this specification and claim(s), the words "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of including, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of including, such as "contains" and "contain") are inclusive or open and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0224] References herein to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, certain terms and phrases are defined below.

[0225] As used herein, reference is made to a group being "attached" or "covalently attached" to a residue of an IL-18 polypeptide or other polypeptide. As used herein, "attached" or "covalently attached" means that the group is tethered to the indicated residue, and such tethering may include a linking group (i.e., a linker). Thus, it is expressly intended that reference to a group being "attached" or "covalently attached" to a residue also encompasses such linking groups.

[0226] As used herein, an "alpha-keto amino acid" or the phrase "alpha-keto" before the name of an amino acid refers to an amino acid or amino acid derivative having a ketone functional group located between the carbon bearing the amino group and the carboxylic acid of the amino acid. The alpha-keto amino acids of the present disclosure have the structure shown in the formula: [ka] having where R is the side chain of any natural or unnatural amino acid. The R functional group can be in either the L or D orientation according to standard amino acid nomenclature. In a preferred embodiment, the alpha-keto amino acid is in the L orientation. When the term "alpha-keto" is used before the name of a conventional natural amino acid (e.g., alpha-ketoleucine, alpha-ketophenylalanine, etc.) or a common unnatural amino acid (e.g., alpha-ketonorleucine, alpha-ketoO-methyl-homoserine, etc.), it is intended that the alpha-keto amino acid referred to corresponds to the above formula and the side chain of the amino acid referred to. When an alpha-keto amino acid residue is described in a peptide or polypeptide sequence herein, it is intended that protected versions of the relevant alpha-keto amino acid are also included (e.g., for sequences terminating with a C-terminal alpha-keto amino acid, the terminal carboxylic acid group may be appropriately capped with a protecting group such as a tert-butyl group, or a ketone group with an acetal protecting group). Other protecting groups that are included are well known in the art.

[0227] Binding affinity refers to the strength of the binding interaction between a single molecule and its ligand / binding partner. A higher binding affinity refers to a stronger binding than a lower binding affinity. In some cases, binding affinity is determined by the dissociation constant (K D ) is measured by K D When comparing values, a binding interaction with a lower value has a higher binding affinity than a binding interaction with a higher value. For protein-ligand interactions, K D is calculated according to the following formula:

number

[0228] In this specification, a particular amino acid sequence (e.g., a polypeptide sequence) is referred to as having a certain percent sequence identity to a reference sequence or referring to residues at positions corresponding to positions in the reference sequence. Sequence identity is measured by the protein-protein BLAST algorithm using the following parameters: Matrix BLOSUM62, Gap Costs Existence: 11, Extension: 1, and composition-adjusted conditional composition score matrix adjustment. This alignment algorithm is also used to assess whether residues are at "corresponding" positions through analysis of the alignment of the two sequences being compared.

[0229] Unless otherwise specified, "protected" forms of amino acids (e.g., those that contain a chemical protecting group attached to the amino acid's functional group, particularly the side chain of the amino acid, as well as at other points on the amino acid) are intended to be considered the same amino acid as the "unprotected" forms for purposes of sequence identity, particularly in the case of chemically synthesized polypeptides. Such protected versions are also intended to be encompassed by the SEQ ID NOs provided herein. Non-limiting examples of protecting groups that may be included include fluorenylmethyloxycarbonyl (Fmoc), triphenylmethyl (trityl or trt), tert-butyloxycarbonyl (Boc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), acetamidomethyl (Acm), tert-butyl (tBu or OtBu), 2,2-dimethyl-1-(4-methoxyphenyl)propane-1,3-diol ketal or acetal, and 2,2-dimethyl-1-(2-nitrophenyl)propane-1,3-diol ketal or acetal. Other protecting groups known in the art are also included. Similarly, modified forms of natural amino acids are also intended to qualify as the natural forms of the amino acids for purposes of sequence identity. For example, amino acids that contain a side chain heteroatom that can be covalently modified (e.g., to add a conjugation handle, optionally via a linker), such as lysine, glutamine, glutamic acid, asparagine, aspartic acid, cysteine, or tyrosine, that have been covalently modified, are counted as basic amino acids (see, e.g., Structure 2 below, which counts as a lysine for purposes of sequence identity and SEQ ID NO:). Similarly, amino acids that contain another group added to the C- or N-terminus are counted as basic amino acids.

[0230] The term "pharmaceutical acceptable" means approved or approvable by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopeia (USP) or other generally recognized pharmacopoeia for use in animals, including humans.

[0231] A "pharmaceutical acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that may be administered to a subject together with a drug and that does not destroy the pharmacological activity of the drug and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the drug.

[0232] A "pharmaceutically acceptable salt" suitable for the present disclosure may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of humans or animals without undue toxicity, irritation, allergic response, or other problems or complications. Such salts include mineral and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Particular pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodine, phenylacetic acid, acetic acid, alkanoic acids such as HOOC-(CH2)n-COOH, where n is 0 to 4. Similarly, pharma- ceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize from this disclosure and knowledge in the art that additional pharma-ceutically acceptable salts include those described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p.1418 (1985). In general, pharma-ceutically acceptable acid or base salts can be synthesized from parent compounds that contain a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or free base forms of these compounds with a stoichiometric amount of an appropriate base or acid in an appropriate solvent.

[0233] It is understood that the ranges provided herein are shorthand notations of all values ​​within the range.For example, the range of 1 to 50 is understood to include any number, combination of numbers, subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal points between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9. For subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range 1 to 50 could include in one direction 1 to 10, 1 to 20, 1 to 30, and 1 to 40, or in the opposite direction 50 to 40, 50 to 30, 50 to 20, and 50 to 10.

[0234] Certain formulas and other figures provided herein depict triazole reaction products resulting from azide-alkyne cycloaddition reactions. Although such formulas generally depict only a single regioisomer of the resulting triazole formed in the reaction, these formulas are intended to encompass both resulting regioisomers. Thus, these formulas do not necessarily depict a single regioisomer (e.g., [ka] Only one positional isomer is shown, but the other positional isomer (e.g., [ka] are also intended to be encompassed.

[0235] The term "subject" refers to an animal that is the object of treatment, observation, or experiment. By way of example, a subject includes a mammal, including, but not limited to, a human or non-human mammal (such as a non-human primate, cow, horse, dog, sheep, or cat).

[0236] The term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0237] The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is often a chemical entity that is embedded in or recognized as appended to a molecule.

[0238] As used herein, the term "number average molecular weight" (Mn) means the statistical average molecular weight of all individual units in a sample and is defined by formula (1).

number

[0239] As used herein, the term "weight average molecular weight" (Mw) means the number defined by formula (2).

number

[0240] As used herein, "peak molecular weight" (Mp) means the molecular weight of the largest peak in a given analytical method (e.g., mass spectrometry, size exclusion chromatography, dynamic light scattering, analytical centrifugation, etc.).

[0241] As used herein, "conjugation handle" refers to a reactive group that can form a bond when contacted with a complementary reactive group. In some cases, the conjugation handle preferably does not have substantial reactivity with other molecules that do not contain the intended complementary reactive group. Non-limiting examples of conjugation handles, their respective complementary conjugation handles, and corresponding reaction products can be found in the table below. Although the table headings place certain reactive groups under the heading of "conjugation handle" or "complementary conjugation handle," any reference to a conjugation handle is instead intended to include the complementary conjugation handle described in the table (e.g., if trans-cyclooctene is the conjugation handle, tetrazine would be the complementary conjugation handle). In some cases, amine conjugation handles and conjugation handles complementary to amines are less preferred for use in biological systems due to the ubiquity of amines in biological systems and increased potential for off-target conjugation. Conjugation Handles Table [Table 2]

[0242] Throughout this application, prefix words are used before the term "conjugation handle" to indicate the functional group to which the conjugation handle is linked. For example, a "protein conjugation handle" is a conjugation handle attached to a protein (either directly or via a linker), an "antibody conjugation handle" is a conjugation handle attached to an antibody (either directly or via a linker), and a "linker conjugation handle" is a conjugation handle attached to a linker group (e.g., a bifunctional linker used to link a synthetic protein to an antibody).

[0243] The term "alkyl" refers to a straight or branched hydrocarbon chain radical having from 1 to 20 carbon atoms and attached to the rest of the molecule by a single bond. Alkyl containing up to 10 carbon atoms is any of C1 to C 10 Similarly, for example, an alkyl group containing up to 6 carbon atoms is a C1-C6 alkyl group. Alkyl groups containing other numbers of carbon atoms (and other moieties defined herein) are similarly represented. Alkyl groups include C1-C 10 Examples of alkyl groups include, but are not limited to, alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl, and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (i-propyl), n-butyl, i-butyl, 5-butyl, n-pentyl, 1,1-dimethylethyl (i-butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, and the like. In some embodiments, the alkyl is methyl or ethyl. In some embodiments, the alkyl is -CH(CH3)2 or -C(CH3)3. Unless otherwise specified in this specification, the alkyl group may be optionally substituted. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the remainder of the molecule to a radical group. In some embodiments, alkylene is -CFF-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, alkylene is -CH2-. In some embodiments, alkylene is -CH2CH2-. In some embodiments, alkylene is -CH2CH2CH2-. Unless stated otherwise in the specification, alkylene groups may be optionally substituted.

[0244] The term "alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain having at least one carbon-carbon double bond connecting the remainder of the molecule to a radical group. In some embodiments, the alkenylene is -CH=CH-, -CHCH=CH-, or -CH=CHCH-. In some embodiments, the alkenylene is -CH=CH-. In some embodiments, the alkenylene is -CHCH=CH-. In some embodiments, the alkenylene is -CH=CHCH-. In some embodiments, the alkenylene is -CH=CHCH-.

[0245] The term "alkynyl" refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. In one embodiment, an alkenyl group has the formula -C≡CR X wherein R x refers to the remainder of the alkynyl group. In some embodiments, R x is H or alkyl. In some embodiments, alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH3, -C≡CCH2CH-, and -CH2C≡CH.

[0246] The term "aryl" refers to a radical that contains at least one aromatic ring, where each of the atoms that form the ring is a carbon atom. An aryl group can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, an aryl is phenyl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group). Unless otherwise stated in the specification, the term "aryl" or the prefix "ar-" (e.g., as in "aralkyl") is meant to include aryl radicals that are optionally substituted. In some embodiments, an aryl group contains a partially reduced cycloalkyl group as defined herein (e.g., 1,2-dihydronaphthalene). In some embodiments, an aryl group contains a fully reduced cycloalkyl group as defined herein (e.g., 1,2,3,4-tetrahydronaphthalene). When an aryl contains a cycloalkyl group, the aryl is attached to the remainder of the molecule via a carbon atom of the aromatic ring. The aryl radical can be a monocyclic or polycyclic (eg, bicyclic, tricyclic, or tetracyclic) ring system, which can include fused, spiro, or bridged ring systems.

[0247] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In some embodiments, the cycloalkyl is saturated or partially unsaturated. In some embodiments, the cycloalkyl is a spirocyclic or bridged compound. In some embodiments, the cycloalkyl is fused to an aromatic ring (in which case the cycloalkyl is attached through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having 3 to 10 carbon atoms, 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. Monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopentyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl. Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetraynyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl, and bicycle[1.1.1]pentyl. Unless stated otherwise in the specification, cycloalkyl groups may be optionally substituted.

[0248] The term "heteroalkylene" or "heteroalkylene chain" refers to a straight or branched divalent heteroalkyl chain that connects the remainder of the molecule to a radical group. Unless stated otherwise in the specification, a heteroalkyl or heteroalkylene group may be optionally substituted as described below. Representative heteroalkylene groups include, but are not limited to, -CH2-O-CH2-, -CH2-N(alkyl)-CH2-, -CH2-N(aryl)-CH2-, -OCH2CHO-, -OCH2CH20CH2CHO-, or -OCH2CHOCH2CHOCH2CHO-.

[0249] The term "heterocycloalkyl" refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, a heterocycloalkyl radical can be a monocyclic or bicyclic ring system, which can include fused (when fused with an aryl or heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized. The nitrogen atom can be optionally quaternized. The heterocycloalkyl radical can be partially or fully saturated. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinoclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of sugars, including but not limited to monosaccharides, disaccharides, and oligosaccharides. Unless otherwise noted, heterocycloalkyls have 2-12 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring and contain 1 or 2 N atoms. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring and contain 3 or 4 N atoms. In some embodiments, heterocycloalkyls have 2-12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring.In some embodiments, a heterocycloalkyl has 2-12 carbons, 1-3 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. When referring to the number of carbon atoms in a heterocycloalkyl, it is understood that the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including heteroatoms) that make up the heterocycloalkyl (i.e., the skeletal atoms of the heterocycloalkyl ring). Unless otherwise stated in the specification, a heterocycloalkyl group may be optionally substituted.

[0250] The term "heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the heteroaryl is monocyclic or bicyclic. Illustrative examples of monocyclic heteroaryl include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. Illustrative examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Illustrative examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, the heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl, or furyl. In some embodiments, the heteroaryl contains 0-6 N atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms in the ring. In some embodiments, the heteroaryl contains 4-6 N atoms in the ring. In some embodiments, the heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl is a C1-C9 heteroaryl.In some embodiments, the monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, the monocyclic heteroaryl is a 5- or 6-membered heteroaryl. In some embodiments, the bicyclic heteroaryl is a C6-C9 heteroaryl. In some embodiments, the heteroaryl group comprises a partially reduced cycloalkyl or heterocycloalkyl group as defined herein (e.g., 7,8-dihydroquinoline). In some embodiments, the heteroaryl group comprises a fully reduced cycloalkyl or heterocycloalkyl group as defined herein (e.g., 5,6,7,8-tetrahydroquinoline). When the heteroaryl comprises a cycloalkyl or heterocycloalkyl group, the heteroaryl is attached to the remainder of the molecule through a carbon or heteroatom of the heteroaromatic ring. The heteroaryl radical can be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) ring system, which can include fused, spiro, or bridged ring systems.

[0251] The term "optionally substituted" or "substituted" means that the group to which it is referred is optionally substituted with one or more additional groups individually and independently selected from D, halogen, -CN, -NH, -NH(alkyl), -N(alkyl), -OH, -COH, -COalkyl, -C(=0)NH, -C(=0)NH(alkyl), -C(=0)N(alkyl), -S(=0)NH, -S(=0)NH(alkyl), -S(=0)N(alkyl), alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, the optional substituents are independently selected from D, halogen, -CN, -NH, -NH(CH), -N(CH), -OH, -COH, -CO(Ci-C4 alkyl), -C(=0)NH, -C(=0)NH(Ci-C4 alkyl), -C(=0)N(Ci-C4 alkyl), -S(=0)NH, -S(=0)NH(Ci-C4 alkyl), -S(=0)N(Ci-C4 alkyl), C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 fluoroalkyl, C1-C4 heteroalkyl, C1-C4 alkoxy, C1-C4 fluoroalkoxy, -SC1-C4 alkyl, -S(=0)C1-C4 alkyl, and -S(=0)2C1-C4 alkyl. In some embodiments, optional substituents are independently selected from D, halogen, -CN, -NH2, -OH, -NH(CH3), -N(CH3)2, -NH(cyclopropyl), -CH3, -CH2CH3, -CF3, -OCH3, and -OCF3. In some embodiments, a substituent is substituted with one or two of the foregoing groups. In some embodiments, optional substituents on an aliphatic carbon atom (acyclic or cyclic) include oxo (=0).

[0252] As used herein, "AJICAP™ technology," "AJICAP™ method," and similar terms refer to a system and method (currently manufactured by Ajinomoto Bio-Pharma Services ("Ajinomoto")) for site-specific functionalization of antibodies and related molecules using affinity peptides to deliver the desired functionalization to a desired site. General protocols for the AJICAP™ methodology can be found at least in PCT Publication No. WO2018199337A1, PCT Publication No. WO2019240288A1, PCT Publication No. WO2019240287A1, PCT Publication No. WO2020090979A1, Matsuda et al., Mol. Pharmaceutics 2021, 18, 4058-4066, and Yamada et al., AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies. Angew. Chem., Int. Ed. 2019, 58, 5592-5597, and in particular in Examples 2-4 of U.S. Patent Publication No. US20200190165A1. In some embodiments, such methodologies site-specifically incorporate a desired functionalization at a lysine residue at a position selected from positions 246, 248, 288, 290, and 317 (EU numbering) of an antibody Fc region (e.g., an IgG1 Fc region). In some embodiments, the desired functionalization is incorporated at residue position 248 (EU numbering) of an antibody Fc region. In some embodiments, position 248 corresponds to the 18th residue (EU numbering) of the human IgG CH2 region. IL-18 Polypeptide Sequence (SEQ ID NO: [Table 3] TIFF2025508823000050.tif235159TIFF2025508823000051.tif236159TIFF2025508823000052.tif235159 TIFF2025508823000053.tif222159TIFF2025508823000054.tif238159TIFF2025508823000055.tif107159

[0253] Additional Exemplary IL-18 Constructs Also provided herein are IL-18 polypeptides that include modifications to SEQ ID NO:1 listed in the table below, each of which is assigned a composition ID, which can be incorporated into the immunocytokine compositions provided herein. In some embodiments, the IL-18 polypeptide of the immunocytokine composition includes the set of amino acid substitutions shown for any one of the constructs shown below. In the constructs shown below, each of the substitutions is listed using SEQ ID NO:1 as a reference sequence. In some embodiments, the IL-18 polypeptide immunocytokine composition includes only the substitutions shown for the construct below relative to SEQ ID NO:1 (i.e., the IL-18 polypeptide has only the set of substitutions shown, with the remaining residues being those represented in SEQ ID NO:1). [Table 4]

[0254] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations could be made therein without departing from the spirit and scope of the present disclosure, as defined in the appended claims.

[0255] The present disclosure is further illustrated in the following examples, which are provided for illustrative purposes and are not intended to limit the present disclosure in any way.

[0256] Working Example Example 1A - Expression and purification of recombinant IL18 Recombinant IL-18 variants suitable for linking to the antibodies or antigen-binding fragments provided herein can be prepared according to the protocols provided below. In some cases, the recombinant IL-18 can contain a cysteine ​​residue at the desired attachment point of the linker or can include an unnatural amino acid (e.g., azidolysine) suitable for attachment of the linker at the desired attachment point.

[0257] Soluble His-SUMO-IL18 mutant E. coli BL21(DE3) containing plasmids encoding N-His-SUMO tagged IL-18 mutant fusions are inoculated into 3 L of LB medium and induced with 0.4 mM IPTG for 6 h at 30 °C. Cells are pelleted and cell lysis is performed by sonication in lysis buffer: PBS, pH 7.4. Soluble proteins are purified with Ni-NTA beads 6FF (wash 1 with PBS, 20 mM imidazole, pH 7.4, wash 2 with PBS, 50 mM imidazole, pH 7.4, elution with PBS, 500 mM imidazole, pH 7.4).

[0258] The fractions containing the protein are pooled and dialyzed into PBS pH 7.4, followed by SUMO digestion.The protein is then purified in two steps by Ni-NTA beads (continuing the flow-through sample) and gel filtration.The fractions containing the protein are pooled and QC is performed using analytical techniques such as SDS-PAGE and analytical SEC.

[0259] Insoluble His-SUMO-IL18 mutant E. coli BL21(DE3) containing plasmids encoding N-His-SUMO tagged IL-18 mutant fusions are inoculated into 10 L of LB medium and induced with 0.4 mM IPTG for 6 h at 30 °C. Cells are pelleted and lysis is performed by sonication in lysis buffer: PBS, 8 M urea, pH 7.4. Proteins are purified with Ni-NTA beads 6FF (wash 1 with PBS, 8 M urea, 20 mM imidazole, pH 7.4, wash 2 with PBS, 8 M urea, 50 mM imidazole, pH 7.4, elution with PBS, 8 M urea, 500 mM imidazole, pH 7.4).

[0260] Fractions containing protein are pooled and dialyzed into PBS pH 7.4 followed by SUMO digestion. Protein is then purified with Ni-NTA beads (column equilibrated with PBS, 8M urea, pH 7.4, washed with PBS, 8M urea, pH 7.4, eluted with PBS, 8M urea, pH 7.4). Fractions containing protein are pooled and dialyzed into PBS pH 7.4 and QC is performed using analytical techniques such as SDS-PAGE and analytical SEC.

[0261] Insoluble untagged IL18 mutant E. coli BL21(DE3) containing a plasmid encoding mIL-18 was inoculated into 2 L of LB medium and induced with 0.4 mM IPTG for 6 h at 30 °C. Cells were pelleted and cell lysis was performed by sonication in lysis buffer: 110 mM Tris, 1.1 M guanidine HCl, 5 mM DTT, pH 8.9. Proteins were purified by Q Sepharose FF (equilibration buffer 20 mM MES, pH 7.0, elution with an increasing gradient of 0 to 1 M NaCl).

[0262] Example 1B - Additional methods for expression and purification of recombinant IL18 The following protocol was used to prepare certain IL-18 polypeptides provided herein, which were then used in any of the assays for conversion to immune cytokine compositions provided herein.

[0263] Expression of IL-18 Polypeptide

[0264] IL-18 polypeptide was produced as an N-terminal fusion to N-His-SUMO-IL18. The gene was synthesized and cloned by a commercial vendor. The plasmid was transformed into E. coli BL21(DE3). Expression was performed in shake flasks containing TB medium. Cells were grown at 37°C until they reached an OD600 of approximately 1.2, after which they were induced with 0.1 mM IPTG and cultured at 18°C ​​for an additional 20 hours. Cells were harvested by centrifugation.

[0265] Purification of IL18 polypeptide

[0266] Cell lysis - Cells were resuspended in lysis buffer (20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole, 1 tablet EDTA-free complete protease inhibitor (Roche, COEDTAF-RO) per liter production) in 100 mL buffer / L medium and disrupted twice using a homogenizer at 1000 bar. The lysate was centrifuged at 40,000 g for 2 x 45 min with flask exchange during, followed by filtration through a 0.22 μm filter to remove debris.

[0267] Affinity purification and endotoxin removal - The lysate was loaded onto Ni NTA resin (Cytiva, 17524802) pre-equilibrated with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole at 5 mL / min and washed with 5 CV of the same buffer. To remove endotoxins, the column was washed with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole, 0.1% Tryton X-114 at 10 mL / min for 30 CV. The column was washed with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole for 5 CV, 5 mL / min, and the protein of interest was eluted by linearly increasing the imidazole concentration. The column was then regenerated with 0.5 M NaOH.

[0268] SUMO Digestion and Dialysis - To cleave the SUMO tag, SUMO protease was added to the elution pool at a 1:250 w / w ratio (protein:SUMO enzyme) and incubated for 18 h at 4° C. Concurrently, the protein was dialyzed (20 mM Tris, pH 8.0, 150 mM NaCl) to reduce the imidazole concentration.

[0269] Purification by reverse IMAC - To remove the cleaved tag and SUMO protease, the digested protein was loaded onto a Ni NTA resin column pre-equilibrated with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole at 5 mL / min. The flow-through was collected.

[0270] Buffer exchange - The flow-through was concentrated to 2.6 mg / mL and buffer exchanged into either 20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, 10% glycerol, pH 7.5 or PBS, 10% glycerol, pH 7.4. Protein was stored at -70°C until further quality control.

[0271] Example 2 - Conjugation of IL-18 Polypeptides with Bifunctional Linking Groups The IL-18 polypeptides provided herein may be conjugated to a bifunctional linking group prior to forming the complete linker of the immunocytokine composition. In some cases, the bifunctional linking group is first attached to the desired residue of the IL-18 polypeptide at the attachment point of the linker. Once attached to the IL-18 polypeptide, the second functional group of the bifunctional linking group is used to attach to a second moiety. An exemplary schematic of such a process is shown in FIG. 6. The process shown in FIG. 6 is not required for all IL-18 polypeptides, such as some IL-18 polypeptides provided herein (e.g., IL-18 with a desired conjugation handle for final attachment to an already attached antibody). An exemplary protocol for IL-18 polypeptides provided herein with a cysteine ​​residue attachment point is described below.

[0272] Conjugated IL-18 polypeptide is stored at a concentration of 2.4 mg / mL in potassium phosphate buffer (pH 7.0) containing 50 mM KCl and 1 mM DTT at -80°C. The sample is thawed on ice to obtain a clear solution. The protein solution is diluted with PBS, pH 7.4. A clear solution with a concentration of approximately 0.4 mg / mL is obtained.

[0273] The protein solution is dialyzed against PBS, pH 7.4 (2 times against 600 mL for 2 h, 1 time against 800 mL for 18 h). After dialysis, a clear solution with no signs of precipitation is obtained. Protein concentration is obtained by BCA protein assay using UV absorbance at 280 nm.

[0274] Prepare a stock solution of bifunctional linking group (e.g., Bromoacetamido-PEG5-azide, CAS:1415800-37-1) in water at a concentration of 20 mM. Mix 500 μL of protein solution with 25 μL of linking group solution. Adjust the pH to 7.5 and react at 20 °C for 3 h.

[0275] The progress of the synthesis is monitored by reverse-phase HPLC on an Aeris WIDEPORE C18 200 Å column (3.6 μm, 150 × 4.6 mm) using a gradient of 5–30% (2.5 min) and 30–75% (7.5 min) of CH3CN containing 0.1% TFA (v / v) at a flow rate of 1 mL / min at 40 °C, and by MALDI-TOF MS.

[0276] Purification - In some cases, ion exchange chromatography is used to purify the conjugated protein. To remove excess probe, the reaction mixture (volume approximately 500 μL) is loaded onto a Hi-Trap-G-FF-1 mL column using 25 mM Tris (pH 7.4) as buffer. The column is eluted with a linear gradient of 0 to 0.35 M NaCl in the same buffer. Fractions containing the target protein are collected, buffer exchanged (25 mM Tris, pH 7.4, 75 mM NaCl, 5% glycerol) and concentrated to 0.4 mg / mL. The concentration of the purified protein is measured by UV absorbance at 280 nm and by BCA protein assay. The protein solution is kept at -80 °C.

[0277] Characterization - The purity and identity of recombinant and conjugated proteins from commercial sources are confirmed by aSEC, HPLC and MALDI-TOF MS.

[0278] Example 3 - Conjugation of IL-18 Polypeptides to Antibodies Preparation of conjugable antibodies

[0279] Modified antibodies (e.g., anti-PD-1 antibodies such as nivolumab or LZM-009) containing a DBCO conjugation handle are prepared using a protocol modified from Examples 2-4 of US Patent Publication No. US2020019165A1. An exemplary illustration of this process, resulting in the attachment of one DBCO conjugation handle, is shown in FIG. 3. Briefly, a CD20 antibody having a free sulfhydryl group attached to a lysine residue side chain in the Fc region is prepared by reacting the antibody with an affinity peptide configured to deliver a protected form of the sulfhydryl group (e.g., a thioester) to the lysine residue. The protecting group is then removed to expose the free sulfhydryl. The free sulfhydryl is then reacted with a bifunctional reagent containing a bromoacetamide group (e.g., bromoacetamide-dPEG®4-amide-DBCO) connected to the DBCO conjugation handle via a linking group. Using this method, antibodies can be generated that have one DBCO group (DAR1) and / or two DBCO groups (DAR2, one DBCO group linked to each Fc of the antibody) attached to them.

[0280] Conjugation of antibodies to IL-18 polypeptides

[0281] The DBCO-modified antibody is then conjugated to an IL-18 polypeptide containing an azide moiety at the desired attachment point (e.g., an IL-18 polypeptide containing an amino acid with an azide side chain, or an IL-18 linked to an azide using a bifunctional linking group as in Example 4). The DBCO-modified antibody with one (DAR1) or two (DAR2) reactive handles is reacted with 2-10 equivalents of azide-containing IL-18 (pH 5.2 buffer, 5% trehalose, room temperature, 24 hours). In an alternative embodiment, an antibody containing two DBCO conjugation handles is reacted with one equivalent of IL-18 containing an azide functional group as an excess of reagent (e.g., 5-10 equivalents) to generate a DAR1 antibody, or an antibody containing two DBCO conjugation handles is reacted with one equivalent of antibody containing an excess of reagent of azide-containing IL-18 (e.g., 5-10 equivalents) to generate a DAR2 antibody. A schematic of this protocol is shown in Figure 7.

[0282] Purification and characterization of antibody-IL-18 immunocytokine

[0283] The resulting immunocytokine is purified by cation exchange chromatography and / or size exclusion chromatography to obtain purified immunocytokine. The antibody-IL-18 polypeptide immunocytokine is purified from unreacted IL-18 and aggregated using desalting columns, CIEX and SEC (GE Healthcare Life Sciences AKTA pure, mobile phase: histidine 5.2 / 150 mM NaCl / 5% trehalose, column: GE Healthcare Life Sciences SUPERDEX™ 200 increase, 3.2 / 300, flow rate: 0.5 mL / min).

[0284] The purity and identity of the antibody-IL-18 polypeptide immunocytokine is confirmed by RP-HPLC (HPLC: ThermoFisher Scientific UHPLC Ultimate 3000, column: Waters BEH C-4 300A, 3.0 μm, 4.6 mm, 250 mm, mobile phase A: 0.05% TFA in water, mobile phase B: 0.05% TFA in a mixture of ACN:IPA:ETOH:HO mixture (5:1.5:2:1.5), flow rate: 0.5 mL / min, injection volume: 10 μg (10 μL injection of 1 mg / mL), gradient: 0% to 20% mobile phase B in 50 min) and SDS-PAGE.

[0285] Exemplary chromatograms and analytical characterization of the DAR1 immunocytokine of IL-18 of SEQ ID NO:60 conjugated via residue C68 and an anti-PD-1 antibody (LZM-009), prepared according to the methods described, are shown in Figures 8-10.

[0286] Additional exemplary chromatograms and analytical characterizations of the DAR1 immunocytokine of IL-18 of SEQ ID NO:30 conjugated via residue C68 to Fc residue K248 of the anti-PD-1 antibody LZM-009 are shown in Figure 11 (RP HPLC), Figure 12 (SEC-HPLC), and Figure 13 (Q-TOF mass spectrometry). The immunocytokine shown is Composition A.

[0287] Example 4 - Characterization of the immunocytokine IL-18 activity The ability of the immunocytokines to carry out various IL-18 activities is measured as provided below, as well as relevant comparisons with unconjugated IL-18 polypeptides.

[0288] Surface plasmon resonance analysis The interaction of immune cytokines, wild-type IL-18, and modified IL-18 polypeptides with human IL-18 receptor subunits is measured using surface plasmon resonance (SPR) technology. Anti-human IgG antibodies are bound by amine coupling onto a CM5 chip for 30 minutes prior to capture to capture 6 μg / mL of Fc-fused human IL-18Rα, 6 μg / mL of Fc-fused human IL-18Rβ, or 2 μg / mL of Fc-fused human IL-18BP isoform a (IL-18BPa). In another setup, 6 μg / mL of alpha and beta IL-18 receptors are mixed and pre-incubated for 30 minutes prior to capture of the alpha / beta heterodimeric IL-18 receptor.

[0289] Kinetic binding of IL-18 analytes and immunocytokines is measured using a Biacore 8K instrument, starting at 1 μM and with two-fold serial dilutions down to 0.98 nM. Regeneration of the surface back to amine-coupled anti-IgG antibodies is performed for each concentration of analyte. To measure protein association to the receptor, samples are injected for 60 seconds at a flow rate of 50 μL / min, followed by 300 seconds of buffer only injection to detect dissociation. The running buffer used is 1xPBS with 0.05% Tween 20. Relative response units (RU, Y-axis) are plotted against time (sec, X-axis) and analyzed with a kinetic 1:1 binding model for monomeric receptor binding and binding to IL-18BP. A kinetic heterogeneous ligand fitting model is applied for alpha / beta heterodimer binding.

[0290] IL-18BP binding alphaLISA assay To determine the binding affinity of each immunocytokine and IL-18 variant to IL-18BP, a human IL-18BP AlphaLISA assay kit was used, which detects the presence of the free form of IL-18BP.

[0291] Sixteen 3-fold serial dilutions of IL-18 analyte are prepared in aMEM medium supplemented with 20% FCS, Glutamax, and 25 μM β-mercaptoethanol in the presence of 5 ng / mL His-tagged human IL-18BP. Final IL-18 analyte concentrations range from 2778 nM to 0.2 pM.

[0292] After 1 hour incubation at room temperature, free IL-18BP levels are measured using a human IFNγ AlphaLISA assay kit. In a 384-well OPTI plate, 5 μL of 5X anti-IL-18BP acceptor beads are added to 7.5 μL of IL-18 / IL-18BP mix. After 30 minutes incubation at room temperature with shaking, 5 μL of biotinylated anti-IL-18BP antibody is added to each well. The plate is incubated for an additional hour at room temperature. Under subdued light, 12.5 μL of 2X streptavidin (SA) donor beads are pipetted into each well and the wells are incubated for an additional 30 minutes at room temperature with shaking. The AlphaLisa signal is then measured on an Enspire plate reader with excitation and emission wavelengths of 680 and 615 nm, respectively. Dissociation constants (K ) are calculated based on a variable slope, 4-parameter analysis using GraphPad PRISM software. D ) to calculate

[0293] IFNγ-induced cell assay The ability of the immunocytokines and IL-18 polypeptides provided herein is assessed for their ability to induce IFNγ in a cellular assay according to the following protocol.

[0294] The NK cell line NK-92 (ATCC® CRL-2407™), derived from a lymphoma patient, was cultured in aMEM medium supplemented with 20% FCS, Glutamax, 25 μM B-mercaptoethanol, and 100 IU / mL recombinant human IL-2.

[0295] On the day of the experiment, cells are harvested and washed with aMEM medium without IL-2 and containing 1 ng / mL recombinant human IL-12. After counting, cells are seeded at 100,000 cells / well in 384-well titer plates and incubated at 37°C / 5% CO2. Sixteen 4-fold serial dilutions of IL-18 analytes were prepared in aMEM medium and 1 ng / mL IL-12 was added to the NK-92 cells. Final IL-18 analyte concentrations ranged from 56 nM to 5x10 -5 up to pM.

[0296] After incubating the cells for 16-20 hours at 37°C / 5% CO2, 5 μL of the supernatant is carefully transferred to a 384-microwell OptiPlate. IFNγ levels are measured using the Human IFNγ AlphaLISA Assay Kit. Briefly, 10 μL of 2.5X AlphaLISA anti-IFNγ acceptor beads and biotinylated antibody anti-IFNγ mix are added to 5 μL of NK-92 supernatant. The mixture is incubated for 1 hour at room temperature with shaking. Under subdued light, 2.5 μL of 2X streptavidin (SA) donor beads are pipetted into each well and the wells are incubated for 30 minutes at room temperature with shaking. The AlphaLISA signal is then measured in an EnSpire™ plate reader using 680 nm and 615 nm as excitation and emission wavelengths, respectively. The half-maximal effective concentration (EC) was calculated based on variable slope and 4-parameter analysis using GraphPad PRISM software. 50 ) to calculate

[0297] IL-18 binding protein inhibition cell assay The NK cell line NK-92 (ATCC® CRL-2407™), derived from a lymphoma patient, was cultured in aMEM medium supplemented with 20% FCS-Glutamax, 25 μM B-mercaptoethanol, and 100 IU / mL recombinant human IL-2.

[0298] On the day of the experiment, cells are harvested and washed with aMEM medium without IL-2 and containing 1 ng / mL recombinant human IL-12. After counting, cells are seeded at 100,000 cells / well in 384-well titer plates and incubated at 37°C / 5% CO2. Sixteen two-fold serial dilutions of Fc-fused human IL-18 binding protein isoform a (IL-18BPa) are prepared in aMEM medium. 1 ng / mL IL-12 containing 2 nM of each modified IL-18 polypeptide variant is added to the NK-92 cells. The final IL-18 analyte concentration was 1 nM, and the final IL-18BPa concentrations ranged from 566 nM to 17 pM. After incubating the cells for 16-20 hours at 37°C / 5% CO2, 5 μL of the supernatant is carefully transferred to a 384-microwell OptiPlate. IFNγ levels are measured using the Human IFNγ AlphaLISA Assay Kit. Briefly, 10 μL of 2.5X AlphaLISA anti-IFNγ acceptor beads and biotinylated antibody anti-IFNγ mix are added to 5 μL of NK-92 supernatant. The mixture is incubated for 1 hour at room temperature with shaking. Under subdued light, 2.5 μL of 2X SA donor beads are pipetted into each well and the wells are incubated for 30 minutes at room temperature with shaking. The AlphaLISA signal is then measured in an EnSpire™ plate reader using 680 nm and 615 nm as excitation and emission wavelengths, respectively. Half-maximal inhibitory concentrations (IC) were calculated based on variable slope and 4-parameter analysis using GraphPad PRISM software. 50 ) to calculate

[0299] IFNγ induction in primary human cells The ability of IL-18 variants to stimulate human peripheral blood mononuclear cells (PBMCs) was assessed according to the following protocol.

[0300] Isolation of lymphocytes: Blood from buffy coats of healthy volunteers was diluted with an equal volume of PBS and slowly poured onto the top of a SepMate tube pre-filled with 15 mL of Histopaque-1077. The tube was centrifuged at 1200 g for 10 min, and the upper layer was collected and washed three times with PBS containing 2% fetal bovine serum. PBMCs were counted and 20 × 10 6 Aliquots of cells were stored frozen.

[0301] Cryopreserved PBMCs were thawed and seeded at 150 000 cells / well in 96w round-bottom 96-well plates. PBMCs were stimulated with a gradient of human IL-18 variants ranging from 0.2 pg / mL to 3600 ng / mL. All stimulations were performed in the presence of hIL-12 (1 ng / ml, Sino Biological, #CT011-H08H) in RPMI containing 10% fetal bovine serum for 24 h.

[0302] Cytokine production after 24 hours of stimulation was measured using a Legendplex bead-based cytokine assay (Biolegend #740930) according to the manufacturer's protocol. GraphPad PRISM software was used to calculate half-maximal effective concentrations (EC) of IFNg released into the culture supernatant based on variable slope and four parameter analysis. 50 ) was calculated.

[0303] IFNγ induction in primary mouse cells The ability of IL-18 mutants to stimulate mouse splenocytes was assessed according to the following protocol.

[0304] Cryopreserved splenocytes isolated from BALB / c and C57BL6 mice were purchased from IQ Biosciences (Berkeley, CA, USA).

[0305] Cryopreserved splenocytes were thawed, treated with DNAseI and seeded at 200 000 cells / well in 96w round-bottom 96-well plates. Splenocytes were stimulated with a gradient of human IL-18 variants ranging from 0.2 pg / mL to 3600 ng / mL. All stimulations were performed in RPMI containing 10% fetal bovine serum in the presence of mIL-12 (1 ng / ml, Peprotech, catalogue no. 210-12) for 24 h.

[0306] Cytokine production after 24 hours of stimulation was measured using a Legendplex bead-based cytokine assay (Biolegend #740622) according to the manufacturer's protocol. GraphPad PRISM software was used to calculate half-maximal effective concentrations (EC) of IFNg released into the culture supernatant based on variable slope and four parameter analysis. 50 ) was calculated.

[0307] Example 5 - Immune cell-associated antigen binding ELISA assay (Figures 14A, 14B, 15A, and 15B) The interaction of unmodified antibodies and corresponding IL-18 immunocytokines with relevant immune cell associated antigens is measured by ELISA assay. For these tests, Corning high-binding half-area plates (Fisher Scientific, Reinach, Switzerland) are coated overnight at 4° C. with 25 μL of unmodified antibodies corresponding to IL-18 immunocytokines at 5 μg / ml in PBS. The plates are then washed four times with 100 μL of PBS-0.02% Tween 20. The plate surface is blocked with 25 μL of PBS-0.02% Tween 20-1% BSA for 1 h at 37° C. The plates are then washed four times with 100 μL of PBS-0.02% Tween 20. Twenty-five microliters (25 μl) of recombinant biotinylated human PD-1 (biotinylated recombinant human PD-1 / CD279-Fc chimera, carrier-free, Biolegend#789406) or PD-L1 (biotinylated human PD-L1 / B7-H1, ACROBiosystems, PD1-H82E5-25UG) protein is added in 7-fold serial dilutions starting at 12 nM down to 0.15 pM in PBS-0.02% Tween20-0.1% BSA and incubated for 2 hours at 37° C. The plate is then washed four times with 100 μl of PBS-0.02% Tween20. 25 microliters of streptavidin-horseradish peroxidase (#RABHRP3, Merck, Buchs, Switzerland) diluted 1:500 in PBS-0.02% Tween20-0.1% BSA are added to each well and incubated for 30 minutes at room temperature. The plate is then washed 4 times with 100 μl of PBS-0.02% Tween20. 50 microliters of TMB substrate reagent (#CL07, Merck, Buchs, Switzerland) are added to each well and incubated for 5 minutes at 37° C. After 5 minutes at 37° C., the horseradish peroxidase reaction is stopped by adding 50 μl / well of 0.5 M H2SO4 stop solution. The ELISA signal is then measured at 450 nm in an ENSPIRE® plate reader from Perkin Elmer (Schwerzenbach, Switzerland). The results of this experiment are shown in the table below. [Table 5]

[0308] Figure 14A and Figure 14B show plots measuring the ability of unmodified and conjugated anti-PD1 antibodies to bind to human PD1 / CD279 ligand, with the figures showing ELISA signal on the y-axis and dose of biotinylated PD-1 protein on the x-axis. The unconjugated reference antibodies are pembrolizumab, LZM-009, nivolumab, atezolizumab, durvalumab, and avelumab. The conjugated antibodies tested in this figure are Composition A and Composition B.

[0309] Figure 15A and Figure 15B show plots measuring the ability of unmodified and conjugated antibodies to bind to human PD-L1 / B7-H1 ligand, with the figures showing ELISA signal on the y-axis and dose of biotinylated PD-L1 protein on the x-axis. The unconjugated reference antibodies are pembrolizumab, LZM-009, nivolumab, atezolizumab, durvalumab, and avelumab. The conjugated antibodies tested in this figure are Composition A and Composition B.

[0310] Example 6 - Kinetic analysis of binding of reference antibodies and immune cytokines to immune cell-associated antigens (Figure 16) The Octet® BLI system, based on Biolayer Interferometry (BLI), allows real-time label-free analysis to determine the kinetics and affinity of a ligand to its receptor. Here, an anti-human IgG FC capture (AHC) sensor is loaded with the test article (IC). The sensor is first immersed in a kinetic buffer for a baseline measurement, then in an analyte solution (here human PD1) to allow association, and again in buffer to allow the analyte to dissociate from the ligand (dissociation). Several concentrations of analyte are run in parallel, allowing the calculation of affinity parameters (Ka, Kd, ​​KD).

[0311] Typically, the sensor is first regenerated by immersing it in a 10 mM glycine solution at pH=2 for 20 seconds, followed by 20 seconds in kinetics buffer, and finally 60 seconds in kinetics buffer for three cycles to establish an initial signal (baseline). The loading column then contains the ligands, here unmodified PD-1 antibody and IL-18 polypeptide-conjugated PD-1 antibody, at fixed concentrations (20ug / mL) determined in a loading scout experiment. Another wash / baseline step then washes away non-immobilized proteins. The association column contains a two-fold dilution series of the analyte (His-tagged human PD1, R&D#8986-PD), including a no-analyte control. The highest concentration is approximately 10 times the KD. Dissociation designates the sensor being returned to the previous baseline column using kinetics buffer. After acquisition, the data is analyzed with Data Analysis Studio software (Sartorius). The data set is first pre-processed by subtracting the reference samples and aligning the curves at the baseline step. A group fit is then applied to the data series and the kinetic parameters are calculated. The results of this experiment are shown in the table below. [Table 6]

[0312] Figure 16 shows plots measuring the ability of unmodified and conjugated antibodies to bind to human PD-L1 / B7-H1 ligand, the figure shows the net BioLayer interferometry shift in nanometers on the y-axis and the incubation dose of biotinylated PD-L1 protein on the x-axis. The figure shows the average ELISA signal on the y-axis and the dose of human Fc gamma receptor on the x-axis. The unconjugated reference antibodies are pembrolizumab and LZM-009. The conjugated antibody tested is Composition A.

[0313] Example 7 - PD-1 / PD-L1 Blocking Assay (Figures 17A and 17B) For immunocytokine compositions containing PD-1 or PD-L1 antibodies or antigen-binding fragments, the experiment outlined below is performed to evaluate the ability of the immunocytokine to interfere with the PD-1 / PD-L1 pathway. The assay is a PD-1 / PD-L1 blockade bioassay from Promega (catalog number J1250, Madison, WI, USA). The PD-1 / PD-L1 blockade bioassay is a bioluminescent cell-based assay based on co-culture of effector cells with target cells that mimics the immune synapse. Jurkat T cells expressing human PD-1 and a luciferase reporter driven by the NFAT response element (NFAT-RE) are activated by CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate the Jurkat cell cognate TCR. The simultaneous interaction PD-1 / PD-L1 inhibits TCR signaling and suppresses NFAT-RE-mediated luminescence. Addition of either anti-PD-1 or anti-PD-L1 antibodies, which block the PD-1 / PD-L1 interaction, releases an inhibitory signal, restoring TCR activity and resulting in a signal in the NFAT-RE luminescent reporter.

[0314] Briefly, PD-L1 aAPC / CHO-K1 target cells were plated in white tissue culture 96-well plates and cultured overnight at 37°C / 5% CO2. Test molecules were measured in 4-fold serial dilutions from 1 μM to 0.002 nM and pre-incubated on the target cells for 10 minutes before addition of freshly thawed PD-1 Jurkat effector cells. After 6 hours at 37°C / 5% CO2, active NFAT-RE luminescent reporter was assessed by addition of Bio-Glo reagent and measured on an ENSPIRE® plate reader (1 second / well) from Perkin Elmer (Schwerzenbach, Switzerland). The results of this experiment are shown in the table below. [Table 7]

[0315] Figures 17A and 17B show plots measuring the ability of unmodified and conjugated anti-PD1 antibodies to interfere with the PD1 / PDL1 pathway, with the figures showing normalized luminescence intensity of effector cell NFAT-Lucia reporter on the y-axis and doses of unmodified and conjugated anti-PD1 antibodies on the x-axis. The unconjugated reference antibodies are pembrolizumab, LZM-009, nivolumab, atezolizumab, durvalumab, and avelumab. The conjugated antibodies tested in this figure are Composition A and Composition B.

[0316] Example 8 - Human FcγR binding assay (Figures 18A and 18B) The interaction of unmodified and conjugated antibodies with human Fc gamma receptor I (FcγRI / CD64), human Fc gamma receptor IIa (FcγRIIa / CD32a), inhibitory human Fc gamma receptor IIb (FcγRIIb / CD32b), and human Fc gamma receptor III FcγRIIIa / CD16 was measured by ELISA.

[0317] Briefly, Corning high-binding half-area plates (Fisher Scientific, Reinach, Switzerland) were coated overnight at 4° C. with 25 μl of unmodified and conjugated anti-PD1 antibodies at 2.5 μg / ml in PBS. The plates were then washed four times with 100 μl of PBS-0.02% Tween 20. The plate surface was blocked with 25 μl of PBS-0.02% Tween 20-1% BSA for 1 h at 37° C. The plates were then washed four times with 100 μl of PBS-0.02% Tween 20. Next, 25 microliters of either recombinant human Fc gamma RI / CD64 protein (R&D systems, 1257-FC-050, CF), recombinant human Fc gamma RIIA / CD32a (H167) protein (R&D systems, 9595-CD-050, CF), recombinant human Fc gamma RIIB / CD32b Avi tag protein (R&D systems, AVI1875-050, CF), or recombinant human Fc gamma RIIIA / CD16a protein (R&D systems, 4325-FC-050, CF) were added to PBS-0.02% Tween20-0.1% BSA in 5-fold serial dilutions ranging from 1000 nM to 0.001 nM and incubated for 2 hours at 37 °C. The plate was then washed four times with 100 μL of PBS-0.02% Tween20. Twenty-five microliters of 1 / 500 HRP-anti-His antibody in PBS-0.02% Tween20-0.1% BSA (R&D systems, anti-HIS-HRP Ab, #MAB050H) was added to each well and the plate was incubated for 1 h at room temperature. Afterwards, the plate was washed four times with 100 μl PBS-0.02% Tween20. Fifty microliters of TMB substrate reagent (#CL07, Merck, Buchs, Switzerland) was added to each well and incubated for 5 min at 37°C. After 5 min at 37°C, the horseradish peroxidase reaction was stopped by adding 50 μl / well of 0.5 M H2SO4 stop solution. The ELISA signal was then measured at 450 nm in an EnSpire plate reader from Perkin Elmer (Schwerzenbach, Switzerland).The results of this experiment are shown in the table below. [Table 8]

[0318] Figures 18A and 18B show plots measuring the ability of unmodified and conjugated antibodies to bind to human Fc gamma receptor I (CD64) in the top panel and to human Fc gamma receptor IIIa (CD16) in the bottom panel. The figures show the average ELISA signal on the y-axis and the dose of human Fc gamma receptor on the x-axis. The unconjugated reference antibodies are LZM-009 and atezolizumab. The conjugated antibodies tested are compositions A and B.

[0319] Example 9 - Human FcRn binding assay (Figure 19) The interaction of unmodified and conjugated anti-PD1 antibodies with human fetal Fc receptor (FcRn) at pH 6 was measured using the AlphaLISA® Human FcRn Binding Kit (AL3095C) from Perkin Elmer (Schwerzenbach, Switzerland). AlphaLISA® detection of FcRn and IgG binding uses IgG-coated AlphaLISA® acceptor beads that interact with captured biotinylated human FcRn on streptavidin-coated donor beads. When the reference IgG binds to FcRn, the donor and acceptor beads are brought into close proximity, allowing the transfer of singlet oxygen that triggers a cascade of energy transfer reactions in the acceptor beads, resulting in a sharp emission peak at 615 nm. Addition of free IgG antibody to the AlphaLISA® mixture results in competition for binding of FcRn to the reference antibody, resulting in a loss of signal.

[0320] Briefly, test molecules were measured in serial dilutions starting from 5 uM to 64 pM and incubated with AlphaLISA® reaction mixture consisting of 800 nM recombinant biotinylated human FcRn, 40 μg / ml human IgG-conjugated acceptor beads, and 40 μg / ml streptavidin-coated donor beads in MES buffer at pH 6. After 90 minutes at 23° C. in the dark, AlphaLISA® signals were measured on an EnSpire plate reader (excitation at 680 nm, emission at 615 nm) from Perkin Elmer (Schwerzenbach, Switzerland). The results from this experiment are shown in the table below. [Table 9]

[0321] Figure 19 shows a plot measuring the ability of unmodified and conjugated antibodies to bind to the human Fc neonatal receptor. The figure shows the average AlphaLISA signal on the y-axis and the dose of human Fc neonatal receptor (FcRn) on the x-axis. The unconjugated reference antibodies are LZM-009 and Atezolizumab. The conjugated antibodies tested are Compositions A and B.

[0322] Example 10 - IFN-gamma secretion assay in NK92 cells (Figures 20 and 21) The IFNγ secretion stimulating activity of unconjugated IL-18 variants and the corresponding IL-18 immunocytokine was evaluated in the NK92 cell line. The NK cell line NK-92 (ATCC, Cat. No. CRL-2407), derived from a lymphoma patient, was cultured in aMEM medium supplemented with 12.5% ​​FCS, 12.5% ​​horse serum (HS), 50 uM B-mercaptoethanol, and 2 ng / ml recombinant human interleukin-2 (IL-2).

[0323] On the day of the experiment, cells were harvested, washed with aMEM medium without IL-2, and resuspended in medium (without IL-2) containing 1 ng / ml recombinant human interleukin-12 (SinoBiologicals, Cat. No. CT011-H08H). After counting, cells were seeded at 100000 cells / well in 384-well titer plates and incubated at 37°C / 5% CO2. Sixteen 4-fold serial dilutions of IL-18 analytes were prepared in aMEM medium-1 ng / mL IL-12 and added to NK-92 cells. Final IL-18 analyte concentrations ranged from 200 nM to 0.01 pM.

[0324] After 16-20 hours of incubation at 37°C / 5% CO2, 5 μL of supernatant was carefully transferred to a 384-microwell OPTIPlate (Perkin Elmer, Cat. No. 6007270) and interferon-gamma (IFNγ) levels were measured using a human IFNγ AlphaLISA assay kit (Perkin Elmer, Cat. No. AL217C). Briefly, 10 μL of 2.5X AlphaLISA anti-IFNγ acceptor beads and biotinylated antibody anti-IFNγ mix were added to 5 μl of NK-92 supernatant and incubated for 1 hour at room temperature with shaking. Under subdued light, 2.5 μl of 2X streptavidin (SA) donor beads were pipetted into each well and incubated for 30 minutes at room temperature with shaking. AlphaLISA signals were then measured on an Enspire plate reader (Perkin Elmer) with excitation and emission wavelengths of 680 nm and 615 nm, respectively. The half maximal effective concentrations (EC50) were calculated based on variable slope and four parameter analysis using GraphPad PRISM software. The results of this experiment are shown in the table below. [Table 10]

[0325] FIG. 20 shows plots measuring PD-1 and PD-L1 surface expression levels on NK92 cells.

[0326] Figure 21 shows plots measuring the ability of unconjugated IL-18 variants and corresponding IL-18 immunocytokines to stimulate secretion of IFN gamma by NK92 cells. The figure shows the average IFNg alphaLISA signal on the y-axis and the dose of unconjugated IL-18 variants and corresponding IL-18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL-18 wild type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. The corresponding IL-18 immunocytokines tested are compositions A, B, C, and D.

[0327] Example 11 - IFN gamma secretion assay in KG-1 cells (Figures 22-24) The IL-18-responsive AML cell line KG-1 shows high expression of IL-18Rα and moderate levels of IL18Rβ, respectively. The KG-1 cell line was used to generate PD-1 expressing cell lines and further to measure IFNγ release upon incubation with IL-18 mutants and the corresponding IL-18 immunocytokines.

[0328] Generation of PD-1 expressing cell line: Briefly, KG-1 cells were transduced using lentiviral particles carrying human PD-1 gene (PDCD1 NM_005018, Origene, CAT number: RC210364L3V) at MOI (multiplicity of infection) 30. Spinfection was performed at 1260g for 90 minutes at 37°C in the presence of 5μg / ml polybrene and 10mM HEPES in complete medium (RPMI, 10% FBS, 1% L-glutamine). Five days after transduction, puromycin was added at a final concentration of 1ug / ml to select PD-1 positive cells. For culture maintenance, the puromycin concentration was reduced to 0.5μg / ml. Stable and homogenous expression of PD-1 was verified by surface staining (BD Pharmingen, #557860).

[0329] IFNγ release was assessed in PD-1 positive (transduced) KG-1 cells and parental PD-1 negative cells. 5Cells were seeded in 96-well U-bottom plates in medium (RPMI, 10% FBS, 1% L-glutamine) and stimulated with IL-18 variants / ICs for 20–24 h. Test articles were diluted to 100 nM in medium, followed by seven 10-fold serial dilutions. The lowest concentration evaluated was 0.05 fM. After incubation, IFNγ release was measured using the LEGENDplex Custom Human Mix and Match Kit (Biolegend LEGENDplex™ Human IFN-γ Capture Beads B5, 13X #740942, LEGENDplex™ HU Essential Immune Response Panel Detection Ab, #740931, LEGENDplex™ Buffer Set A #740368). For this purpose, cell culture supernatants were collected and diluted 1:1 with assay buffer. Fluorescence measurements were performed on a Quanteon flow cytometer from Acea Biosciences. For analysis, MFI values ​​(median fluorescence intensity) were exported and plotted against the concentrations used. EC 50 Values ​​(half maximal effective concentrations) were calculated based on a variable slope and four parameter analysis using GraphPad PRISM software version 9. The results of this experiment are shown in the table below. [Table 11]

[0330] FIG. 22 shows plots measuring PD-1 and PD-L1 surface expression levels on KG-1 cells.

[0331] FIG. 23 shows that unconjugated IL-18 variants and the corresponding IL-18 immunocytokine inhibited the parental PD-1 陰性 and engineered PD-1 陽性Figure 1 shows a plot measuring the ability to stimulate the secretion of IFN gamma by KG-1 cells. The figure shows the average IFNg legendplex signal on the y-axis and the dose of unconjugated IL-18 variants and the corresponding IL-18 immunocytokine on the x-axis. The unconjugated IL-18 variants are native IL-18 wild type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. The corresponding IL-18 immunocytokines tested are compositions A, B, C, and D.

[0332] Example 12 - IL-18 Binding Protein alphaLISA Assay (Figure 24) Wild-type or modified IL-18 polypeptide samples were diluted to 5.6 μM in 1× alphaLISA immunoassay buffer solution provided with the alphaLISA IFNγ Detection Kit and diluted applying 3-fold serial dilutions down to 1.7 pM in a 384 deep-well plate. A 10 ng / ml human IL-18BP-His solution was prepared in 1× alphaLISA immunoassay buffer. IL-18 / IL-18BP complex formation was performed by incubating 30 μL of IL-18BP solution against the IL-18 sample titrations for 1 hour at 20°C. IL-18BP standards were pre-diluted from the stock solution provided with the alphaLISA IFNγ Detection Kit at 100 ng / ml using 1× alphaLISA buffer and applying 2-fold serial dilutions to prepare the titrations. The following solutions were prepared: a 50 μg / ml solution of anti-IL-18BP alphaLISA acceptor beads, a 5 nM solution of biotinylated anti-IL18BP antibody, and an 80 μg / ml light-protected solution of streptavidin donor beads in 1xalphaLISA immunoassay buffer. To detect unbound IL-18BP in IL-18 / IL-18P complex samples, 5 μl of premixed acceptor bead solution was transferred on top of 7.5 μL of sample in a 384-well Optiplate, followed by a short centrifugation step at 150 g and incubation for 30 min at 20° C. with shaking at 750 rpm.

[0333] 5 μl of biotinylated anti-IL-18BP antibody was added, followed by a short centrifugation step at 150 g and incubation at 20° C. for 60 min with shaking at 750 rpm. Under subdued light, 12.5 μl of premixed donor beads were added, followed by a short centrifugation step at 150 g and incubation at 20° C. for 30 min without light and shaking at 750 rpm. AlphaLISA signal was then measured on an Enspire plate reader (Perkin Elmer) with excitation and emission wavelengths of 680 nm and 615 nm, respectively. Unbound IL-18BP concentration was interpolated from the standard signal-concentration curve using GraphPad Prism. The results of this experiment are shown in the table below. [Table 12]

[0334] Figure 24 shows a plot measuring the ability of unconjugated IL-18 variants and the corresponding IL-18 immunocytokines to bind to human IL-18 binding protein (IL-18BP). The figure shows the average free IL-18BP AlphaLISA signal on the y-axis and the dose of unconjugated IL-18 variants and the corresponding IL-18 immunocytokines on the x-axis. The unconjugated IL-18 variants are native IL-18 wild type (SEQ ID NO: 1), SEQ ID NO: 30, and SEQ ID NO: 31. The corresponding IL-18 immunocytokines tested are compositions A, B, C, and D.

[0335] Example 13 - Cellular IL-18 binding protein resistance assay (Figure 25) The NK cell line NK-92 (ATCC, Cat. No. CRL-2407), derived from a lymphoma patient, was cultured in aMEM medium supplemented with 20% FCS-Glutamax, 25 μM B-mercaptoethanol, and 100 IU / ml recombinant human interleukin 2 (IL-2). On the day of the experiment, cells were harvested and washed with αMEM medium without IL-2. After counting, cells were seeded at 100000 cells / well in 384-well titer plates and incubated at 37°C / 5% CO2. Sixteen two-fold serial dilutions of F...

Claims

1. 1. An immunocytokine composition comprising: IL-18 polypeptide, and an antibody or antigen-binding fragment thereof specific for an immune cell-associated antigen, and Linker wherein the linker comprises a first attachment point to the IL-18 polypeptide, and a second point of attachment to the antibody or antigen-binding fragment thereof, The immunocytokine composition.

2. The immunocytokine composition of claim 1 , wherein the linker comprises a polymer.

3. The immunocytokine composition of claim 2 , wherein the polymer comprises a water-soluble polymer.

4. The immunocytokine composition of claim 3, wherein the water soluble polymer comprises a poly(alkylene oxide), a polysaccharide, a poly(vinylpyrrolidone), a poly(vinyl alcohol), a polyoxazoline, a poly(acryloylmorpholine), or a combination thereof.

5. The immunocytokine composition of any one of claims 2 to 4, wherein the polymer has a weight average molecular weight of at least about 0.1 kDa, at least about 0.5 kDa, at least about 1 kDa, at least about 5 kDa, at least about 10 kDa, at least about 20 kDa, at least about 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, at least 120 kDa, at least 130 kDa, at least 140 kDa, at least 150 kDa, or more.

6. The immunocytokine composition of any one of claims 1 to 5, wherein the first point of attachment is at a residue that is neither N-terminal nor C-terminal of the IL-18 polypeptide.

7. 7. The immunocytokine composition of any one of claims 1 to 6, wherein the first point of attachment is to a residue within the region of residues 2 to 156 of the IL-18 polypeptide, the numbering of residue positions being based on SEQ ID NO:

1.

8. The immunocytokine composition of claim 7, wherein the first point of attachment is to a residue within the region of residues 38-144 of the IL-18 polypeptide.

9. 9. The immunocytokine composition of claim 7 or 8, wherein the first attachment point is at residue 38, 68, 69, 70, 76, 78, 85, 86, 95, 98, 121, 127, or 144 of the IL-18 polypeptide.

10. The immunocytokine composition of any one of claims 7 to 9, wherein the first attachment point is at residue 68, 69, 70, 85, 86, 95, or 98 of the IL-18 polypeptide.

11. The immunocytokine composition of any one of claims 7 to 10, wherein the first attachment point is at residue 68 of the IL-18 polypeptide.

12. The immunocytokine composition of any one of claims 1 to 11, wherein the IL-18 polypeptide comprises an amino acid substitution at the first attachment point.

13. The immunocytokine composition of claim 12, wherein the amino acid substitution at the first attachment point is for a non-natural amino acid.

14. The immunocytokine composition of claim 12, wherein the amino acid substitution at the first attachment point is to a naturally occurring amino acid.

15. 13. The immunocytokine composition of claim 12, wherein the amino acid substitution at the first attachment point is E69C, K70C, E85C, M86C, T95C, or D98C.

16. The immunocytokine composition of any one of claims 1 to 15, wherein the second point of attachment is at an amino acid residue in the Fc region of the antibody or antigen-binding fragment.

17. The immunocytokine composition of claim 16, wherein the Fc region comprises an amino acid sequence having 90% or greater identity to the amino acid sequence of SEQ ID NO:

105.

18. 18. The immunocytokine composition of claim 17, wherein the second attachment point is (a) amino acid residues 16-18 of SEQ ID NO:260, (b) amino acid residues 58-60 of SEQ ID NO:260, or (c) amino acid residue 87 of SEQ ID NO:

260.

19. The immunocytokine composition of any one of claims 1 to 18, wherein the second point of attachment is to a lysine residue on the antibody or antigen-binding fragment.

20. The immunocytokine composition of any one of claims 1 to 19, wherein said IL-18 polypeptide exhibits reduced binding to IL-18 binding protein (IL-18BP) compared to the wild-type IL-18 polypeptide of SEQ ID NO:

1.

21. 21. The immunocytokine composition of any one of claims 1 to 20, wherein said IL-18 polypeptide contains one or more amino acid substitutions located at residue positions selected from Y01, F02, E06, V11, C38, K53, D54, S55, T63, E69, K70, E85, C76, M86, T95, D98, and C127, and the numbering of the residue positions of said IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence.

22. 22. The immunocytokine composition of claim 21, wherein the one or more amino acid substitutions in the IL-18 polypeptide are selected from Y01G, F02A, E06K, V11I, C38S, C38A, K53A, D54A, S55A, T63A, C68S, C68A, E69C, K70C, C76S, C76A, E85C, T95C, D98C, C127S, and C127A.

23. The immunocytokine composition of any one of claims 1 to 22, wherein the IL-18 polypeptide contains E06K and K53A amino acid substitutions.

24. The immunocytokine composition of any one of claims 1 to 23, wherein the IL-18 polypeptide contains a T63A amino acid substitution.

25. The immunocytokine composition of any one of claims 1 to 24, wherein the IL-18 polypeptide comprises a V11I amino acid substitution.

26. The IL-18 polypeptide is (a) a homoserine residue located at any one of residues 26 to 36; (b) a homoserine residue located at any one of residues 45 to 67; (c) a homoserine residue located at any one of residues 70 to 80; (d) a homoserine residue located at any one of residues 110 to 130; (e) a norleucine or O-methyl-homoserine residue located at any one of residues 28 to 38; (f) a norleucine or O-methyl-homoserine residue located at any one of residues 46 to 56; (g) a norleucine or O-methyl-homoserine residue located at any one of residues 54 to 64; (h) a norleucine or O-methyl-homoserine residue located at any one of residues 80 to 90; (i) a norleucine or O-methyl-homoserine residue located at any one of residues 108 to 118, and (j) a norleucine or O-methyl-homoserine residue located at any one of residues 145 to 155; 26. The immunocytokine composition of any one of claims 1 to 25, comprising one or more amino acid substitutions selected from:

27. 27. The immunocytokine composition of claim 26, wherein the IL-18 polypeptide comprises one or more amino acid substitutions selected from homoserine (Hse)31, norleucine (Nle)33, O-methyl-homoserine (Omh)33, Hse50, Nle51, Omh51, Hse57, Nle60, Omh60, Hse63, Hse67, Hse75, Nle86, Omh86, Hse106, Nle113, Omh113, Hse116, Hse121, Nle150, and Omh150.

28. The immunocytokine composition of any one of claims 1 to 27, wherein the antibody or antibody-binding fragment thereof is a monoclonal antibody, a humanized antibody, a grafted antibody, a chimeric antibody, a human antibody, a deimmunized antibody, or a bispecific antibody.

29. The antibody or antigen-binding fragment thereof, the antigen-binding fragment, wherein the antigen-binding fragment is a Fab, Fab', F(ab')2, bispecific F(ab')2, trispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), dsFv, bispecific scFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, AVIMER®, minibody, diabody, bispecific diabody, triabody, tetrabody, minibody, maxibody, camelid, VHH, minibody ...

29. The immunocytokine composition of any one of claims 1 to 28, comprising a nibody, an intrabody, a fusion protein comprising an antibody portion (domain antibody), a single chain binding polypeptide, a scFv-Fc, a Fab-Fc, a bispecific T cell engager (BiTE), a tetravalent tandem diabody (TandAb), a dual affinity retargeting antibody (DART), a bispecific antibody (bscAb), a single domain antibody (sdAb), a fusion protein, or a bispecific disulfide stabilized Fv antibody fragment (dsFv-dsFv').

30. The immunocytokine composition of any one of claims 1 to 29, wherein the antibody or antigen-binding fragment thereof comprises or is derived from IgG, IgM, IgE, IgA, IgD.

31. 31. The immunocytokine composition of claim 30, wherein the antibody or antigen-binding fragment thereof comprises an IgG, wherein the IgG is or is derived from IgG1, IgG4.

32. 32. The immunocytokine composition of any one of claims 1 to 31, wherein the immune cell associated antigen is 4-IBB, CD3, CCR8, CD8A, CD8B, CD16A, CD28, CD80, CD86, CD96, CD226, CTLA-4, D40, GITR, ICOS, LAG-3, MHCI, MHCII, NKG2A, NKG2D, NKp30, NKp44, NKp46, OX40, PD-1, PD-L1, PD-L2, SIRPA, TCR, TIGIT, TIM-3, or VISTA.

33. The immunocytokine composition according to any one of claims 1 to 32, wherein the immune cell associated antigen is PD-1.

34. The antibody or antigen-binding fragment thereof is selected from the group consisting of tislelizumab, baizean, 0KVO411B3N, BGB-A317, hu317-1 / IgG4mt2, sintilimab, tyvyt, IBI-308, toripalimab, TeRuiPuLi, telepryl, tuoyi, JS-001, TAB-001, tamrelizumab, HR-301210, INCSHR-01210, SHR-1210, temiprimab, cemiplimab-rwlc, 6QVL057INT, H4H7798N, REGN-2810, SAR-439684, lambrolizumab, pembrolizumab, Mab, MK-3475, SCH-900475, h409A11, nivolumab, nivolumab BMS, BMS-936558, MDX-1106, ONO-4538, prorugolimab, forteca, BCD-100, penprimab, AK-105, zimberelimab, AB-122, GLS-010, WBP-3055, balstilimab, 1Q2QT5M7EO, AGEN-2034, AGEN-2034w, genolinzumab, geptanolimab, APL-501, CBT-501, GB-226, dostallimab, ANB-011, GSK-4057190A, P0GVQ9A4S5, TSR-042, WBP-285, selplulimab, HLX-10, CS-1003, retifanlimab, 2Y3T5IF01Z, INCMGA-00012, INCMGA-0012, MGA-012, sasanlimab, LZZ0IC2EWP, PF-06801591, RN-888, spartalizumab, PDR-001, QOG25L6Z8Z, retatolimab / nivolumab, BMS-986213, cetolimab, JNJ-3283, JNJ-63723283, LYK98WP91F, tebotelimab, MGD-013, BCD -217, BAT-1306, HX-008, MEDI-5752, JTX-4014, cadnilimab, AK-104, BI-75409 1. Pidilizumab, CT-011, MDV-9300, YBL-006, AMG-256, RG-6279, RO-7284755, BH -2950, ​​IBI-315, RG-6139, RO-7247669, ONO-4685, AK-112, 609-A, LY-3434 172, T-3011, AMG-404, IBI-318, MGD-019, ONCR-177, LY-3462817, RG-7769,RO-7121661, F-520, XmAb-23104, Pd-1-pik, SG-001, S-95016, Sym-021, LZM-009, budigalimab, 6VDO4TY3OO, ABBV-181, PR-1648817, CC-90006, XmAb-20717, 2661380, AMP-224 , B7-DCIg, EMB-02, ANB-030, PRS-332, [89Zr]deferoxamide-pembrolizumab, 89Zr-Df-pembrolizumab, [89Zr]Df-pembrolizumab, STI-1110, STI-A1110, CX-188, mPD-1, MCLA-134, 244C8, ENUM 224C8, ENUM C8, 388D4, ENUM 388D4, ENUM D4, MEDI0680, NVP-LZV-184, or AMP-514, or modified forms thereof.

35. 34. The immunocytokine composition of claim 33, wherein the antibody or antigen-binding fragment thereof comprises nivolumab, pembrolizumab, LZM-009, or cemiplimab, or modified forms thereof.

36. The immunocytokine composition according to any one of claims 1 to 32, wherein the immune cell associated antigen is PD-L1.

37. The antibody is selected from the group consisting of avelumab (Bavencio, 451238, KXG2PJ551I, MSB-0010682, MSB-0010718C, PF-06834635, CAS1537032-82-8), durvalumab (Imfinzi, 28X28X9OKV (UNII code), MEDI-4736, CAS1428935-60-7), atezolizumab (Tecentriq, 52CMI0WC3Y, MPDL-3280A, RG-7446, RO-5541267, CAS1380723-44-3), and suge Malimab (CS-1001, WBP-3155), KN-046 (CAS2256084-03-2), APL-502 (CBT-502, TQB-2450), embafolimab (3D-025, ASC-22, KN-035, hu56V1-Fc-m1, CAS2102192-68-5), vintrafusp alfa (M-7824, MSB-0011359C, NW9K8C1JN3, CAS1918149-01-5), STI-1014 (STI-A1014, ZKAB-001), PD-L1 t-haNK, A-167 (HBM-9167, KL-A167), IMC-001 (STI-3031, STI-A-1015, STI-A1015, s), HTI-1088 (SHR-1316), IO-103, CX-072 (CytomX Therapeutics), AUPM-170 (CA-170), GS-4224, ND-021 (NM21-1480, PRO-1480), BNT-311 (DuoBody-PD-L1x4-1BB, GEN-1046), BGB-A333, IBI-322, NM-01, LY-3434172, LDP, CDX-527, IBI-318, 89Zr-DFO-REGN3504, ALPN-202 (CD80 vIgD-Fc), INCB-086550, LY-3415244, SHR-1701, JS-003 (JS003-30, JS003-SD), HLX-20 (PL2#3), E S-101 (INBRX-105, INBRX-105-1), MSB-2311, PD-1-Fc-OX40L (SL-279252, TAK-252), FS-118, FS118 mAb2, LAG-3 / PD-L1mAb2), FAZ-053 (LAE-005), lodapolimab (LY-3300054, NR4MAD6PPB, CAS 2118349-31-6), MCLA-145, BMS-189, cosibelimab (CK-301, TG-1501, CAS 2216751-26-5), IL-15Ralpha-SD / IL-15 (KD-033), WP-1066 (CAS 8 57064-38-1), BMS-936559 (MDX-1105), BMS-986192, RC-98, CD-200AR-L (CD200AR-L), ATA-3271, IB C-Ab002, BMX-101, AVA-04-VbP, ACE-1708, KY-1043, ACE-05 (YBL-013), ONC-0055 (ONC0055, PRS-344 37. The immunocytokine composition of claim 36, wherein the cytokine is selected from the group consisting of 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-29, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-36, IL-37, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-59, IL-59, IL-59, IL-59, IL-60, IL-61, IL-62, IL-63, IL-74, IL-75, IL-85, IL-91, IL-92, IL-93, IL-94, IL-95, IL-95, IL-95, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-37, IL-38, IL-39, IL-41, IL-42, IL-43, IL-54, IL-55, IL-14, IL-15, IL-15,

38. 37. The immunocytokine composition of claim 36, wherein the antibody is durvalumab, atezolizumab, or avelumab, or a modified form thereof.

39. The immunocytokine composition of any one of claims 1 to 38, wherein the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence of Table 1.

40. 40. The immunocytokine composition of any one of claims 1 to 39, wherein the IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:

1.

41. The immunocytokine composition of any one of claims 1 to 40, wherein the IL-18 polypeptide of the immunocytokine provided herein comprises the amino acid sequence of any one of SEQ ID NOs: 2 to 67.

42. The immunocytokine composition of any one of claims 1 to 40, wherein the IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO:

30.

43. The immunocytokine composition of any one of claims 1 to 40, wherein the IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 68 to 72.

44. The immunocytokine composition comprises a binding affinity (K Ｄ 44. The immunocytokine composition of any one of claims 1 to 43, which exhibits a binding affinity within 10-fold of that of the antibody.

45. The immunocytokine composition is characterized in that it is capable of measuring the binding affinity (K Ｄ 45. The immunocytokine composition of any one of claims 1 to 44, which exhibits a binding affinity within 5-fold of the antibody binding affinity of the antibody of claim 1.

46. The immunocytokine composition has a half maximal effective concentration (EC) that is within about 100-fold of the corresponding IL-18 polypeptide not included in the immunocytokine composition. ５０ ) and the immune cytokine composition exhibits a lower EC ５０ The immunocytokine composition according to any one of claims 1 to 45, which exhibits the following:

47. The immunocytokine composition of any one of claims 1 to 46, wherein said immunocytokine composition exhibits improved anti-tumor growth inhibition compared to said antibody alone.

48. 48. The immunocytokine composition of any one of claims 1 to 47, wherein said immunocytokine composition exhibits improved anti-tumor growth inhibition compared to said antibody and said IL-18 polypeptide administered in combination.

49. The immunocytokine composition according to any one of claims 1 to 48, further comprising a second linker, said second linker being a third point of attachment to the antibody or antigen-binding fragment thereof; and a fourth attachment point for an additional cytokine; The immunocytokine composition comprising:

50. 50. The immunocytokine composition of claim 49, wherein the additional cytokine is selected from a second IL-18 polypeptide, an IL-7 polypeptide, or an IL-2 polypeptide.

51. The immunocytokine composition of claim 49 or 50, wherein the additional cytokine is a second IL-18 polypeptide.

52. 52. The immunocytokine composition of claim 51, wherein said second IL-18 polypeptide is substantially identical to said IL-18 polypeptide.

53. The immunocytokine composition of any one of claims 49 to 52, wherein the third attachment point is to a different heavy chain than the second attachment point of the antibody or antigen-binding fragment thereof.

54. a) an immunocytokine composition according to any one of the preceding claims, and b) one or more pharma- ceutically acceptable carriers or excipients; 13. A pharmaceutical composition comprising:

55. 55. The pharmaceutical composition of claim 54, wherein the pharmaceutical composition is formulated for parenteral or enteral administration.

56. 56. The pharmaceutical composition of claim 54 or 55, wherein the pharmaceutical composition is formulated for intravenous or subcutaneous administration.

57. The pharmaceutical composition according to any one of claims 54 to 56, wherein the pharmaceutical composition is in a lyophilized form.

58. 58. The pharmaceutical composition of any one of claims 54-57, wherein the one or more pharma- ceutically acceptable carriers or excipients comprise one or more of each of the following: carbohydrates, inorganic salts, antioxidants, surfactants, buffering agents, or any combination thereof.

59. 59. The pharmaceutical composition of any one of claims 54 to 58, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more excipients.

60. 60. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an immunocytokine composition according to any one of claims 1 to 53 or a pharmaceutical composition according to any one of claims 54 to 59.

61. 61. The method of claim 60, wherein the cancer is melanoma, lung cancer, bladder cancer (BC), microsatellite instability-high (MSI-H) / mismatch repair deficient (dMMR) solid tumor, high tumor mutation burden (TMB-H) solid tumor, triple-negative breast cancer (TNBC), gastric cancer (GC), cervical cancer (CC), pleural mesothelioma (PM), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), or a combination thereof.

62. 61. The method of claim 60, wherein the cancer is adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colon cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal cancer tumors, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, childhood cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureter cancer, uterine cancer, vaginal cancer, or vulvar cancer.

63. 63. The method of claim 62, wherein the cancer is a carcinoma, the carcinoma comprising cutaneous squamous cell carcinoma (CSCC), urothelial carcinoma (UC), renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), head and neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma (ESCC), gastroesophageal junction (GEJ) cancer, endometrial carcinoma (EC), Merkel cell carcinoma (MCC), or a combination thereof.

64. 61. The method of claim 60, wherein the cancer is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, AIDS-related lymphoma, multiple myeloma, plasma cell tumor, post-transplant lymphoproliferative disorder, or Waldenstrom's macroglobulinemia.

65. 65. The method of claim 64, wherein the cancer is leukemia, and the leukemia is acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, small lymphocytic lymphoma, or chronic myelogenous leukemia.

66. 66. The method of claim 65, wherein the cancer is a lymphoma, comprising Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma, cutaneous lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma, peripheral T-cell lymphoma, Waldenstrom's macroglobulinemia, lymphedema lymphoma, marginal zone lymphoma, B-cell cutaneous lymphoma, extranodal natural killer T-cell lymphoma, T-cell lymphoblastic lymphoma, peripheral T-cell lymphoma, or T-cell cutaneous lymphoma.

67. A method for producing an immunocytokine composition according to any one of claims 1 to 53, comprising the steps of: a) covalently linking a reactive group to a specific residue of said antibody or antigen-binding fragment thereof; b) contacting said reactive group with a complementary reactive group attached to said IL-18 polypeptide; and c) forming said composition; The method comprising:

68. 1. A method of making a composition, comprising: The composition, IL-18 polypeptide, and an antibody or antigen-binding fragment thereof specific for an immune cell antigen, and Linker wherein the linker comprises a first attachment point to the IL-18 polypeptide, and A second attachment point for the antibody or antigen-binding fragment thereof. wherein the method comprises: a) providing the antibody or antigen-binding fragment, the antibody or antigen-binding fragment comprising at least one acceptor amino acid residue that reacts with the linker in the presence of a coupling enzyme; and b) reacting the antibody or antigen-binding fragment with the linker comprising a primary amine in the presence of an enzyme capable of causing the formation of a covalent bond between the at least one acceptor amino acid residue and the linker, wherein the linker comprises a reactive group (R); the covalent bond is not at the R moiety, and the method is performed under conditions sufficient to cause the at least one acceptor amino acid residue to form a covalent bond to the reactive group through the linker, the covalent bond comprising the second attachment point of the linker.

69. 69. The method of claim 68, wherein the enzyme comprises a transaminase.

70. 70. The method of claim 69, wherein the enzyme comprises transglutaminase.

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