Novel PD1-targeting IL-2 immune cytokine and VioKine fusion

Abstract

The present disclosure provides novel PD1Ab-IL-2 immune cytokine and VioKine compositions that are aimed at directly targeting IL-2, which is attenuated in efficacy or is bioactivatable, to tumor-infiltrating lymphocytes to reduce toxicity based on systemic mechanisms and result in a wider therapeutic utility of IL-2 for the treatment of cancer. For PD1Ab-IL-2VitoKine, IL-2 will remain inert until locally activated by an upregulated protease in diseased tissue, which will prevent excessive activation of the pathway and reduce undesirable "extra-tissue" in-target "toxicity and significantly reduce potential antigen or target submergence and thereby extend in vivo half-life and result in improved biodistribution, bioavailability and therapeutic efficacy. In addition, the PD1 antibody capable of blocking PD1 and reversing non-reactivity or depletion of T cells can also act synergistically with IL-2 anti-cancer immune response.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 404,608, filed on September 8, 2022, which is hereby incorporated by reference in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (SeqListing-CUGENE PD1 AB-IL-2.xml; size: 216 kilobytes; generated on September 5, 2023) are hereby incorporated by reference in its entirety. Technical Field

[0005] Although cancer has traditionally been treated with chemotherapy, radiation therapy, targeted therapy, and surgery, the fifth pillar of cancer treatment, immunotherapy, has emerged in recent years and revolutionized the war on cancer. The benchmark for immunotherapy drugs was established by the development of T cell checkpoint (CTLA-4 and PD1 / PD-L1) inhibitors. It has been shown that these therapies effectively expand and reactivate the pool of tumor-specific T cells, resulting in an objective response rate of up to 50% in patients with certain cancers.

[0006] Interleukin 2 (IL-2) is the first described growth factor for T cells. The ability of IL-2 to expand lymphocyte populations in vivo and increase the effector functions of these cells confers anti-tumor effects of IL-2 and led to the approval of high-dose recombinant IL-2 for certain metastatic cancers. Although IL-2 cancer immunotherapy has shown durable responses in approximately 10% of patients, IL-2 cancer immunotherapy is associated with more than one problem, including severe toxicity caused by the induction of vascular leak syndrome (VLS), tumor tolerance caused by the induction of activation-induced cell death (AICD), and immunosuppression caused by Treg cell activation.

[0007] Several approaches have been taken to overcome the challenges inherent in IL-2 immunotherapy. One such approach to counteract systemic toxicity involves targeting cytokine activity to cancer cells and their surrounding tissues through a tumor-targeted IL-2 immunocytokine, which is constructed by fusing IL-2 with an antibody specific for a tumor-associated antigen. However, this strategy lacks the ability to specifically target effector T cells associated with anti-cancer immunity within the tumor microenvironment (TME). This gap in intratumoral T cell targeting can be filled by fusing IL-2 with an anti-programmed cell death protein 1 (PD1) antibody. PD1 (also known as CD279) is highly expressed on tumor-infiltrating lymphocytes (TILs), and the PD1 antibody IL-2 immunocytokine enables IL-2 to directly target TILs. It exhibits elevated affinity for intratumoral CD8+ T cells rather than Treg cells or peripheral CD4+ and CD8+ T cells. Thus, this strategy further improves IL-2 anti-cancer immunity while reducing systemic toxicity.

[0008] In addition to directly targeting TILs with IL-2 to improve IL-2 anti-cancer immunity, PD1 antibodies that can block PD1 and reverse T cell anergy or exhaustion can act in synergy with IL-2 to further enhance the anti-cancer immune response of IL-2. Therefore, it is desirable to construct a PD1 Ab-IL-2 immunocytokine with a PD1 antibody having superior target-binding and PD1-blocking capabilities. Among the various PD1-blocking antibodies commercially available globally, pembrolizumab ( Merck Sharp & Dohme Corp.), which has revolutionized the field of cancer immunotherapy, has received significant attention due to its high efficacy and approval for treating many cancer types. Although pembrolizumab exhibits superior target-binding and blocking capabilities, it has several sequence deficiencies, including a relatively low degree of humanness that may cause immunogenicity issues and high hydrophobicity that tends to increase its aggregation propensity. Therefore, it is preferred to optimize pembrolizumab to mitigate its sequence deficiencies while fully maintaining its biological activity. The resulting optimized sequence is expected to improve the developability of the PD1 Ab-IL-2 fusion protein.

[0009] Importantly, the fusion of the PD1 Ab with the fully active IL-2 moiety can override the intended antibody-mediated targeting, localizing the fusion protein to IL-2 receptor-expressing cells in the periphery rather than tumor-infiltrating lymphocytes (TILs) in the tumor. Thus, to improve target specificity and selectivity, one approach is to prepare fusions using an IL-2 moiety with attenuated IL-2Rβγ activity to establish a stoichiometric balance between the cytokine and antibody components. Additionally, reducing cytokine potency can potentially mitigate pathway overactivation as well as antigen sink and target-mediated deposition.

[0010] Another related but more complex strategy to improve target specificity and selectivity is to apply the VitoKine platform disclosed by the present inventors in WO2019246392 and WO2021119516. In the VitoKine construct, the activity of the IL-2 moiety will remain inert or minimal until locally activated by proteases upregulated in or around the tumor. By doing so, binding of the IL-2 moiety to its receptors in the periphery of non-diseased cells or on the cell surface can be significantly restricted. This can help prevent pathway overactivation and reduce undesired "off-tissue" "on-target" toxicity, and the improved safety profile of VitoKine can permit human dose levels within the effective range of the PD1 antibody. Additionally, the inertness of the IL-2 moiety prior to protease activation will significantly reduce potential antigen sink or target sink, and thereby extend the in vivo half-life and result in improved biodistribution and bioavailability at the intended site of treatment.

[0011] Disclosure of the Invention

[0012] In one aspect, the present invention provides a novel PD1-targeted bioactivatable IL-2 immunocytokine (referred to herein as PD1 Ab-IL-2 VitoKine) that is designed to directly target bioactivatable IL-2 to tumor-infiltrating lymphocytes. The activity of the IL-2 moiety will remain nearly inert or minimal until locally activated by proteases upregulated in the tumor, which will restrict binding of the IL-2 moiety to its receptors in the periphery of non-diseased cells or normal tissues or on the cell surface. This can help prevent pathway overactivation, reduce undesired "off-tissue" "on-target" toxicity, and minimize undesired target sink.

[0013] In another aspect, the present invention provides novel PD1-targeted IL-2 immunocytokines that are designed to directly target an activity-modulated IL-2 domain to tumor-infiltrating lymphocytes. Attenuated IL-2 activity is expected to facilitate establishment of a stoichiometric balance between the cytokine and antibody arms, help mitigate pathway overactivation, and reduce antigen sink and target-mediated deposition.

[0014] This strategy specifically targets effector T cells associated with anti-cancer immunity within the tumor microenvironment (TME). By implementing this strategy, the ability of IL-2 to expand lymphocyte populations and enhance their effector functions synergizes with the function of PD1-blocking antibodies in reversing T cell anergy or exhaustion. This approach, especially when using attenuated or bioactivatable IL-2, reduces toxicity based on systemic mechanisms, leading to a broader therapeutic use of IL-2 for cancer treatment and improving biodistribution and bioavailability at the intended treatment site.

[0015] In various embodiments, the PD1-targeted bioactivatable IL-2 immunocytokine is referred to herein as PD1Ab-IL-2 VitoKine. In various embodiments, the VitoKine platform disclosed by the inventors in WO2019246392 and WO2021119516 is defined by the construct depicted in FIG. 1 and one of the proposed activation methods depicted as Figure 2 In various embodiments, the PD1 Ab-IL-2 VitoKine of the present invention is more specifically defined by the construct illustrated in FIG. 3A. Referring to FIG. 3A, the PD1 Ab-IL-2 VitoKine of the present invention comprises a PD1-blocking antibody, a monovalent IL-2 domain (active moiety domain) whose N-terminus is fused to the C-terminus of the heterodimeric Fc chain of the PD1 antibody via an L1 linker, and whose C-terminus is fused to the N-terminus of the IL-2Rα sushi domain (masking moiety domain) via an L2 linker.

[0016] In various embodiments, the variable domain of the PD1-blocking antibody of the present invention is optimized from the variable domain of pembrolizumab by introducing germline sequence substitutions into CDR residues, introducing germline sequence substitutions into framework somatic mutations, and / or employing the most prevalent and better-performing VH3 human germline family sequence as the receptor framework. In various embodiments, the PD1-blocking antibody has high affinity for the human PD1 protein listed in SEQ ID NO:1, inhibits PD1 with equal or comparable potency to pembrolizumab, exhibits a higher sequence similarity score to its closest human germline sequence than pembrolizumab, thereby indicating a higher degree of human origin, and is predicted to have lower hydrophobicity than pembrolizumab, which in turn reduces its tendency to aggregate.

[0017] In various embodiments, the PD1-blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:7. In various embodiments, the PD1-blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:9. In various embodiments, the PD1-blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:11. In various embodiments, the PD1-blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:13. In various embodiments, the PD1-blocking antibody comprises a light chain variable region having the sequence set forth in SEQ ID NO:3 and a heavy chain variable region having the sequence set forth in SEQ ID NO:18.

[0018] In various embodiments, the PD1-targeted IL-2 immunocytokine is defined by the construct depicted in Figure 3B. In various embodiments, the potency-modulated IL-2 of the PD1-targeted IL-2 immunocytokine is an IL-2 variant (or mutant) that comprises a sequence derived from the mature human IL-2 polypeptide (also referred to herein as huIL-12 or IL-2 wild type (w / t)), such as the sequence set forth in SEQ ID NO:116 that comprises one or more amino acid substitutions, deletions, or insertions. In various embodiments, the amino acid change is one or more amino acid substitutions at position 19, 65, 125, or 126 of SEQ ID NO:116. In various embodiments, the amino acid change is a substitution of L to D or H or N or P or Q or R or S or Y at position 19, P to G or E or H or R or A or K or N or Q at position 65, C to I at position 125, Q to A or D or E or F or G or H or I or K or L or M or N or P or R or S or T or V or W or Y at position 126 of the mature human IL-2 sequence, or any combination of these substitutions. In various embodiments, the IL-2 variant has reduced / eliminated binding to IL-2Rα compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant has reduced binding activity to the IL-2Rβγ receptor compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant has reduced / eliminated binding to IL-2Rα and modulated binding activity to the IL-2Rβγ receptor compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant is selected from the group of sequences set forth in SEQ ID NO:117-180.

[0019] In various embodiments, the active portion of the PD1 Ab-IL-2 VitoKine is an IL-2 domain comprising a sequence of a mature human IL-2 polypeptide as set forth in SEQ ID NO:116. In various embodiments, the IL-2 domain is an IL-2 variant (or mutant) comprising a sequence having one or more amino acid substitutions, deletions or insertions from the sequence of the mature human IL-2 polypeptide as set forth in SEQ ID NO:116. In various embodiments, the amino acid change is one or more amino acid substitutions at position 19, 65, 125 or 126 of SEQ ID NO:116. In various embodiments, the amino acid change is a substitution of L to D or H or N or P or Q or R or S or Y at position 19, P to G or E or H or R or A or K or N or Q at position 65, C to I at position 125, Q to A or D or E or F or G or H or I or K or L or M or N or P or R or S or T or V or W or Y at position 126 of the mature human IL-2 sequence, or any combination of these substitutions. In various embodiments, the VitoKine construct will comprise an IL-2 portion designed to have reduced / eliminated binding to IL-2Rα. In various embodiments, the IL-2 variant has reduced binding activity to IL-2Rβγ compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant has reduced / eliminated binding to IL-2Rα and altered binding activity to IL-2Rβγ compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant in the VitoKine construct can modulate the intrinsic basal activity of the IL-2 VitoKine to achieve optimal anti-tumor efficacy while minimizing undesired systemic toxicity to widen the therapeutic window. In various embodiments, the IL-2 domain is selected from the group of sequences set forth in SEQ ID NOs:117 - 180.

[0020] In various embodiments, the masking moiety domain is a cognate receptor / binding partner, or any binding partner identified for IL-2. In various embodiments, the masking moiety domain is the extracellular domain of IL-2Rα having the sequence listed in SEQ ID NO:181 or a functional fragment thereof. In various embodiments, the extracellular domain of IL-2Rα or a functional fragment thereof is the sushi domain of IL-2Rα having the sequence listed in SEQ ID NO:182. In various embodiments, the masking moiety domain is a variant (mutant) of the IL-2Rα Sushi domain. In various embodiments, the amino acid change is one or more amino acid substitutions at positions 36, 38, 42, or 43 of SEQ ID NO:182. In various embodiments, the amino acid change is an R to A substitution at position 36, a K to E substitution at position 38, an L to G substitution at position 42, and a Y to A substitution at position 43. In various embodiments, the variant (mutant) of the IL-2Rα Sushi domain is designed to facilitate dissociation and diffusion upon proteolytic cleavage. In various embodiments, the variant (mutant) of the IL-2Rα Sushi domain is selected from the group consisting of the sequences listed in SEQ ID NO:183 - 185.

[0021] In various embodiments, both the L1 linker and the L2 linker of the PD1 Ab-IL-2 VitoKine construct are protease-cleavable peptide linkers. In various embodiments, the L1 of the PD1 Ab-IL-2 VitoKine construct is a protease-cleavable peptide linker and the L2 is a non-cleavable peptide linker. In various embodiments, the L1 of the PD1 Ab-IL-2 VitoKine construct is a non-cleavable peptide linker and the L2 is a protease-cleavable peptide linker. In various embodiments, both the L1 linker and the L2 linker of the PD1 Ab-IL-2 VitoKine construct are protease-non-cleavable peptide linkers. In various embodiments, the non-cleavable linker is rich in G / S content (e.g., at least about 60%, 70%, 80%, 90% or more of the amino acids in the linker are G or S). Each peptide linker sequence can be independently selected. In various embodiments, the protease-cleavable linker is selected from the group of sequences listed in SEQ ID NO: 54-77. In various embodiments, the protease-cleavable linker can have additional peptide spacers of variable length at the N-terminus of the cleavable linker or at the C-terminus of the cleavable linker or at both ends of the cleavable linker to improve the accessibility of enzymatic cleavage. In various embodiments, the protease-cleavable linker having additional peptide spacers of variable length at the N-terminus or C-terminus or both ends of the cleavable linker is selected from the group of sequences listed in SEQ ID NO: 78-94. In various embodiments, the non-cleavable linker is selected from the group of sequences listed in SEQ ID NO: 95-115. In various embodiments, the linker is flexible or rigid and has various lengths.

[0022] In various embodiments, the IL-2 domain (D2) and the IL-2Rα domain (D3) of the VitoKine construct are placed at the C-terminus of the PD1 Ab domain (D1), as depicted in FIG. 1A. In various embodiments, the D2 domain and the D3 domain of the VitoKine construct are placed at the N-terminus of the D1 domain, as depicted in FIG. 1B.

[0023] In various embodiments, the PD1 blocking Ab, the IL-2 domain, and the IL-2Rα domain of the PD1 Ab-IL-2 VitoKine construct can be monomers, or dimers, or a combination of dimers and monomers, such as the PD1 blocking Ab is a dimer and the IL-2 domain and the IL-2Rα domain are monomers.

[0024] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma or any cancer.

[0025] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, stem cell transplantation, cell therapy including chimeric antigen receptor (CAR)-T, CAR-NK, induced pluripotent stem cell (iPS)-induced CAR-T or iPS-induced CAR-NK, and vaccines such as Bacille Calmette-Guerine (BCG). In various embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of immunotherapy, which includes, but is not limited to, treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic, or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec-7, Siglec-8, Siglec-9, Siglec-15, and VISTA; treatment with bispecific T cell engager antibodies Treatments such as blinatumomab; treatments involving administration of biologic response modifiers such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatments using therapeutic vaccines such as sipuleucel-T; treatments using dendritic cell vaccines or tumor antigen peptide vaccines; treatments using CAR-T cells; treatments using CAR-NK cells; treatments using tumor-infiltrating lymphocytes (TIL); treatments using adoptively transferred anti-tumor T cells (ex vivo expanded T cells and / or TCR transgenic T cells); treatments using TALL-104 cells; and treatments using immune stimulants such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatments using vaccines such as BCG; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., there is a synergy between the VitoKine construct and the immunotherapy when co-administered.

[0026] In another aspect, the present disclosure provides the use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating cancer.

[0027] In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding the pharmaceutical composition of the present disclosure. In another aspect, the present disclosure provides a vector comprising the nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising the nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising the expression vector of the present disclosure. In another aspect, a method of preparing a VitoKine construct is provided by culturing a host cell under conditions that promote the expression of the protein or polypeptide.

[0028] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated pharmaceutical composition of the present invention admixed with a pharmaceutically acceptable carrier. Brief Description of the Drawings

[0030] Figure 1 depicts representative forms of the VitoKine construct. Figure 1A depicts a VitoKine construct in which D2 (active domain) and D3 (masking domain) are placed at the C-terminus of D1 (targeting domain). Figure 1B depicts a VitoKine construct in which the D2 domain and the D3 domain are placed at the N-terminus of the D1 domain.

[0031] Figure 2Depicts the proposed activation mechanism of the PD1 Ab-IL-2 VitoKine construct of the present invention. Exemplary VitoKine constructs contain two protease-cleavable linkers; protease 1 activation resulting from cleavage of the L1 linker produces active form 1; protease 2 activation resulting from cleavage of the L2 linker produces active form 2; activation of both proteases resulting from cleavage of the L1 and L2 linkers produces active form 3. After protease cleavage, the masking partial domain (D3) will be released and diffused from the active partial domain (D2). If the L1 linker is the only protease-cleavable linker, then active form 1 will be the only activated form. Similarly, if the L2 linker is the only protease-cleavable linker, then active form 2 will be the single activated form.

[0032] Figure 3A depicts a representative PD1 Ab-IL-2 VitoKine construct of the present invention. The monomeric IL-2 or IL-2 variant as the active partial domain (D2) is fused at its N-terminus with the C-terminus of the PD1 antibody heterodimer Fc (D1) using the L1 linker; the C-terminus of the IL-2 portion is fused with the N-terminus of the IL-2Rα or IL-2Rα variant as the masking partial domain (D3) using the L2 linker. Figure 3B depicts a representative PD1 Ab-IL-2 immunocytokine, which also serves as a non-VitoKine immunocytokine counterpart.

[0033] Figure 4 depicts a comparison of PD1 blocking activities between a reference antibody (P-0734) and a pembrolizumab (PBL) biosimilar in a luciferase reporter assay. Figures 4A and 4B depict the dose-dependent increases in luminescence signal and induction fold, respectively. P-0734 and the PBL biosimilar share the same variable domains and have IgG1 and IgG4 isotypes, respectively.

[0034] Figure 5 depicts the (A) ELISA binding and (B-C) PD1 blocking activities of the PD1 blocking antibodies P-1148, P-1150, P-1151, and P-1153 compared to the reference antibody (P-0734), as tested in a luciferase reporter assay. Figures 5B and 5C depict the dose-dependent increases in luminescence signal and induction fold, respectively.

[0035] Figure 6 depicts the PD1 blocking activities of the PD1 blocking antibodies P-1127, P-1129, and P-1174 compared to the reference antibody (P-0734). They were tested in a luciferase reporter assay, and the dose-dependent increase in luminescence signal is illustrated.

[0036] Figure 7 depicts the PD1 blocking activity of PD1 blocking antibodies P-1175 and P-1181 compared to a reference antibody (P-0734), as tested in a luciferase reporter assay. Figures 7A and 7B depict the dose-dependent increases in luminescence signal and fold induction, respectively.

[0037] Figure 8 depicts the PD1 blocking activity of PD1 blocking antibodies P-1175, P-1176, P-1177, and P-1178 compared to a reference antibody (P-0734), as tested in a luciferase reporter assay. Figures 8A and 8B depict the dose-dependent increases in luminescence signal and fold induction, respectively.

[0038] Figure 9 depicts the PD1 blocking activity of PD1 blocking antibodies P-1198, P-1199, and P-1201 compared to a reference antibody (P-0734), as tested in a luciferase reporter assay. Figures 9A and 9B depict the dose-dependent increases in luminescence signal and fold induction, respectively. An isotype control antibody was included as a negative control.

[0039] Figure 10 depicts the PD1 blocking activity of PD1 blocking antibodies P-1194, P-1201, and P-1238 compared to a reference antibody (P-0734), as tested in a luciferase reporter assay. Figures 10A and 10B depict the dose-dependent increases in luminescence signal and fold induction, respectively.

[0040] Figure 11 depicts the binding of PD1 blocking antibodies P-1174, P-1193, P-1198, P-1199, and P-1201 to PD1 + HEK293 cells compared to a reference antibody (P-0734). Figures 11A and 11C depict the dose-dependent increases in the percentage of positive cells, and Figures 11B and 11D depict the dose-dependent increases in mean fluorescence intensity (MFI).

[0041] Figure 12 Depicts the ELISA binding of IL-2Rα Sushi variants P-0751, P-0752, and P-0753 to IL-2. P-0757 contains wild-type IL-2Rα Sushi and was included for comparison.

[0042] Figure 13 depicts the assessment of the activity of Fc IL-2 VitoKine with wild-type IL-2Rα Sushi as the D3 domain (P-0701) or an IL-2Rα Sushi variant as the D3 domain (P-0754, P-0755, and P-0756). P-0704, an IL-2 P65R variant Fc fusion protein, was included as a fully active IL-2 control. Activity was assessed by analyzing the induction of Ki67 expression on A) CD8+ T cells and B) NK cells of human PBMCs using flow cytometry.

[0043] Figure 14 depicts the ELISA binding of IL-2 variants to IL-2Rα. Each IL-2 variant contains a different amino acid substitution at position P65 (see Table 18 for details of molecular information). P-0531 and P-0689 were used as wild-type IL-2 controls in the bivalent form and monovalent form, respectively.

[0044] Figure 15 depicts the potency of IL-2 P65 variants to stimulate STAT5 phosphorylation in CD4+ Treg cells (see Table 18 for details of molecular information). P-0531 and P-0689 were used as wild-type IL-2 controls in the bivalent form and monovalent form, respectively. Similarly, the benchmarks (D) and Benchmark were the bivalent form and monovalent form of the IL-2F42A / Y45A / L72G variant, respectively.

[0045] Figure 16 depicts the activity of IL-2 P65 variants against IL-2Rβγ by analyzing A) ELISA binding to the recombinant expressed IL-2 receptor subunits β and γ complex and B) induction of Ki67 expression on CD8+ T cells in fresh human PBMCs using flow cytometry. For detailed information on the molecular information of IL-2 variants, see Table 18. P-0531 and P-0689 were used as wild-type IL-2 controls in the bivalent form and monovalent form, respectively.

[0046] Figure 17 depicts the assessment of the activity of various alternative murine PD1Ab-IL-2 VitoKines P-0800, P-0830, P-0831, and P-0802 compared to the non-VitoKine immunocytokine counterpart P-0782. This was done by analyzing the induction of Ki67 expression on A) CD8+ T cells and B) NK cells of human PBMCs. The four IL-2 antibody VitoKines differ only in the binding strength of their IL-2 partial domain to IL-2Rα.

[0047] Figure 18 depicts the activity assessment of IL-2 variants P-0731, P-0759, and P-0761, which contain mutations that disrupt their interaction with IL-2Rβ. This assessment is based on their effect on inducing Ki67 expression on A) CD8+ T cells and B) NK cells of human PBMC. All these IL-2 variants also have the P65R mutation that eliminates binding to IL-2Rα. P-0704 is used as a fully active IL-2 control.

[0048] Figure 19 depicts the activity assessment of IL-2 variants containing mutations that interfere with their binding to γc. This assessment is based on their effect on inducing Ki67 expression on CD8+ T cells (A, C, and E) and NK cells (B, D, and F) of human PBMC. All these IL-2 variants also have the P65R mutation that eliminates IL-2Rα binding. P-0704 serves as a fully active IL-2 control.

[0049] Figure 20 depicts the activity assessment of P-1247, an IL-2 variant containing mutations targeting both IL-2Rβ and γc, compared to P-1158, which contains only γc-interfering mutations. The assessment is performed by analyzing the effect on inducing Ki67 expression on A) CD8+ T cells and B) NK cells of human PBMC. P-0704 is used as an IL-2 full agonist control.

[0050] Figure 21 depicts the PD1 blocking activity of PD1 blocking antibodies P-1174, P-1238, and P-1271 compared to their respective PD1 Ab-IL-2 VitoKine P-1197, P-1239, and P-1272 in a luciferase reporter assay. Figures 21A and 21B depict the dose-dependent increase in luminescence signal and induction fold, respectively.

[0051] Figure 22 depicts the assessment of the intrinsic basal IL-2 activity of PD1 Ab-IL-2 VitoKine P-0872, P-1197, and P-1272 compared to their corresponding non-VitoKine immunocytokine counterparts P-0879 and P-1271. The activity assessment is based on the analysis of the induction of the proliferation marker Ki67 on CD8+ T cells (A and C) and NK cells (B and D) of human PBMC. The three VitoKines differ only in the composition of the D1 domain, i.e., they contain different PD1 blocking antibodies. P-1174 (the component PD1 antibody of P-1197) is included as a negative control for this assay.

[0052] Figure 23 depicts the proteolytic cleavage and activation of PD1 Ab-IL-2 VitoKine. The figure includes A) a reducing SDS-PAGE gel showing both the full-length and active forms of P-1272, and B) a display of the dose-dependent induction of Ki67 expression on CD8+ T cells by VitoKine P-1272 compared to its non-VitoKine counterpart P-1273, C) VitoKine P-0831 compared to its non-VitoKine counterpart P-0838, and D) VitoKine P-1345 compared to its non-VitoKine counterpart P-0838.

[0053] Figure 24 Serum concentrations of murine PD1 Ab-IL-2 VitoKine (P-0831) and its non-VitoKine immunocytokine equivalent P-0838 are depicted following a single intraperitoneal injection in C57B / L6 mice. Blood was collected from the mice at multiple time points after dosing, and the serum levels of the compounds were determined using an ELISA assay.

[0054] Figure 25 depicts the dose- and time-dependent effects of a single dose of P-0831, a murine PD1 Ab-IL-2 VitoKine, on the expansion of A) CD8+ T cells, B) granzyme B+ CD8+ T cells, C) NK cells, and D) granzyme B+ NK cells in the peripheral blood of C57B / L6 mice. P-0838, its non-VitoKine immunocytokine equivalent, was included for comparison. Blood was collected on days 0, 3, 5, 7, and 10 for lymphocyte phenotyping by FACS analysis. Data are represented as mean ± SEM.

[0055] Figure 26 depicts the effects of a single dose of P-0831, a murine PD1 Ab-IL-2 VitoKine, on A) the dose-dependent increase in the serum level of the inflammatory marker IFNγ and B) the change in body weight across different dose levels in naïve C57BL / 6 mice. P-0838, its non-VitoKine immunocytokine equivalent, was included for comparison. Additionally, the vehicle (PBS) and its component murine PD1 antibody P-0722 were used as negative controls.

[0056] Figure 27 depicts the anti-tumor effect of P-0831, a murine PD1 Ab-IL-2 VitoKine, in an established MC38 murine colon cancer model after two doses administered every 10 days (Q10D). Growth curves of MC38 tumors in individual mice are presented for A) 3 mg / kg of P-0831, B) 6 mg / kg of P-0831, C) 9 mg / kg of P-0831, and D) 1 mg / kg of P-0838 (a non-VitoKine immunocytokine equivalent of P-0831). For comparison, the change in mean tumor volume ± standard error of the mean (SEM) of the vehicle group over time is plotted with a dashed line. The change in mean tumor volume ± SEM of each treatment group over time is shown in Figure 27E, and the change in body weight of each treatment group over time is shown in Figure 27F.

[0057] Figure 28 Immunohistochemical (IHC) analysis depicting the effect of P-0831, a murine PD1 Ab-IL-2 VitoKine, in tumor tissue isolated 5 days after treatment at a dose of 6 mg / kg. Tissue sections were fixed in 10% formalin, paraffin-embedded, processed, and stained with antibodies by HistoWiz to evaluate immune cells in the tumor tissue. For comparison, P-0722, its component murine PD1 antibody, was included at a dose of 6 mg / kg, and P-0838, its non-VitoKine immunocytokine counterpart, was included at a dose of 1 mg / kg.

[0058] Figure 29 depicts the ex vivo activity of P-0831 in human PBMC and its in vivo anti-tumor efficacy in mice with established CT26 murine tumors, compared to its non-cleavable VitoKine counterpart P-0877. In vitro assessment measured the induction of the proliferation marker Ki67 on A) CD8+ T cells and B) NK cells in fresh human PBMC. In vivo analysis included C) mean tumor volume ± SEM and D) the change in body weight over time of each treatment group in the CT26 murine model after two doses of 10 mg / kg Q12D. P-0879 was used as a control for fully active IL-2 immunocytokine in the human PBMC assay. P-0722 (the component murine PD1 antibody of P-0831) administered at 10 mg / kg was included as a control in the tumor model.

[0059] Figure 30Depicts the in vivo anti-tumor efficacy of P-0831 in mice with established CT26 murine tumors compared to the off-target VitoKine counterpart of P-0831, P-0871, and its component murine PD1 antibody, P-0722. After two doses of 10 mg / kg Q12D, the change in mean tumor volume ± SEM over time for each treatment group in the CT26 murine model was plotted.

[0060] Figure 31 depicts the assessment of the activities of murine PD1 Ab-IL-2 immunocytokines P-0782, P-0783, and P-0786 by analyzing their effects on A) inducing Ki67 expression on human CD8+ T cells, B) inducing Ki67 expression on human NK cells, and C) the proliferation of murine CTLL-2 cells.

[0061] Figure 32 depicts the dose-dependent and time-pharmacodynamic effects of several murine PD1 Ab-IL-2 immunocytokines. This includes A) the expansion of peripheral CD8+ T cells, B) the expansion of peripheral NK cells, and C) the change in body weight after a single dose of 2 mg / kg in C57B / L6 mice. Blood was collected on days 0, 3, 5, 7, and 10 for lymphocyte phenotyping by FACS analysis. Data are presented as mean ± SEM.

[0062] Figure 33 depicts the anti-tumor efficacy of several murine PD1 Ab-IL-2 immunocytokines in the established MC38 murine colon cancer model after two Q12D treatments of 0.5 mg / kg. The change in mean tumor volume ± SEM over time for each treatment group is shown in Figure 33A, and the individual tumor volumes in mice at day 26 after the first dose are shown in Figure 33B.

[0063] Figure 34 depicts the anti-tumor efficacy of two murine PD1 Ab-IL-2 immunocytokines, P-0783 and P-0786, in the established MC38 murine colon cancer model after two Q10D doses of 0.3 mg / kg. The change in mean tumor volume ± SEM over time for each treatment group is shown in Figure 34A. The individual growth curves of murine subcutaneous MC38 tumors are shown for B) P-0783 and C) P-0786.

[0064] Figure 35 depicts the anti-tumor efficacy of P-0786, a murine PD1 Ab-IL-2 immunocytokine, in an established MC38 murine colon cancer model after two Q10D treatments at 1 mg / kg. This includes A) mean tumor volume ± SEM and B) body weight over time for each treatment group. The murine PD1 antibody P-0722 administered at 9 mg / kg was included for comparison.

[0065] Figure 36 depicts the dose-dependent anti-tumor efficacy of P-0786, a murine PD1 Ab-IL-2 immunocytokine, after two Q14D treatments at different dose levels in an established MC38 murine colon cancer model. The change in mean tumor volume ± SEM over time for each treatment group is shown in Figure 36A . Individual tumor growth curves for doses of B) 0.03 mg / kg, C) 0.1 mg / kg, D) 0.3 mg / kg, and E) 1 mg / kg are shown. The change in mean tumor volume ± SEM over time for the vehicle group (represented as a dashed line) is included for comparison.

[0066] Figure 37 Depicts that there was no tumor recurrence after re-challenge implantation of MC38 colon cancer cells in tumor-free mice treated with P-0786. This is in contrast to the successful regeneration of the same type of tumor in age-matched naïve mice used as controls.

[0067] Figure 38 depicts the efficacy of P-0786 as a single agent in inhibiting tumor growth in two additional subcutaneous syngeneic tumor models. These models include A) the CT26 murine colon cancer tumor model and B) the B16F10 murine melanoma tumor model.

[0068] Methods for practicing the present disclosure

[0069] In one aspect, the present disclosure provides a PD1Ab-IL-2 VitoKine construct comprising three domains: an optimized PD1-blocking antibody as a TIL-targeting moiety, an IL-2 variant as an active moiety domain, and an IL-2Rα sushi variant as a shielding moiety domain. Importantly, the IL-2Rα sushi variant domain is capable of shielding or attenuating the functional activity of the IL-2 domain until it is activated at the intended site of treatment.

[0070] The PD1-blocking antibody directs VitoKine to TILs in the tumor microenvironment and confines the activation of VitoKine locally to improve the therapeutic index. In various embodiments, the PD1-blocking antibody is optimized by modifications in the variable domains of pembrolizumab. In various embodiments, the modifications involve germline sequence substitutions of CDR residues, germline sequence substitutions of framework residues, and the adoption of a VH3 human germline family sequence as the receptor framework. In various embodiments, these modifications are implemented singly or in combination to develop optimized PD1-blocking antibodies. In various embodiments, these optimized PD1-blocking antibodies exhibit high binding affinity for PD1, inhibit PD1 with the same or comparable potency as pembrolizumab, have a higher sequence similarity score with their closest human germline sequence, result in an increased degree of humanization compared to pembrolizumab, and are predicted to have lower hydrophobicity, resulting in a reduced tendency to aggregate. In various embodiments, PD1 Ab-IL-2 VitoKine constructs based on these optimized PD1-blocking antibodies have enhanced developability characteristics.

[0071] In various embodiments, the IL-2 domain is the active moiety but remains inert until locally activated by proteases upregulated in diseased tissue; this will limit the binding of the active moiety to receptors in the periphery of non-diseased cells or tissues or on the cell surface to prevent overactivation of the pathway and reduce unwanted "off-tissue" "on-target" toxicity. The improved safety profile of VitoKine may permit human dose levels within the effective range of the PD1 antibody. Additionally, the inertness of the VitoKine active moiety prior to protease activation will significantly reduce potential antigen sink, and thereby prolong the in vivo half-life, as well as result in improved biodistribution, bioavailability, and efficacy at the intended site of treatment.

[0072] In various embodiments, incorporating an IL-2 variant with attenuated potency as the active moiety domain (such IL-2 variants achieved by disrupting the IL-2Rβγ interaction) can also fine-tune the intrinsic basal activity and post-activation activity of VitoKine. In various embodiments, such VitoKine with an IL-2 variant with attenuated potency as the active moiety domain can additionally expand the therapeutic index.

[0073] In various embodiments, the unique and non-signaling α-subunit of the IL-2 receptor is used as a shielding moiety domain via a protease-cleavable linker to reversibly shield cytokine activity. The shielding α-subunit may preferably dissociate upon protease cleavage of the linker. Thus, amino acid modifications of the α-receptor to modulate the binding affinity for IL-2 may be beneficial.

[0074] In various embodiments, in the PD1 Ab-IL-2 VitoKine construct, the three domains are connected using two linkers with variable length and rigidity, and are optionally coupled with protease cleavable sequences. These protease cleavable sequences are peptide substrates of specific protease subtypes with elevated or dysregulated expression at the disease site, thus allowing the functional IL-2 domain to be displayed or released at the site of the disease. The linker length and composition are fine-tuned to ensure optimal shielding of the IL-2 domain from its receptor, thereby minimizing systemic involvement. At the same time, the stability of the VitoKine construct in the blood circulation is maintained, while allowing effective cleavage when encountering a specific protease at the intended treatment site.

[0075] On the other hand, the present disclosure provides novel PD1-targeted IL-2 immunocytokines that are intended to directly target the activity-regulated IL-2 domain to tumor-infiltrating lymphocytes. In various embodiments, the PD1 blocking antibodies are optimized by modifications in the variable domains of pembrolizumab. In various embodiments, the targeted IL-2 immunocytokines based on the optimized PD1 blocking antibodies are predicted to have enhanced developability characteristics.

[0076] In various embodiments, the activity-regulated IL-2 domain (monomer) is fused to the C-terminus of the heterodimer PD1 antibody heavy chain. In various embodiments, the IL-2 domain in the PD1-targeted IL-2 immunocytokine is IL-2Rβγ selective and attenuated. In various embodiments, the weakening of IL-2 potency is conducive to establishing a stoichiometric balance between cytokines and antibody components, helping to alleviate overactivation of pathways, and alleviating antigen sinking and target-mediated deposition. In various embodiments, the use of IL-2 variants with reduced potency (such variants have impaired interactions with γc) in PD1-targeted IL-2 immunocytokines can provide additional benefits in alleviating antigen sinking, and then lead to an extended half-life in vivo, which may be due to the influence of γc receptors in the signal cascade leading to cell expansion.

[0077] definition

[0078] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. Generally, the nomenclature and techniques described herein in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are those that are commonly used and well known in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), which is incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature, as well as experimental procedures and techniques described herein in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry are those that are commonly used and well known in the art. Standard techniques are used for chemical synthesis, chemical analysis, the preparation, formulation, and delivery of drugs, and the treatment of subjects.

[0079] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acid residues. In various embodiments, a “peptide,” “polypeptide,” and “protein” is a chain of amino acids whose α-carbons of the amino acids are joined by peptide bonds. Thus, the terminal amino acid at one end (amino terminus) of the chain has a free amino group, while the terminal amino acid at the other end (carboxyl terminus) of the chain has a free carboxyl group. As used herein, the term “amino terminus” (abbreviated N-terminus) refers to the free α-amino group on the amino acid at the amino terminus of a peptide, or to the α-amino group of an amino acid at any other position in the peptide (the amino group when participating in a peptide bond). Similarly, the term “carboxyl terminus” (abbreviated C-terminus) refers to the free carboxyl group on the carboxyl terminus of a peptide, or to the carboxyl group of an amino acid at any other position in the peptide. Peptides also include substantially any polyamino acid, including but not limited to peptide mimetics such as amino acids joined by ether bonds rather than amide bonds.

[0080] The polypeptides of the present disclosure include polypeptides that have been modified in any way and for any reason, e.g., to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity, and (5) confer or alter other physicochemical or functional properties.

[0081] As used herein, an amino acid "substitution" refers to the replacement of one amino acid at a specific position in the parental polypeptide sequence of a polypeptide with a different amino acid. Amino acid substitutions can be generated using genetic or chemical methods well known in the art. For example, single amino acid substitutions or more than one amino acid substitution (e.g., conservative amino acid substitutions) can be made in a naturally occurring sequence (e.g., in a portion of the polypeptide outside of one or more domains that form intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid that is functionally similar. The following six groups each contain amino acids that are conservative substitutions for one another:

[0082] 1) Alanine (A), Serine (S), and Threonine (T)

[0083] 2) Aspartic acid (D) and Glutamic acid (E)

[0084] 3) Asparagine (N) and Glutamine (Q)

[0085] 4) Arginine (R) and Lysine (K)

[0086] 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

[0087] 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0088] A "non-conservative amino acid substitution" refers to the replacement of a member of one of these classes with a member from another class. In making such a change, the hydropathic index of the amino acid can be considered according to various embodiments. Based on the hydrophobicity and charge characteristics of amino acids, each amino acid has been assigned a hydropathic index. They are: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / Cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamic acid (-3.5); Glutamine (-3.5); Aspartic acid (-3.5); Asparagine (-3.5); Lysine (-3.9); and Arginine (-4.5).

[0089] Those skilled in the art understand the importance of the hydrophilic amino acid index in conferring biological functions of protein interactions (see, for example, Kyte et al., 1982, J. Mol. Biol. 157: 105 - 131). It is known that certain amino acids can be replaced by other amino acids with similar hydrophilic indices or scores and still retain similar biological activities. In making changes based on the hydrophilic index, in various embodiments, substitutions of amino acids with a hydrophilic index within ±2 are included. In various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0090] Those skilled in the art also understand that similar amino acid substitutions can be effectively made based on hydrophilicity, particularly in cases where the resulting biologically functional protein or peptide is intended for use in immunological embodiments as disclosed herein. In various embodiments, the maximum local average hydrophilicity of a protein (determined by the hydrophilicity of its adjacent amino acids) is related to its immunogenicity and antigenicity, i.e., to the biological properties of the protein.

[0091] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamic acid (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based on similar hydrophilicity values, in various embodiments, substitutions of amino acids with a hydrophilicity value within ±2 are included, in various embodiments, those within ±1 are included, and in various embodiments, those within ±0.5 are included.

[0092] Exemplary amino acid substitutions are listed in Table 1.

[0093] Table 1

[0094]

[0095]

[0096] A person skilled in the art will be able to use well-known techniques to determine suitable polypeptide variants as listed herein. In various embodiments, a person skilled in the art can identify suitable regions of a molecule that can be altered without disrupting activity by targeting regions that are considered unimportant for activity. In other embodiments, a person skilled in the art can identify residues and portions of a molecule that are conserved among similar polypeptides. In additional embodiments, even regions that may be important for biological activity or for structure can undergo conservative amino acid substitutions without disrupting biological activity or unduly affecting polypeptide structure.

[0097] In addition, a person skilled in the art can review structure - function studies that identify residues important for activity or structure in similar polypeptides. Given such comparisons, a person skilled in the art can predict the importance of amino acid residues in a polypeptide corresponding to amino acid residues important for activity or structure in similar polypeptides. A person skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0098] A person skilled in the art can also analyze the three - dimensional structure and amino acid sequence related to that structure in similar polypeptides. Given such information, a person skilled in the art can predict the arrangement of amino acid residues of a polypeptide in terms of its three - dimensional structure. In various embodiments, a person skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the polypeptide, as such residues may be involved in important interactions with other molecules. In addition, a person skilled in the art can generate test variants that contain a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to a person skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is found that a change in a particular amino acid residue results in disrupted, undesirably reduced, or inappropriate activity, variants having such a change can be avoided. In other words, based on the information collected from such routine experiments, a person skilled in the art can readily determine the amino acids at which further substitutions, either alone or in combination with other mutations, should be avoided.

[0099] As used herein, the terms "polypeptide fragment" and "truncated polypeptide" refer to a polypeptide having an amino-terminal deletion and / or a carboxyl-terminal deletion as compared to the corresponding full-length protein. In various embodiments, the length of the fragment can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids. In various embodiments, the length of the fragment can also be, for example, at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids. The fragment can also contain one or more additional amino acids at either or both of its termini, for example, an amino acid sequence from a different naturally occurring protein (e.g., Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., artificial linker sequence).

[0100] As used herein, the terms "polypeptide variant", "hybrid polypeptide", and "polypeptide mutant" refer to a polypeptide that contains an amino acid sequence in which one or more amino acid residues have been inserted into, deleted from, and / or substituted into the amino acid sequence as compared to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. The hybrids of the present disclosure include fusion proteins.

[0101] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, such as conjugation to another chemical moiety (such as, for example, polyethylene glycol, albumin (e.g., human serum albumin)), phosphorylation, and glycosylation.

[0102] The terms “% sequence identity” and “% identity” are used interchangeably herein and refer to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity determined by a defined algorithm and means that a given sequence is at least 80% identical to another sequence of another length. In various embodiments, the % identity is selected from, for example, sequence identity of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or greater with a given sequence. In various embodiments, the % identity is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 95% to about 99%.

[0103] The terms “% sequence homology” and “% homology” are used interchangeably herein and refer to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% homology determined by a defined algorithm means the same as 80% sequence homology and thus a homolog of a given sequence has a sequence homology greater than 80% relative to the length of the given sequence. In various embodiments, the % homology is selected from, for example, sequence homology of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% or greater with a given sequence. In various embodiments, the % homology is in the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95% or about 95% to about 99%.

[0104] Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, a suite of BLAST programs publicly available on the Internet at the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990 (specifically refer to the publicly disclosed default settings, i.e., parameters w = 4, t = 17) and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. When evaluating a given amino acid sequence relative to GenBank protein sequences and amino acid sequences in other public databases, the BLASTP program is typically used for sequence searches. The BLASTX program is preferably used to search for nucleic acid sequences that have been translated in all reading frames against amino acid sequences in GenBank protein sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0 and an extended gap penalty of 1.0 and using the BLOSUM-62 matrix.

[0105] In addition to calculating the percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the minimum total probability (P(N)), which gives an indication of the probability that a match between two nucleotide sequences or amino acid sequences would occur by chance. For example, if in a comparison of a test nucleic acid with a reference nucleic acid, the minimum total probability is, for example, less than about 0.1, less than about 0.01, or less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0106] As used herein, the term "modification" refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modification of a polypeptide (e.g., glycosylation).

[0107] As used herein, the term "knob-into-hole modification" refers to a modification within the interface between the CH3 domains of two immunoglobulin heavy chains. In one embodiment, a "knob-into-hole modification" comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one antibody heavy chain, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. The knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).

[0108] As used herein, the term "bioactivatable drug" or "VitoKine" means a compound that is a prodrug and that, upon administration to a subject, releases a drug in vivo via some chemical or physiological process such that the bioactivatable drug is converted into a product that is active against a target tissue. A bioactivatable drug is any compound that undergoes bioactivation and then exhibits its pharmacological action. Thus, a bioactivatable drug can be regarded as a drug that contains a specialized non-toxic protective group that is used in a temporary manner to modify or eliminate undesirable properties of the parent molecule.

[0109] As used herein, the term "immunoconjugate" or "fusion protein" refers to a molecule that comprises an antibody or an antigen-binding fragment thereof that is conjugated (or linked) directly or indirectly to an effector molecule. The effector molecule can be a detectable label, an immunotoxin, a cytokine, a chemokine, a therapeutic agent or a chemotherapeutic agent. The antibody or an antigen-binding fragment thereof can be conjugated to the effector molecule via a peptide linker. The immunoconjugate and / or fusion protein retains the immunoreactivity of the antibody or antigen-binding fragment, e.g., the antibody or antigen-binding fragment has an ability to bind to an antigen that is approximately the same or only slightly reduced after conjugation as before conjugation. As used herein, an immunoconjugate can also be referred to as an antibody-drug conjugate (ADC). Because immunoconjugates and / or fusion proteins are initially prepared from two molecules with separate functions, such as an antibody and an effector molecule, they are sometimes also referred to as "chimeric molecules".

[0110] "Linker" refers to a molecule that covalently or through ionic bonds, van der Waals forces or hydrogen bonds links two other molecules, such as a nucleic acid molecule that hybridizes to a complementary sequence at the 5' end and hybridizes to another complementary sequence at the 3' end to link two non-complementary sequences. "Cleavable linker" refers to a linker that can be degraded, digested or otherwise cleaved to separate two components linked by the cleavable linker. Cleavable linkers are typically cleaved by enzymes, usually peptidases, proteases, nucleases, lipases, etc. Cleavable linkers can also be cleaved by changes in environmental factors such as temperature, pH, salt concentration, etc.

[0111] As used herein, the term "peptide linker" refers to a peptide containing one or more amino acids, typically about 1 to 30 amino acids. Peptide linkers are known in the art or are described herein. Suitable non-immunogenic linker peptides include, for example, (G 4 S) n , (SG 4 ) n or G 4 (SG 4 ) n peptide linkers. "n" is typically a number between 1 and 10, usually between 2 and 4.

[0112] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. The pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to the amount of an agent effective to produce the desired pharmacological result. "Pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, vehicle, buffer and excipient, such as phosphate buffered saline solution, aqueous solution of 5% dextrose, and emulsions such as oil / water emulsion or water / oil emulsion, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington’s Pharmaceutical Sciences, 21st Edition 2005, Mack Publishing Co, Easton. "Pharmaceutically acceptable salt" is a salt of a compound that can be formulated for pharmaceutical use, including, for example, salts of metals (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or salts of organic amines.

[0113] As used herein, "treatment" (and its grammatical variations such as "treat" or "treating") refers to a clinical intervention that attempts to alter the natural course of a disease in an individual being treated, and the clinical intervention can be used prophylactically or during a clinical pathologic process. Desirable treatment outcomes include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathologic consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and ameliorating or improving the prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of the symptoms of the disease, disorder, or condition. Additionally, reference to "treatment" herein includes reference to curative, palliative, and prophylactic treatment.

[0114] As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as to improve, alleviate, reduce, and / or delay one or more of its symptoms. With reference to cancer or other undesired cell proliferation, an effective amount includes an amount sufficient to effect: (i) a reduction in the number of cancer cells; (ii) a reduction in tumor size; (iii) inhibition, to some extent, of the infiltration of cancer cells into peripheral organs, with delay and preferably cessation; (iv) inhibition (i.e., slowing to some extent and preferably cessation) of tumor metastasis; (v) inhibition of tumor growth; (vi) prevention or delay of the occurrence and / or recurrence of a tumor; and / or (vii) alleviation, to some extent, of one or more cancer-related symptoms. The effective amount can be administered in one or more administrations.

[0115] The phrase "administer" or "cause to be administered" refers to the action of a medical professional (e.g., a physician) or a person controlling the medical care of a patient in controlling and / or allowing the administration of the agent / compound under discussion to the patient. Causing administration can include diagnosing and / or determining a suitable treatment regimen, and / or prescribing a particular agent / compound for the patient. Such prescribing can include, for example, drafting a prescription form, annotating medical records, etc. "Causing administration" is also contemplated when administration is described herein.

[0116] The terms "patient", "individual", and "subject" are used interchangeably and refer to a mammal, preferably a human or non-human primate, but also a domestic mammal (e.g., canine or feline), a laboratory mammal (e.g., mouse, rat, rabbit, hamster, guinea pig), and an agricultural mammal (e.g., equine, bovine, swine, ovine). In various embodiments, the patient can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other healthcare worker in a hospital, a psychiatric care facility such as an outpatient clinic, or other clinical setting. In various embodiments, the patient can be an immunocompromised patient or a patient with a weakened immune system, including but not limited to patients with primary immunodeficiency, AIDS; cancer patients and transplant patients taking certain immunosuppressive drugs; and patients with genetic diseases affecting the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including but not limited to bladder cancer, lung cancer, melanoma, and other cancers reported to have a high mutation rate (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0117] The term "immunotherapy" refers to cancer treatment including but not limited to the following: treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic antibodies, antagonistic antibodies, or blocking antibodies against co-stimulatory molecules or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD1, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec 15, VISTA, CD276, CD272, TIM-3, and B7-H4; treatment with bispecific T cell engaging antibodies Treatments such as blinatumomab; treatments involving administration of a biological response modifier (such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, IL-22, GM-CSF, IFN-α, IFN-β, IFN-γ, a TGF-β antagonist or a TGF-β trap); treatments using a therapeutic vaccine such as sipuleucel-T; treatments using a therapeutic virus (including but not limited to an oncolytic virus such as T-vec); treatments using a dendritic cell vaccine or a tumor antigen peptide vaccine or a neoantigen vaccine; treatments using NK cells; treatments using chimeric antigen receptor (CAR)-T cells; treatments using CAR-NK cells; treatments using DC or T cells; treatments using iPS-induced NK cells; treatments using iPS-induced T cells; treatments using a vaccine such as bacillus Calmette-Guérin (BCG); treatments using tumor-infiltrating lymphocytes (TIL); treatments using adoptively transferred anti-tumor T cells (ex vivo expanded T cells and / or TCR-T cells); treatments using TALL-104 cells; and treatments using immunostimulants such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9 and imiquimod.

[0118] "Tolerant or refractory cancer" refers to tumor cells or cancer that do not respond to a previous anti-cancer therapy, the previous anti-cancer therapy including, for example, chemotherapy, surgery, radiotherapy, stem cell transplantation and immunotherapy. Tumor cells can be tolerant or refractory at the start of treatment, or they can become tolerant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment, or tumors that initially respond for a short period of time but are unable to respond to treatment. Refractory tumor cells also include tumors that respond to treatment with an anti-cancer therapy but are unable to respond to subsequent rounds of therapy. For the purposes of the present invention, refractory tumor cells also include tumors that show suppression upon treatment with an anti-cancer therapy but recur up to 5 years, sometimes up to 10 years or longer after treatment has stopped. The anti-cancer therapy can use a single chemotherapeutic agent, radiotherapy alone, targeted therapy alone, surgery alone or a combination thereof. For ease of description and not by way of limitation, it should be understood that refractory tumor cells are interchangeable with tolerant tumors.

[0119] The term "neoantigen" refers to, for example, a cell surface antigen to which the immune system has not been previously exposed, especially a cell surface antigen generated by altering a host antigen by radiation, chemotherapy, viral infection, oncogenic transformation / mutation, drug metabolism, etc., which is selectively expressed or overexpressed in cancer cells relative to most normal cells.

[0120] As used herein, the term "antibody" is used in the broadest sense and encompasses a variety of antibody structures (IgG1, 2, 3, or 4, IgM, IgA, IgE), including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or bifunctional antibodies), and antibody fragments, provided that they exhibit the desired antigen-binding activity.

[0121] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies.

[0122] As used herein, the term "Fab fragment" refers to an immunoglobulin fragment that comprises the VL domain and the constant domain (CL) of the light chain and the VH domain and the first constant domain (CH1) of the heavy chain.

[0123] As used herein, the term "variable region" or "variable domain" refers to the domain of an immunoglobulin or antibody heavy or light chain that generally participates in binding of the immunoglobulin or antibody to an antigen. The variable domains of the heavy and light chains of an immunoglobulin or antibody (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FRs) and three complementarity-determining regions (CDRs).

[0124] The term "complementarity-determining region" or "CDR" contains the antigen-contact residues ("antigen contacts"). Generally, an antibody comprises six CDRs: three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). The CDRs occur at amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although the CDRs vary between antibodies, only a limited number of amino acid positions within the CDRs directly participate in antigen binding. These positions within the CDRs are referred to as specificity-determining residues (SDRs).

[0125] A "single-chain antibody" is an Fv molecule in which the heavy-chain variable region and the light-chain variable region have been joined by a flexible linker to form a single polypeptide chain that forms the antigen-binding region. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649, U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference.

[0126] As used herein, a "human immunoglobulin" is an immunoglobulin having an amino acid sequence corresponding to that of an immunoglobulin produced by a human or a human cell or derived from a non-human source using a human immunoglobulin repertoire or other human immunoglobulin-encoding sequences. This definition of human immunoglobulin specifically excludes humanized immunoglobulins that contain non-human antigen-binding residues.

[0127] As used herein, the term "humanized antibody" refers to an antibody that contains a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody can have a limited number of substitutions of amino acids taken from the donor framework, and such substitutions are referred to herein as back mutations. A humanized antibody or other monoclonal antibody can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions.

[0128] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region contains the IgG CH2 and IgG CH3 domains. The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "knob" in one of its chains and a corresponding introduced "hole" in the other of its chains; see U.S. Patent No. 5,821,333, which is specifically incorporated herein by reference). Such variant CH3 domains can be used to facilitate heterodimerization of two different immunoglobulin heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system.

[0129] As used herein, the term "effector function" refers to those biological activities attributable to the Fc region of an immunoglobulin, which varies with immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0130] As used herein, "specifically binds" means that the binding to an antigen is selective and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art such as surface plasmon resonance (SPR) techniques.

[0131] As used herein, the term "affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of molecule X for its partner Y can generally be presented by the dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). The specific method for measuring affinity is SPR.

[0132] As used herein, the term "immunogenicity" refers to the ability of an antibody or antigen-binding fragment to elicit an immune response (humoral or cellular response) when administered to a recipient, and includes, for example, a human anti-mouse antibody (HAMA) response. The HAMA response is initiated when T cells from a subject mount an immune response to the administered antibody. The T cells then recruit B cells to produce specific "anti-antibody" antibodies.

[0133] As used herein, the term "immune cell" means any cell of the hematopoietic lineage that is involved in regulating an immune response against an antigen (e.g., a self-antigen). In various embodiments, the immune cell is, for example, a T cell, B cell, dendritic cell, monocyte, natural killer cell, macrophage, Langerhans cell, or Kupffer cell.

[0134] As used herein, the term "reduced binding" refers to a decrease in the affinity of the respective interaction, as measured, for example, by SPR. Conversely, "increased binding" refers to an increase in the binding affinity of the respective interaction.

[0135] As used herein, the term "polymer" generally includes, but is not limited to, homopolymers; copolymers such as, for example, block, graft, random, and alternating copolymers; and terpolymers; as well as mixtures and modifications thereof. Further, unless otherwise expressly defined, the term "polymer" shall include all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and atactic symmetries.

[0136] "Polynucleotide" refers to a polymer containing nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") and nucleic acid analogs. Nucleic acid analogs include those containing: non-naturally occurring bases, nucleotides that engage with other nucleotides by linkages other than the naturally occurring phosphodiester bond, or nucleotides containing bases attached by linkages other than the phosphodiester bond. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, dithiophosphates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using automated DNA synthesizers. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, usually no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the RNA sequence (i.e., A, U, G, C) in which "U" replaces "T".

[0137] Conventional symbols are used herein to describe polynucleotide sequences: the left-hand end of a single-stranded polynucleotide sequence is the 5'-end; the left-hand direction of a double-stranded polynucleotide sequence is called the 5'-direction. The direction of adding nucleotides to a nascent RNA transcript from 5' to 3' is called the transcription direction. The DNA strand having the same sequence as the mRNA is called the "coding strand"; the sequence at the 5' that is 5' of the DNA strand having the same sequence as the mRNA transcribed from that DNA and at the 5'-end of the RNA transcript is called the "upstream sequence"; the sequence at the 3' that is 3' of the DNA strand having the same sequence as the RNA and at the 3'-end of the coding RNA transcript is called the "downstream sequence".

[0138] "Complementary" refers to the topological compatibility or matching together of the interaction surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and further, the contacting surface features are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially the same as the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.

[0139] "Vector" is a polynucleotide that can be used to introduce another nucleic acid linked thereto into a cell. One type of vector is a "plasmid", which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), into which additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell and thus replicate with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0140] "Regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, or location of expression) of a nucleic acid operably linked thereto. A regulatory sequence can, for example, exert its effect directly on the nucleic acid being regulated or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Additional examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res. 23: 3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (e.g., level, timing, or location of expression).

[0141] "Host cell" is a cell that can be used to express a polynucleotide of the present disclosure. The host cell can be a prokaryote, such as Escherichia coli (E. coli), or the host cell can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell, or insect cell), or a hybridoma. Generally, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to denote a host cell that has been transformed or transfected with the nucleic acid to be expressed. The host cell can also be a cell that contains the nucleic acid but does not express the nucleic acid at a desired level, unless regulatory sequences are introduced into the host cell such that the regulatory sequences become operably linked to the nucleic acid. It should be understood that the term host cell refers not only to a particular subject cell but also to the progeny or potential progeny of such a cell. Since certain modifications may occur in subsequent generations due to, for example, mutation or environmental influences, such progeny may not actually be identical to the parental cell but are still included within the scope of the term as used herein.

[0142] The term "isolated molecule" (wherein the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that by virtue of its origin or source of derivation (1) is not associated with the components that are naturally associated with it in its native state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cell system different from the cell of its native origin will be "isolated" from its naturally associated components. Molecules can also be made substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be determined in many ways well known in the art. For example, the purity of a polypeptide sample can be determined using techniques well known in the art using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0143] A protein or polypeptide is "substantially pure", "substantially homogeneous" or "substantially purified" when at least about 60% to 75% of the sample appears as a single species of polypeptide. The polypeptide or protein can be monomeric or polymeric. A substantially pure polypeptide or protein will generally comprise about 50%, 60%, 70%, 80% or 90% w / w of the protein sample, more usually about 95% and preferably will be in excess of 99% pure. Protein purity or homogeneity can be indicated by many means well known in the art, such as polyacrylamide gel electrophoresis of the protein sample followed by visualization of individual polypeptide bands after staining the gel with a stain well known in the art. For certain purposes, higher resolution can be provided by using HPLC or other means well known in the art for purification.

[0144] As used herein, the term "label" or "labeled" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, e.g., a polypeptide incorporating a radiolabeled amino acid or attached to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin incorporating a fluorescent marker or an enzyme activity that can be detected by optical methods or calorimetry). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or a toxin. A variety of methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I); fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores); enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pairing sequences, binding sites of second antibodies, metal binding domains, epitope tags); magnetic agents, such as gadolinium chelates; toxins such as pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogs or homologs. In various embodiments, the label is attached via spacer arms of various lengths to reduce potential steric hindrance.

[0145] As used herein, the term "heterologous" refers to a combination or state that is not natural or does not exist in nature, which combination or state can be achieved, for example, by replacing existing natural components or states with components or states from another source. Similarly, protein expression in an organism other than the organism in which the protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0146] It should be understood that the aspects and embodiments of the present disclosure described herein include "consisting of these aspects and embodiments" and / or "consisting essentially of these aspects and embodiments".

[0147] As used herein, the recitation of "about" a value or parameter includes (and describes) variations that are directed to that value or parameter itself. For example, the recitation of "about X" includes the description of "X".

[0148] Unless the context clearly dictates otherwise, the singular forms "a", "or", and "the" as used herein and in the appended claims include plural referents. It should be understood that the aspects and variations of the present disclosure described herein include "consisting of these aspects and variations" and / or "consisting essentially of these aspects and variations".

[0149] PD1 blocking antibody

[0150] In one aspect, the PD1-blocking antibody directs the IL-2 portion of VitoKine to tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) and confines the activation of VitoKine locally to improve the therapeutic index. In another aspect, the PD1-blocking antibody directs the IL-2 portion of the immunocytokine to TILs in the TME. In various embodiments, the PD1-blocking antibody is optimized by modifications in the variable domains of pembrolizumab. In various embodiments, the modifications involve germline sequence substitutions of CDR residues, germline sequence substitutions of framework residues, and adoption of the most prevalent and better-performing VH3 human germline family sequence as the receptor framework. In various embodiments, these modifications are implemented singly or in combination to develop an optimized PD1-blocking antibody. In various embodiments, these optimized PD1-blocking antibodies exhibit high binding affinity for PD1, inhibit PD1 with the same or comparable potency as pembrolizumab, have a higher sequence similarity score with their closest human germline sequence, result in a higher degree of human origin compared to pembrolizumab, and are predicted to have lower hydrophobicity, resulting in a lower tendency to aggregate than pembrolizumab. In various embodiments, the PD1 Ab-IL-2VitoKine constructs and PD1-targeted IL-2 immunocytokines based on these optimized PD1-blocking antibodies are predicted to have enhanced developability characteristics. In various embodiments, the PD1 antibody comprises a light chain variable region having a sequence selected from the group consisting of the sequences listed in SEQ ID NO: 3-5 and a heavy chain variable region having a sequence selected from the group consisting of the sequences listed in SEQ ID NO: 7-18. In various embodiments, the PD1 antibody comprises the light chain sequence listed in SEQ ID NO: 44 and a heavy chain having a sequence selected from the group consisting of the sequences listed in SEQ ID NO: 45-49.

[0151] IL-2 domain

[0152] Interleukin-2 (IL-2) is a classical Th1 cytokine that is produced by T cells upon activation through the T cell antigen receptor and the co-stimulatory molecule CD28. Regulation of IL-2 occurs through signal transduction pathways and activation of transcription factors that act on the IL-2 promoter to generate new gene transcription, but also involves regulation of the stability of IL-2 mRNA. IL-2 binds to a multi-chain receptor, including a highly regulated α-chain and β and γ chains that mediate signal transduction through the Jak-STAT pathway. IL-2 delivers activation, growth, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important in mediating the cell death of activated-induced T cells, a function that provides a key mechanism for terminating the immune response. An unglycosylated human recombinant IL-2 product, aldesleukin (available from Prometheus Laboratories Inc., San Diego, Calif. as des-alanyl-1, serine-125 human interleukin-2 under the trademark ), has been approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been proposed for administration in patients with or infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, the short half-life and severe toxicity limit the optimal dosing of IL-2.

[0153] As used herein, the terms "native IL-2" and "native interleukin-2" in the context of a protein or polypeptide refer to any naturally-occurring mammalian interleukin-2 amino acid sequence, including immature or precursor forms and mature forms. Non-limiting examples of GenBank accession numbers for the amino acid sequences of native mammalian interleukin-2 of various species include NP_032392.1 (Mus musculus, immature form), NP_001040595.1 (Macaca mulatta, immature form), NP_000577.2 (human, precursor form), CAA01199.1 (human, immature form), and AAD48509.1 (human, immature form). In various embodiments of the invention, native IL-2 is the immature or precursor form of naturally-occurring mammalian IL-2. In other embodiments, native IL-2 is the mature form of naturally-occurring mammalian IL-2. In various embodiments, native IL-2 is the precursor form of naturally-occurring human IL-2. In various embodiments, native IL-2 is the mature form of naturally-occurring human IL-2. In various embodiments, the IL-2 in the VitoKine and immunocytokine constructs of the invention is derived from the amino acid sequence of the mature human IL-2 polypeptide sequence set forth in SEQ ID NO:116:

[0154] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCL

[0155] EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:116)

[0156] In various embodiments, the IL-2 domain will be an IL-2 variant (or mutant) comprising a sequence derived from the mature human IL-2 polypeptide sequence set forth in SEQ ID NO:116. In various embodiments, the IL-2 variant comprises a single C125I amino acid substitution which generally enhances the developability of the protein while fully retaining its biological activity. In various embodiments, the IL-2 variant comprising a single C125I mutation has the amino acid sequence set forth in SEQ ID NO:117.

[0157] In various embodiments, compared to the native IL-2 sequence, the sequence of the IL-2 variant has at least one amino acid change, e.g., a substitution or deletion, such a change results in IL-2 agonist or antagonist activity. IL-2 agonists are exemplified by having comparable or increased biological activity compared to wild-type IL-2. IL-2 antagonists are exemplified by having decreased biological activity compared to wild-type IL-2 or by the ability to inhibit IL-2-mediated responses. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO:117 with altered binding to IL-2Rα. In various embodiments, the IL-2 variant with altered binding to IL-2Rα comprises the amino acid sequences listed in SEQ ID NO:118 - 125. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO:117 with reduced / eliminated binding to IL-2Rα to selectively activate and proliferate effector T cells (Teff) for cancer treatment. In various embodiments, the IL-2 variant with reduced / eliminated binding to IL-2Rα comprises the amino acid sequences listed in SEQ ID NO:118 - 122. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO:117 with reduced binding to IL-2Rβ and / or γc. In various embodiments, the IL-2 variant with reduced binding to IL-2Rβ and / or γc comprises the amino acid sequences listed in SEQ ID NO:126 - 150. In various embodiments, the IL-2 variant has an amino acid sequence derived from SEQ ID NO:117 with reduced / eliminated binding to IL-2Rα and reduced binding to IL-2Rβ and / or γc. In various embodiments, the IL-2 variant with reduced / eliminated binding to IL-2Rα and reduced binding to IL-2Rβ and / or γc comprises the amino acid sequences listed in SEQ ID NO:151 - 180. As will be understood by those skilled in the art, all mutations can optionally and independently be combined in any way to achieve optimal affinity and activity modulation.

[0158] IL-2Rα domain (masked partial domain in PD1 Ab-IL-2 VitoKine)

[0159] The interleukin-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells such as lymphocytes, which binds and responds to IL-2. IL-2R has three subunits: α (CD25), β (CD122), and γ (CD132 or common γ chain (γc)), which is a chain shared with five other cytokine receptors, IL-4R, IL-7R, IL-9R, IL-15R, and IL-21R. The α chain (alias: Tac antigen or p55) of the human receptor is encoded by the gene IL-2RA on chromosome 10p14-15. The gene for the β chain (IL-2RB, CD122) of the human receptor is located on chromosome 22q11.2-12, while the gene for the human γ chain (IL-2RG) is located on chromosome Xq13. The assembly of all three subunits of the receptor is important for signal transduction into B cells and T cells. IL-2R is present (temporarily or permanently) on the cell surface of almost all hematopoietic cells, including lymphoid lineage T, B, and NK cells, as well as myeloid cells such as macrophages, monocytes, and neutrophils. Signals are transmitted into the cell via Janus kinases—Jak1 and Jak3. Phosphorylation of the cytoplasmic portion of the β chain of the receptor enables the STAT-3 and STAT-5 factors to form homodimers. The homodimers of STAT-3 and STAT-5 show increased affinity for the nucleus, where they bind to specific DNA elements, enhancing the transcription of IL-2-dependent genes.

[0160] As used herein, the terms “native IL-2Rα” and “native interleukin-2 receptor α” in the context of a protein or polypeptide refer to any naturally occurring mammalian interleukin-2 receptor α (“IL-2Rα”) amino acid sequence, including immature or precursor forms and mature forms, as well as naturally occurring isotypes. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various native mammalian IL-2Rα include NP_032393.3 (Mus musculus), CAK26553.1 (human), and NP_000408.1 (human). In various embodiments, the IL-2Rα domain is derived from the amino acid sequence of the human IL-2Rα sequence set forth in SEQ ID NO:181:

[0161] MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRI

[0162] KSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQ

[0163] PVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKM

[0164] THGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAA

[0165] TMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI(SEQ ID NO:181)

[0166] In various embodiments, the masking moiety domain (D3) is used to reversibly mask the activity of the IL-2 domain in the PD1 Ab-IL-2 VitoKine construct. In various embodiments, the masking moiety domain is the extracellular domain of IL-2Rα or a functional fragment thereof. In various preferred embodiments, the masking moiety domain is the IL-2Rα Sushi domain, which comprises the amino acid sequence of the mature human IL-2Rα polypeptide as set forth in SEQ ID NO:182. In various preferred embodiments, the masking moiety domain is a variant of the IL-2Rα Sushi domain.

[0167] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDN

[0168] QCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENE

[0169] ATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG

[0170] (SEQ ID NO:182)

[0171] In various embodiments, the PD1 Ab-IL-2 VitoKine comprises IL-2RαSushi (SEQ ID NO:182) as a shielding partial domain to shield the activity of IL-2 (including IL-2 variants). Although wild-type IL-2Rα binds to IL-2 with a moderate affinity of 30 nM, there is a possibility that IL-2Rα may not dissociate when the cleavage linker is cleaved. The association between the cleaved IL-2Rα and IL-2 can reduce the activity of IL-2 and / or tilt the balance of T cell subsets towards an undesirable outcome. As affinity-reducing mutations are introduced into IL-2RαSushi, the IL-2Rαsushi domain may dissociate from IL-2 when the linker is cleaved. In various embodiments, the shielding partial domain in the PD1 Ab-IL-2 VitoKine is an IL-2RαSushi variant comprising an IL-2 binding-attenuating mutation, such as, R36A, K38E, L42G, or Y43A, or any combination of substitutions. In various embodiments, the IL-2RαSushi variant can effectively shield the activity of the IL-2 partial domain, although its affinity for IL-2 is reduced. In various embodiments, due to the reduced affinity of the IL-2RαSushi variant for IL-2, it is expected that the IL-2RαSushi variant dissociates from IL-2 and diffuses away when the linker is cleaved.

[0172] In various embodiments, the PD1 Ab-IL-2 VitoKine construct of the present invention comprises a shielding partial domain that is one of the IL-2RαSushi domain variants comprising the amino acid sequences listed in SEQ ID NOs: 183-185.

[0173] The L1 linker and L2 linker in PD1 Ab-IL-2 VitoKine

[0174] Cleavable Linker

[0175] Cleavable linkers or linkers susceptible to disease-related enzymes can contain moieties, such as protein substrates, that can be specifically cleaved by proteases present at elevated levels at the disease site compared to non-diseased tissue. The literature contains numerous reports of increased levels of enzymes with known substrates in various types of cancer (e.g., solid tumors). See, e.g., La Rocca et al., Brit. J. Cancer 90:1414-1421 and Ducry et al., Bioconjug. Chem. 21:5-13, 2010, each of which is incorporated herein by reference in its entirety. In various embodiments, the proteases capable of cleaving the protease-cleavable linker are selected from the group consisting of: metalloproteases such as matrix metalloproteases (MMP) 1-28, serine proteases such as urokinase-type plasminogen activator (uPA) and matriptase, cysteine proteases such as podoplanin, aspartic proteases, and cathepsin proteases. Exemplary protease sequences are provided in Table 2:

[0176] Table 2

[0177]

[0178]

[0179] Exemplary protease substrate peptide sequences that can be used as protease-cleavable linkers with or without peptide spacers are provided in Table 3:

[0180] Table 3

[0181]

[0182]

[0183] In various embodiments, the protease is MMP-9 or MMP-2. In additional specific embodiments, the protease is matriptase. In additional specific embodiments, the protease is MMP-14. In additional specific embodiments, the protease is podoplanin. In various embodiments, the protease-cleavable linker may contain two or more protease substrate sequences. In various embodiments, the protease is MMP-2 / MMP-9 and matriptase. In various embodiments, the protease-cleavable linker contains the protease recognition sequence 'GPLGMLSQ' (SEQ ID NO:61). In various embodiments, the protease-cleavable linker contains the protease recognition sequence 'SGRSENIRTA' (SEQ ID NO:60). In various embodiments, the protease-cleavable linker contains the protease recognition sequence 'GPTNKVR' (SEQ ID NO:69). In various embodiments, the protease-cleavable linker contains the protease recognition sequence 'PMAKK' (SEQ ID NO:74). In various embodiments, the protease-cleavable linker contains the protease recognition sequence 'GPLGMLSQPMAKK' (SEQ ID NO:76). In various embodiments, the protease-cleavable linker contains the protease recognition sequence 'PMAKKGPLGMLSQ' (SEQ ID NO:77).

[0184] In various embodiments, a peptide spacer can be incorporated on either side or both sides of the protease-cleavable sequence, or as a non-cleavable linker that does not have protease substrate sites. The peptide spacer is used to position the cleavable linker to make it more accessible to the enzyme responsible for cleavage. The length and composition of the peptide spacer can be fine-tuned to balance the accessibility of enzymatic cleavage and the steric constraints required to reversibly mask the D2 domain from exerting its biological activity. The peptide spacer can include from 1 to 100 amino acids. Suitable peptide spacers are known in the art and include, but are not limited to, peptide linkers containing flexible amino acid residues such as glycine and serine. In various embodiments, the peptide spacer can contain from 1 to 12 amino acids, including the motifs G, S, GSGG (SEQ ID NO:104), GGSS (SEQ ID NO:105), GSGS (SEQ ID NO:109), GSGSGS (SEQ ID NO:110), GSGSGSGS (SEQ ID NO:111), GSGSGSGSGS (SEQ ID NO:112), or GSGSGSGSGSGS (SEQ ID NO:113). In other embodiments, the peptide spacer can contain the motif (GGGGS) (SEQ ID NO:106) n, where n is an integer from 1 to 10. In other embodiments, the peptide spacer may also contain amino acids other than glycine and serine. The peptide spacer is stable under physiological conditions and at the site of disease, such as at the site of cancer.

[0185] Exemplary protease-cleavable linkers are provided in Table 4, which have a peptide spacer flanking a protease substrate peptide (underlined):

[0186] Table 4

[0187] Protease-Cleavable Linker SEQ ID NO: <![CDATA[GGGSGGGGSGGGGS LSGRSDNH GGSGGGGS]]> 78 <![CDATA[GSS SGRSENIRTA GT]]> 79 <![CDATA[GGGGSGGGGSGGGS LGGSGRSANAILE GGSGGGGS]]> 80 <![CDATA[GGGGSGGGGS LGGSGRSANAILE GGGGS]]> 81 <![CDATA[GGGGS LGGSGRSANAILE GGS]]> 82 <![CDATA[GGGS GPTNKVR GGS]]> 83 <![CDATA[GGS GPLGMLSQ GGGS]]> 84 <![CDATA[G GPLGMLSQ S]]> 85 <![CDATA[GG GPLGMLSQ GGS]]> 86 <![CDATA[G GPTNKVR GS]]> 87 <![CDATA[G RQARAVGG S]]> 88 <![CDATA[GGG SGRSENIRTA GG]]> 89 <![CDATA[SGGP GPAGMKGL PGS]]> 90 <![CDATA[GGGGS PMAKK GGGGS]]> 91 <![CDATA[G GPLGMLSQPMAKK S]]> 92 <![CDATA[GGS GPLGMLSQPMAKK GGGS]]> 93 <![CDATA[GGG PMAKKGPLGMLSQ GGGS]]> 94

[0188] Non-Cleavable Linker

[0189] Non-cleavable linkers provide a covalent connection and additional structural and / or spatial flexibility between protein domains. As is known in the art, peptide linkers containing flexible amino acid residues such as glycine and serine can be used as non-cleavable linkers. In various embodiments, the non-cleavable linker may contain 1 - 100 amino acids. In various embodiments, the spacer may contain the motifs GSGG (SEQ ID NO:104), GGSS (SEQ ID NO:105), GSGS (SEQ ID NO:109), GSGSGS (SEQ ID NO:110), GSGSGSGS (SEQ ID NO:111), GSGSGSGSGS (SEQ ID NO:112), or GSGSGSGSGSGS (SEQ ID NO:113). In other embodiments, the spacer may contain the motif (GGGGGS)(SEQID NO:106) n , where n is an integer from 1 to 10. In other embodiments, the linker may also contain amino acids other than glycine and serine. In another embodiment, the non-cleavable linker may be a simple chemical bond, such as an amide bond (e.g., chemical conjugation via PEG). The non-cleavable linker is stable under physiological conditions and at the site of disease, such as at the site of cancer.

[0190] Exemplary non-cleavable linkers are provided in Table 5:

[0191] Table 5

[0192]

[0193] Combinations of Cleavable and Non-Cleavable Linkers

[0194] In various embodiments, both the L1 linker and the L2 linker can be cleavable or a combination of a cleavable linker and a non-cleavable linker to generate different forms of the active portion of the IL-2 domain to achieve specific therapeutic goals, optimize the risk-benefit ratio, or align with different properties of the cytokine. Exemplary active forms released by linker cleavage are depicted in Figure 2 . Both active form 1 derived from L1 linker cleavage and active form 3 derived from L1 and L2 linker cleavage are short-acting cytokines due to their release from the targeting antibody upon proteolysis. The presence or absence of the shielding domain will result in different activities of these two active forms in the local environment. After local action, the short-acting active forms can be rapidly eliminated from the systemic circulation, resulting in reduced toxicity. In contrast, active form 2 derived from L2 linker cleavage ( Figure 2 depicted) is a fully functional IL-2 fused to the PD1 Ab at or near the disease site. This active form is capable of cis-activating IL-2R signaling on PD1-expressing T cells at or near the disease site, which synergistically enhances both pathways and potentiates the anti-cancer immune response while minimizing systemic toxicity.

[0195] Polynucleotide

[0196] In another aspect, the present disclosure provides isolated nucleic acid molecules that comprise polynucleotides of the IL-2, IL-2 variants, IL-2Rα, IL-2Rα variants, PD1 blocking antibodies, antibody fragments, PD1 Ab-IL-2 VitoKine constructs, or PD1-targeted IL-2 immunocytokines of the present disclosure. The subsequent paragraphs of this sub-section "Polynucleotide" will utilize the PD1-targeted IL-2 VitoKine (VitoKine) construct as an illustrative example, however these concepts should equally apply to the PD1-targeted IL-2 immunocytokines of the present invention.

[0197] The subject nucleic acid can be single-stranded or double-stranded. Such nucleic acids can be DNA or RNA molecules. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA encoding a VitoKine construct is obtained from genomic libraries that are available for many species. Synthetic DNA can be obtained by chemically synthesizing overlapping oligonucleotide fragments and then assembling the fragments to reconstruct part or all of the coding region and flanking sequences. RNA can be obtained from prokaryotic expression vectors that direct high-level synthesis of mRNA, such as vectors using the T7 promoter and RNA polymerase. The DNA molecules of the present disclosure include full-length genes as well as polynucleotides and fragments thereof. Full-length genes can also contain sequences encoding N-terminal signal sequences. Such nucleic acids can be used, for example, in methods for preparing new VitoKine constructs.

[0198] In various embodiments, an isolated nucleic acid molecule comprises a polynucleotide described herein and further comprises a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker described herein.

[0199] In various embodiments, the recombinant nucleic acids of the present disclosure can be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are recognized in the art and are selected to direct the expression of the VitoKine construct. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Generally, the one or more regulatory nucleotide sequences can include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. The present disclosure contemplates constitutive or inducible promoters known in the art. The promoter can be a naturally occurring promoter or a hybrid promoter that combines elements of more than one promoter. The expression construct can be present on an episome such as a plasmid in a cell, or the expression construct can be inserted into a chromosome. In various embodiments, the expression vector contains a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary depending on the host cell used.

[0200] In another aspect of the present disclosure, the subject nucleic acids are provided in an expression vector comprising a nucleotide sequence encoding a VitoKine construct operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector used for expressing a polypeptide from a polynucleotide sequence. Vectors suitable for expression in a host cell are readily available and utilize standard recombinant DNA techniques to insert nucleic acid molecules into the vector. Such vectors can include a variety of expression control sequences that, when operably linked to a DNA sequence, control the expression of that DNA sequence and can be used in these vectors to express the DNA sequence encoding the VitoKine construct. Such useful expression control sequences include, for example, the early and late promoters of SV40, the tet promoter, the adenovirus- or cytomegalovirus-mediated early promoters, the RSV promoter, the lac system, the trp system, the TAC or TRC systems, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage λ, the control region of the fd coat protein, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter of an acid phosphatase such as PhoS, the promoter of yeast a-mating factor, the polyhedron promoter of the baculovirus system, and other sequences known to control gene expression in prokaryotic or eukaryotic cells or their viruses and various combinations thereof. It should be understood that the design of the expression vector may depend on factors such as the choice of host cell to be transformed and / or the type of protein desired to be expressed. In addition, consideration should also be given to the copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector such as antibiotic markers. Exemplary expression vectors suitable for the expression of VitoKine are pDSRa and its derivatives comprising the VitoKine polynucleotide, and any other suitable vectors known in the art or described hereinafter.

[0201] The recombinant nucleic acids of the present disclosure can be produced by ligating a cloned gene or a portion thereof to a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammalian), or both. Expression mediators for producing the recombinant VitoKine construct include plasmids and other vectors. For example, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as Escherichia coli.

[0202] Some mammalian expression vectors contain both prokaryotic sequences that facilitate the replication of the vector in bacteria and one or more eukaryotic transcription units for expression in eukaryotic cells. Examples of mammalian expression vectors suitable for transfection of eukaryotic cells are vectors derived from pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg. Some of these vectors are modified with sequences from bacterial plasmids such as pBR322 to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Optionally, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. The various methods employed in the preparation of plasmids and the transformation of host organisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells and for general recombinant procedures, see Chapters 16 and 17 of Molecular Cloning A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press, 1989) by Sambrook, Fritsch, and Maniatis. In some instances, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include vectors derived from pVL (such as pVL1392, pVL1393, and pVL941), vectors derived from pAcUW (such as pAcUW1), and vectors derived from pBlueBac (such as pBlueBac III containing B-gal).

[0203] In various embodiments, vectors designed to produce the subject VitoKine constructs in Chinese hamster ovary (CHO) cells or human embryonic kidney 293 (HEK293) cells will be used, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.). It will be apparent that the subject gene constructs can be used to cause expression of the subject VitoKine constructs in cells growing in culture, for example to produce proteins for purification, including fusion proteins or variant proteins.

[0204] The present disclosure also relates to host cells transfected with a recombinant gene comprising a nucleotide sequence encoding an amino acid sequence of one or more of the subject VitoKine constructs. The host cell can be a prokaryotic cell or a eukaryotic cell. For example, the VitoKine constructs of the present disclosure can be expressed in bacterial cells such as Escherichia coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art, such as CHO cells or HEK293 cells.

[0205] Accordingly, the present disclosure also relates to methods of producing the subject VitoKine constructs. For example, host cells transfected with an expression vector encoding a VitoKine construct can be cultured under appropriate conditions that permit expression of the VitoKine construct. The VitoKine construct can be secreted and isolated from a mixture of the cells and the culture medium containing the VitoKine construct. Optionally, the VitoKine construct can be retained in the cytoplasm or in a membrane fraction, and the cells can be harvested, lysed, and the protein isolated. Cell cultures include host cells, culture medium, and other by-products. Suitable culture media for cell culture are well known in the art.

[0206] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques well known to those skilled in the art. These techniques include, at one level, a crude fractionation of protein fractions and non-protein fractions. After the peptide or polypeptide has been separated from other proteins, chromatographic and electrophoretic techniques can be utilized to further purify the peptide or polypeptide of interest to achieve partial or complete purification (or purification to homogeneity). As used herein, the terms "isolated polypeptide" or "purified polypeptide" are intended to refer to a composition separable from other components, wherein the polypeptide is purified to any degree relative to its naturally occurring state. Thus, a purified polypeptide also refers to a polypeptide removed from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has undergone fractionation to remove a variety of other components, and the polypeptide composition substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the majority of the components of the composition, such as about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the proteins constituting the composition.

[0207] A variety of techniques suitable for purification will be well known to those skilled in the art. These techniques include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), etc. or by heat denaturation followed by centrifugation; chromatography, such as affinity chromatography (Protein-A column), ion exchange, gel filtration, reverse phase, hydroxyapatite, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is generally known in the art, it is contemplated that the order of performing multiple purification steps may be altered, or certain steps may be omitted, and still result in a suitable method for preparing a substantially purified polypeptide.

[0208] Pharmaceutical composition

[0209] The subsequent paragraphs of this sub-section "Pharmaceutical composition" will utilize the PD1-targeted IL-2 VitoKine (VitoKine) construct as an illustrative example, however these concepts should apply equally to the PD1-targeted IL-2 immunocytokines of the present invention.

[0210] In another aspect, the present disclosure provides pharmaceutical compositions comprising a VitoKine construct admixed with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those of ordinary skill in the art and have been widely described (see, e.g., Remington’s Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers may be included for purposes of modifying, maintaining or preserving, for example, pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or permeability of the composition. Such pharmaceutical compositions can affect the physical state, stability, rate of in vivo release and rate of in vivo clearance of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring agents; flavoring agents and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides (preferably sodium chloride or potassium chloride), mannitol and sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0211] The primary vehicle or carrier in a pharmaceutical composition can be substantially aqueous or non-aqueous. For example, suitable vehicles or carriers can be water for injection, saline solution, or artificial cerebrospinal fluid, which may be supplemented with other materials commonly used in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are additional exemplary vehicles. Other exemplary pharmaceutical compositions contain a Tris buffer at about pH 7.0 - 8.5 or an acetate buffer at about pH 4.0 - 5.5, which may also contain sorbitol or a suitable alternative. In one embodiment of the present disclosure, the composition can be prepared by mixing a selected composition having a desired degree of purity with an optional formulation agent (Remington’s Pharmaceutical Sciences, supra) for storage in the form of a lyophilized cake or an aqueous solution. Additionally, the therapeutic composition can be formulated as a lyophilized product using a suitable excipient such as sucrose. The optimal pharmaceutical composition will be determined by one of ordinary skill in the art depending on, for example, the intended route of administration, the delivery form, and the desired dose.

[0212] When parenteral administration is contemplated, the therapeutic pharmaceutical composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired VitoKine construct in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the polypeptide is formulated as a sterile, isotonic solution for suitable storage. In various embodiments, pharmaceutical formulations suitable for injectable administration can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks solution, Ringer solution, or physiological buffered saline. Aqueous injectable suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compound can be prepared as suitable oily injectable suspensions. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound and allow the preparation of highly concentrated solutions.

[0213] In various embodiments, the therapeutic pharmaceutical composition can be formulated for targeted delivery using colloidal dispersion systems. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, and lipid-based systems include oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include lecithin, dipalmitoyl phosphatidylcholine, and distearoyl phosphatidylcholine. Targeting of liposomes can also be based on, for example, organ specificity, cell specificity, and organelle specificity and is known in the art.

[0214] In various embodiments, oral administration of the pharmaceutical composition is contemplated. The pharmaceutical composition administered in this form can be formulated with or without carriers commonly used in the compounding of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more of the therapeutic compounds of the present disclosure can be mixed with one or more pharmaceutically acceptable carriers such as sodium citrate or dicalcium phosphate and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin wax; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition can also contain buffering agents. Similar types of solid compositions can also be used as fillers in gelatin capsules that are soft-filled and hard-filled with excipients such as lactose or milk sugar and high molecular weight polyethylene glycol. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifying agents, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (particularly, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of dehydrated sorbitol and mixtures thereof. In addition to the inert diluent, oral compositions can also contain adjuncts such as wetting agents, emulsifying agents, and suspending agents, sweetening agents, flavoring agents, coloring agents, perfuming agents, and preservatives.

[0215] In various embodiments, topical administration of a pharmaceutical composition to the skin or to mucous membranes is contemplated. The topical formulation may also contain one or more of a number of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of such agents are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methanol or isopropanol, dimethyl sulfoxide, and azone. Additional agents may also be included to make the formulation cosmetically acceptable. Examples of such agents are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratolytic agents, such as those known in the art, may also be included. Examples are salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed with a pharmaceutically acceptable carrier and with any preservatives, buffers, or propellants as may be required, under sterile conditions. In addition to the subject compounds of the present disclosure (e.g., VitoKine constructs), ointments, pastes, creams, and gels may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide or mixtures thereof.

[0216] Additional pharmaceutical compositions contemplated for use herein include formulations that comprise a polypeptide in a sustained release or controlled release formulation. In various embodiments, the pharmaceutical composition can be formulated as nanoparticles, as a sustained release hydrogel, or incorporated into an oncolytic virus. Such nanoparticle approaches include, for example, encapsulation in nanoparticles comprising a polymer having a hydrophobic backbone and hydrophilic side chains as a drug carrier, encapsulation in microparticles, insertion into liposomes as an emulsion, and conjugation with other molecules. Examples of nanoparticles include mucoadhesive nanoparticles coated with chitosan and carbomer (Takeuchi et al., Adv. Drug Deliv. Rev. 47(1):39-54, 2001) and nanoparticles comprising a combination of charged polyesters, poly(2-sulfoethyl vinyl alcohol), and poly(D,L-lactic-co-glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1):147-160, 2000). Albumin-based nanoparticle compositions have been developed as drug delivery systems for delivering hydrophobic drugs such as taxanes. See, e.g., U.S. Patent Nos. 5,916,596; 6,506,405; 6,749,868; 6,537,579; 7,820,788; and 7,923,536. (Albumin-stabilized paclitaxel nanoparticle formulation) was approved in the United States in 2005 and subsequently approved in several other countries for the treatment of metastatic breast cancer.

[0217] Techniques for formulating a variety of other sustained release or controlled release vehicles such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections are also known to those of skill in the art.

[0218] The effective amount of the pharmaceutical composition to be used for treatment will depend, for example, on the treatment context and the treatment objective. Those skilled in the art will understand that the appropriate dosage level for treatment will thus vary, in part, depending on the molecule being delivered, the indication for which the polypeptide is used, the route of administration, and the size (body weight, body surface, or organ size) and condition (age and general health) of the patient. Accordingly, the clinician can adjust the dosage and change the route of administration to obtain the best therapeutic effect. Typical dosages can range from about 0.0001 mg / kg to up to about 100 mg / kg or more, depending on the factors mentioned above. The polypeptide composition can be preferably administered by injection or intravenously. Depending on the half-life and clearance rate of the particular formulation, the long-acting pharmaceutical composition can be administered at intervals of every three to four days, weekly, bi-weekly, tri-weekly, monthly, or even longer. The dosing frequency will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Generally, the composition is administered until a dose is reached that achieves the desired effect. Thus, the composition can be administered as a single dose or as multiple doses over time (at the same or different concentrations / doses) or as a continuous infusion. Further refinement of the appropriate dose is routinely carried out. The appropriate dose can be determined by using appropriate dose-response data.

[0219] The route of administration of the pharmaceutical composition is according to known methods, for example, oral; by intravenous, intraperitoneal, intratumoral, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, intravesicular, transdermal, subcutaneous or intraperitoneal injection; and intranasal, enteral, topical, sublingual, urethral, vaginal or rectal means; by sustained release systems or by implant devices. When desired, the composition can be administered by bolus injection, or continuously by infusion, or by implant device. Optionally or additionally, the composition can be administered locally by implantation of a membrane, sponge, or another suitable material to which the desired molecule has been adsorbed or encapsulated. When using an implant device, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be by diffusion administration, sustained release bolus, or continuous administration.

[0220] Therapeutic use

[0221] The subsequent paragraphs of this sub-section "Therapeutic use" will use the PD1-targeted IL-2 VitoKine (VitoKine) construct as an illustrative example, however these concepts should equally apply to the PD1-targeted IL-2 immunocytokines of the present invention.

[0222] The present disclosure provides methods for treating cancer cells in a subject, comprising administering to the subject a therapeutically effective amount (as a single therapy or in a combination therapy regimen) of a VitoKine construct of the present disclosure in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of cancer cells. In particular, the VitoKine constructs of the present disclosure are useful in treating disorders characterized by cancer. Such disorders include, but are not limited to, solid tumors such as breast cancer, respiratory cancer, brain cancer, cancers of the reproductive organs, cancers of the digestive tract, cancers of the urinary tract, eye cancer, liver cancer, skin cancer, head and neck cancer, thyroid cancer, parathyroid cancer, and their distant metastases, lymphomas, sarcomas, multiple myeloma, and leukemias. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of respiratory cancer include, but are not limited to, small cell lung cancer and non-small cell lung cancer, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancer include, but are not limited to, brainstem and hypothalamic gliomas, cerebellar and cerebral astrocytomas, neuroblastoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumors. Tumors of the male reproductive organs include, but are not limited to, prostate cancer and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, and vulvar cancer, as well as sarcomas of the uterus. Tumors of the digestive tract include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, liver cancer, breast cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer. Tumors of the urinary tract include, but are not limited to, bladder cancer, penile cancer, kidney cancer, renal pelvic cancer, ureteral cancer, and urethral cancer. Eye cancer includes, but is not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and combined hepatocellular cholangiocarcinoma. Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancer includes, but is not limited to, nasopharyngeal cancer and cancers of the lip and oral cavity. Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and hairy cell leukemia.

[0223] In various embodiments, the VitoKine construct can be used as a single agent for treating all types of cancer, including but not limited to non-small cell lung cancer, small cell lung cancer, melanoma, renal cell carcinoma, urothelial carcinoma, liver cancer, breast cancer, pancreatic cancer, colorectal cancer, gastric cancer, prostate cancer, and sarcoma.

[0224] A "therapeutically effective amount" or "therapeutically effective dose" refers to the amount of a therapeutic agent that will relieve one or more symptoms of the disorder being treated to some extent.

[0225] The therapeutically effective dose can be initially evaluated by determining the IC 50 (half maximal inhibitory concentration) from cell culture assays. Then, the dose can be formulated in an animal model to achieve a circulating plasma concentration range that includes the IC 50 determined in the cell culture. Such information can be used to more precisely determine the dose useful in humans. The levels in plasma can be measured, for example, by HPLC. The exact composition, route of administration, and dose can be selected by an individual physician in view of the condition of the subject.

[0226] The dosage regimen can be adjusted to provide the optimum desired response (e.g., a therapeutic response or a prophylactic response). For example, a single bolus can be administered, several divided doses (multiple or repeated or maintenance) can be administered over time, and the dose can be proportionally decreased or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as a single dose for the mammalian subject to be treated; each unit contains a predetermined quantity of the active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure will be determined primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0227] Thus, those skilled in the art will understand that, based on the disclosure provided herein, the dosage and dosing regimen are adjusted according to methods well known in the art of therapy. That is, the maximum tolerable dose can be readily determined, and the effective amount that provides a detectable therapeutic benefit to the subject can also be determined, as can the time requirements for administering each agent to provide a detectable therapeutic benefit to the subject. Thus, although certain dosages and dosing regimens are exemplified herein, these examples are in no way limiting as to the dosages and dosing regimens that can be provided to a subject in practicing the present disclosure.

[0228] It should be noted that the dosage value can vary with the type and severity of the condition to be alleviated and can include a single dose or more than one dose. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges listed herein are only exemplary and are not intended to limit the scope of the claimed composition or practice. In addition, the dosage regimen of the compositions of the present disclosure can be based on a number of factors, including the type of disease, the age, weight, sex, medical condition, severity of the condition and the route of administration of the subject. Thus, the dosage regimen can vary widely but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects such as toxic effects and / or experimental values. Thus, the present disclosure includes intra-subject dose-escalation within a subject as determined by a person skilled in the art. Determining the appropriate dosage and regimen is well known in the relevant art and will be understood to be within the grasp of a person skilled in the art once the teachings disclosed herein are provided.

[0229] Exemplary, non-limiting, daily dosage ranges for a therapeutically effective amount or a prophylactically effective amount of VitoKine or a VitoKine variant of the present disclosure can be from 0.0001 to 100 mg / kg body weight, from 0.0001 to 90 mg / kg body weight, from 0.0001 to 80 mg / kg body weight, from 0.0001 to 70 mg / kg body weight, from 0.0001 to 60 mg / kg body weight, from 0.0001 to 50 mg / kg body weight, from 0.0001 to 40 mg / kg body weight, from 0.0001 to 30 mg / kg body weight, from 0.0001 to 20 mg / kg body weight, from 0.0001 to 10 mg / kg body weight, from 0.0001 to 5 mg / kg body weight, from 0.0001 to 4 mg / kg body weight, from 0.0001 to 3 mg / kg body weight, from 0.0001 to 2 mg / kg body weight, from 0.0001 to 1 mg / kg body weight, from 0.001 to 50 mg / kg body weight, from 0.001 to 40 mg / kg body weight, from 0.001 to 30 mg / kg body weight, from 0.001 to 20 mg / kg body weight, from 0.001 to 10 mg / kg body weight, from 0.001 to 5 mg / kg body weight, from 0.001 to 4 mg / kg body weight, from 0.001 to 3 mg / kg body weight, from 0.001 to 2 mg / kg body weight, from 0.001 to 1 mg / kg body weight, from 0.010 to 50 mg / kg body weight, from 0.010 to 40 mg / kg body weight, from 0.010 to 30 mg / kg body weight, from 0.010 to 20 mg / kg body weight, from 0.010 to 10 mg / kg body weight, from 0.010 to 5 mg / kg body weight, from 0.010 to 4 mg / kg body weight, from 0.010 to 3 mg / kg body weight, from 0.010 to 2 mg / kg body weight, from 0.010 to 1 mg / kg body weight, from 0.1 to 50 mg / kg body weight, from 0.1 to 40 mg / kg body weight, from 0.1 to 30 mg / kg body weight, from 0.1 to 20 mg / kg body weight, from 0.1 to 10 mg / kg body weight, from 0.1 to 5 mg / kg body weight, from 0.1 to 4 mg / kg body weight, from 0.1 to 3 mg / kg body weight, from 0.1 to 2 mg / kg body weight, from 0.1 to 1 mg / kg body weight, from 1 to 50 mg / kg body weight, from 1 to 40 mg / kg body weight, from 1 to 30 mg / kg body weight, from 1 to 20 mg / kg body weight, from 1 to 10 mg / kg body weight, from 1 to 5 mg / kg body weight, from 1 to 4 mg / kg body weight, from 1 to 3 mg / kg body weight, from 1 to 2 mg / kg body weight, or from 1 to 1 mg / kg body weight. It should be noted that dosage values can vary with the type and severity of the condition to be alleviated. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition, and the dosage ranges listed herein are only exemplary and are not intended to limit the scope of the claimed composition or practice.

[0230] The toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure can be determined by standard pharmacological procedures in cell cultures or experimental animals, such as those for determining the LD 50 (dose lethal to 50% of the population) and the ED 50 (dose therapeutically effective in 50% of the population) of standard pharmacological procedures. The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the therapeutic index can be expressed as the ratio LD 50 / ED 50 . Compositions that exhibit a large therapeutic index are generally preferred.

[0231] The dosing frequency of administration of the VitoKine construct pharmaceutical composition depends on the nature of the therapy and the particular disease being treated. The subject can be treated at regular intervals, such as weekly or monthly, until the desired therapeutic outcome is achieved. Exemplary dosing frequencies include, but are not limited to: once a week without interruption; once a week every other week; once every 2 weeks; once every 3 weeks; once a week without interruption for 2 weeks and then once a month; once a week without interruption for 3 weeks and then once a month; once a month; once every two months; once every 3 months; once every 4 months; once every 5 months; or once every 6 months, or once a year.

[0232] Combination therapy

[0233] The subsequent paragraphs of this sub-section "Combination therapy" will utilize the PD1-targeted IL-2 VitoKine (VitoKine) construct as an illustrative example, however these concepts should apply equally to the PD1-targeted IL-2 immunocytokines of the present invention.

[0234] As used herein, when referring to the VitoKine constructs of the present disclosure and one or more other therapeutic agents, the terms "co - administration", "co - administered", and "in combination with" are intended to mean, and do in fact mean and include the following: such combinations of the VitoKine constructs of the present disclosure and one or more therapeutic agents are administered simultaneously to a subject in need of treatment, where such components are formulated together into a single dosage form that releases the components to the subject at substantially the same time; such combinations of the VitoKine constructs of the present disclosure and one or more therapeutic agents are administered substantially simultaneously to a subject in need of treatment, where such components are formulated separately into separate dosage forms that are taken by the subject at substantially the same time, at which time the components are released to the subject substantially at the same time; such combinations of the VitoKine constructs of the present disclosure and one or more therapeutic agents are administered sequentially to a subject in need of treatment, where such components are formulated separately into separate dosage forms that are taken by the subject at successive times with a significant time interval between each administration, at which time the components are released to the subject at substantially different times; and, such combinations of the VitoKine constructs of the present disclosure and one or more therapeutic agents are administered sequentially to a subject in need of treatment, where such components are formulated together into a single dosage form that releases the components in a controlled manner, at which time the components are released to the subject simultaneously, continuously, and / or overlappingly at the same and / or different times, where each portion can be administered by the same or different routes.

[0235] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention in combination with a second therapy, the second therapy including but not limited to immunotherapy, cytotoxic chemotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, and stem cell transplantation. For example, such a method can be used for prophylactic cancer prevention, prevention of cancer recurrence and metastasis after surgery, and as an adjunct to other conventional cancer therapies. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced by the use of the combination methods described herein.

[0236] A large number of conventional compounds have been shown to have anti-cancer activity. These compounds have been used as agents in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant T cells in leukemia or myeloid malignancies. Although chemotherapy is effective in treating various types of malignancies, many anti-cancer compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments can act synergistically and allow for a reduction in the dose of each treatment, thereby reducing the harmful side effects produced by each compound at higher doses. In other cases, malignancies that are refractory to treatment can respond to combination therapies of two or more different treatments.

[0237] In various embodiments, a second anti-cancer agent, such as a chemotherapeutic agent, will be administered to a patient. A list of exemplary chemotherapeutic agents includes, but is not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, taxanes (such as paclitaxel and docetaxel) and / or anthracyclines, and combinations of agents, such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX. In various embodiments, the doses of such chemotherapeutic agents include, but are not limited to, about 10 mg / m 2 ² 2 、30 mg / m 2 ² 2 、50 mg / m 2 ² 2 、75 mg / m 2 ² 2 、90 mg / m 2 ² 2 、100 mg / m 2 ² 2 、150 mg / m 2 ² 2 、200 mg / m 2 ² 2 、230 mg / m 2, 240 mg / m 2 , 250 mg / m 2 , 260 mg / m 2 and 300 mg / m 2 or any one of them.

[0238] In various embodiments, the combination therapy methods of the present disclosure may further include administering to a subject a therapeutically effective amount of an immunotherapy, which includes but is not limited to, treatment using depleting antibodies against specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic antibodies, antagonistic antibodies or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints), such as including but not limited to antibodies against CTLA-4, PDL-1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPα, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec9, Siglec 15, VISTA, CD276, CD272, TIM-3 and B7-H4; treatment using bispecific T cell engager antibodies such as blinatumomab; treatment involving administration of a biological response modifier (such as IL-7, IL-10, IL-12, IL-15, IL-21, IL-22, GM-CSF, IFN-α, IFN-β, IFN-γ, TGF-β antagonist or TGF-β trap); treatment using a therapeutic vaccine (including but not limited to an oncolytic virus, such as T-vec) or treatment using a therapeutic vaccine such as sipuleucel-T; treatment using a dendritic cell vaccine or a tumor antigen peptide or a neoantigen vaccine; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using NK cells; treatment using iPS-induced NK cells; treatment using iPS-induced T cells; treatment using iPS-induced CAR-T cells or iPS-induced CAR-NK cells; treatment using tumor-infiltrating lymphocytes (TIL); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded T cells and / or TCR-T cells); treatment using TALL-104 cells; and treatment using immune stimulants such as Toll-like receptor (TLR) agonists CpG, TLR7, TLR8, TLR9 and vaccines such as Bacillus Calmette-Guérin (BCG) and imiquimod; wherein the combination therapy provides increased effector cell killing of tumor cells, that is, there is a synergistic effect between the VitoKine construct and the immunotherapy when co-administered.

[0239] In various embodiments, the combination therapy includes co-administering the VitoKine construct and the second agent composition either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the VitoKine construct composition and the second agent composition are administered sequentially, i.e., the VitoKine construct composition is administered before or after the second agent composition. In various embodiments, the administration of the VitoKine construct composition and the second agent composition is simultaneous, i.e., the administration time periods of the VitoKine construct composition and the second agent composition overlap with each other. In various embodiments, the administration of the VitoKine construct composition and the second agent composition is non-simultaneous. For example, in various embodiments, the administration of the VitoKine construct composition is terminated and then the second agent composition is administered. In various embodiments, the administration of the second agent composition is terminated before the VitoKine construct composition is administered.

[0240] The following examples are provided to more fully illustrate the present disclosure but should not be construed as limiting the scope of the present disclosure.

[0241] Example 1

[0242] Sequence Optimization of the Variable Domains of Pembrolizumab

[0243] The present invention aims to optimize the variable domain sequences of pembrolizumab to enhance the similarity score with the human germline sequence (a measure for "human origin"). This enhancement can potentially reduce the risk of immunogenicity. Additionally, the inventors used the human VH3 family germline sequence as an alternative receptor framework, which, although less homologous to pembrolizumab, is more prevalent and performs better. The purpose of doing so is to improve the biophysical properties of the resulting humanized antibody, ensure its full activity is retained, and enhance its sequence human origin.

[0244] Pembrolizumab was humanized by CDR grafting using the most homologous human antibody sequences available in the RCSB Protein Data Bank as acceptor human frameworks. The frameworks of accession numbers AB063829 (SEQ ID NO:40) and M29469 (SEQ ID NO:41) found in GenBank were used as the acceptor human frameworks for the heavy chain variable domain (VH) and light chain variable domain (VL), respectively (Carven GJ et al., US8354509B2). However, based on a comparison of the variable region exons using the International Immunogenetics Information System (IMGT) DomainGapAlign tool (www.imgt.org), pembrolizumab shares only 79.6% sequence identity with its closest human germline IGHV1-2. In 2014, the World Health Organization's (WHO) International Nonproprietary Name (INN) group proposed a similarity score to the human germline sequence as a defining criterion for therapeutic antibodies, assuming that a higher similarity might indicate reduced immunogenicity. The low similarity score or "degree of humanization" of the pembrolizumab heavy chain (Abhinandan KR et al., J Mol Biol (2007) 369:852-62) may be due to poor conservation between the murine CDRs and their human sequence counterparts, the need to retain some structurally important murine framework residues to recapitulate antigen binding, and unique somatic mutations in the human framework sequence AB063829.

[0245] To enhance the degree of humanization of pembrolizumab, certain CDR residues were targeted for replacement with their equivalent residues from the closest human germline sequences. This method is referred to herein as CDR germlining. While avoiding CDR perturbation has traditionally been a central tenet of humanized Ab design, only a limited number of CDR residues are involved in direct antigen interaction. Therefore, certain CDR residues can be replaced without compromising antibody activity. According to the Kabat numbering scheme, the CDRs are defined as amino acid residues 24-34 (CDR-L1), 50-56 (CDR-L2), 89-97 (CDR-L3), 31-35b (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3).

[0246] For the CDR3 sequences, a portion of the light chain CDR3 (CDR-L3) and the entire heavy chain CDR3 (CDR-H3) are not part of the variable exon V regions of the germline sequences. Thus, there are no human germline residues available to replace the mouse CDR counterparts. Additionally, CDR3, particularly CDR-H3, is highly variable and crucial for antigen binding and functional activity, making it essential to maintain the conformation of CDR3. Therefore, the CDR germlining process does not include the CDR-L3 (QHSRDLPLT; SEQ ID NO:25) and CDR-H3 (RDYRFDMGFDY; SEQ ID NO:33) of pembrolizumab.

[0247] The sequences of pembrolizumab's CDR-L1 and CDR-L2 were aligned with the counterparts from the closest human germline IGKV3D-11 (GenBank accession number X17264; SEQ ID NO:39). The alignment is shown in Table 6A. Similarly, the alignment of pembrolizumab's CDR-H1 and CDR-H2 sequences with the closest human germline sequence IGHV1-2 (GenBank accession number X62106; SEQ ID NO:37) is presented in Table 6B.

[0248] Table 6A

[0249] Alignment of pembrolizumab's CDR-L1 sequence and CDR-L2 sequence with IGKV3D-11

[0250]

[0251] Table 6B

[0252] Alignment of pembrolizumab's CDR-H1 sequence and CDR-H2 sequence with IGHV1-2

[0253]

[0254] “-” indicates a sequence gap.

[0255] Residues in bold and italics represent the residues that interact with PD1 according to the complex structure (Horita S. et al., Sci Rep

[0256] (2016)6:35297).

[0257] Underlined residues undergo the CDR germlining process.

[0258] Multiple pembrolizumab CDR residues are directly involved in polar interactions with PD1, e.g., hydrogen bonds and salt bridges (Horita, S. et al. Sci.Rep (2016). 6:35297). Contact residues in or around VL and VH CDR1 and CDR2 include L Ser28, L Tyr30, L Tyr49, L Tyr53, H Tyr33, H Tyr35, H Asn52, H Ser53, H Asn54, H Thr57, H Asn58 (where the superscript letter "L" denotes the light chain and "H" denotes the heavy chain). Except for L Tyr49, which is not a CDR residue and is not shown, all the above antigen-interacting CDR residues are bold and italicized in Tables 6A and 6B. Among the CDR residues that differ from the germline sequences, the CDR-L1 residues L Lys27, L His34, L Leu54, L Glu55, H Phe59, H Asn60, H Glu61, H Lys64 and H Asn65 (underlined in Tables 6A and 6B) were selected for CDR germlining. They were replaced with the following amino acids by their respective human germline equivalents: L K27Q, L H34A, L L54R, L E55A, H F59Y, H N60A, H E61Q, H K64Q and H N65G, alone or in combination. Other CDR residues were retained to avoid any disruption of antigen-interacting residues.

[0259] For CDR-H1 (NYYMY; SEQ ID NO:26), only a single residue HAsn31 conforms to CDR germlining and is replaced by the equivalent residue glycine in the human germline sequence. Other residues are retained because they participate in direct antigen interaction with PD1 ( H Tyr33 and H Tyr35) or because they are conserved between the mouse and human germline sequences ( H Tyr32 and H Met34). However, considering the relatively short length of CDR-H1 with only 5 amino acids and the fact that two residues interact directly with the antigen, any amino acid change may potentially disrupt the CDR conformation and affect the antibody's activity. Therefore, H Asn31 was not modified by CDR germlining, and CDR-H1 was fully retained.

[0260] In addition to the low level of conservation between the mouse CDRs and their corresponding human germline sequences, the pembrolizumab heavy chain framework (FR) also contains multiple non-germline residues. Their generation is due to the retention of unique somatic mutations in the receptor framework sequence AB063829. These somatic mutations, including H Val9 in FR-H1, H Thr76, H Lys82a, H Gln83, H Phe84 in FR-H3, and H Thr108 in FR-H4, are considered structurally unimportant. Replacing them with their respective germline counterparts H V9A, H T76S, H K82aS, H Q83R, H F84S, H T108L results in a significant increase in the similarity score to the human germline sequence without disturbing the CDR conformation or altering the antibody's activity.

[0261] In addition, IGHV3-23 (SEQ ID NO:38) was used as an alternative receptor framework to investigate whether the use of a human receptor framework with significantly lower sequence homology but superior biophysical properties could enhance the biophysical properties of the resulting humanized antibody without compromising its functional activity. IGHV3-23 belongs to the human germline VH3 family of antibody heavy chains, which is the most common VH family in the human repertoire. It is also the most prevalent in commercially available human monoclonal antibodies and is widely recognized for its superior drug-like properties. Given the extraordinary sensitivity of CDR conformation to the chemical environment of the surrounding framework, some structurally important framework residues in pembrolizumab that differ from the VH3 germline family counterparts were selected for backmutation to their corresponding pembrolizumab equivalents. In addition, several CDR-H2 residues were targeted for CDR germlining using the IGHV3-23 CDR-H2 as a template to increase the similarity score to the human germline sequence.

[0262] The alignment of the CDR-H1 and CDR-H2 sequences of pembrolizumab with IGHV3-23 is shown in Table 6C. Six CDR-H2 residues H Phe59, H Asn60, H Glu61, H Lys62, H Phe63 and H Asn65 (underlined in Table 6C; the superscript letter "L" indicates the light chain and "H" indicates the heavy chain) were selected for CDR germlining with the following amino acid substitutions: H F59Y, H N60A, H E61D, H K62S, H F63V and H N65G. Due to the reasons described above, the CDR germlining process did not include CDR-H1.

[0263] Table 6C

[0264] Alignment of the CDR-H1 and CDR-H2 sequences of pembrolizumab with IGHV3-23

[0265]

[0266] Residues in bold and italics represent the residues that interact with PD1 according to the complex structure (Horita S. et al., Sci Rep

[0267] (2016) 6:35297). Underlined residues are subject to the CDR germlining process.

[0268] The underlined residues are subject to CDR germlining.

[0269] Two framework residues, H Thr30 and H Arg94, are considered structurally important and are retained without changing to their corresponding germline equivalents H Ser30 and H Lys94. Five additional IGHV3 framework residues H Val48, H Ser49, H Ile69, H Arg71 and H Asn73 belong to the Vernier region (U.S. Patent Nos. 5,821,337 and 5,859,205) and may be structurally important and are reverted individually or in combination to their corresponding pembrolizumab residues H Met48, H Gly49, H Leu69, H Thr71 and H Ser73. The importance of specific framework amino acid residues is evaluated experimentally. The number of revertant mutations is minimized to ensure the highest similarity score to the germline sequence without negatively affecting antibody activity.

[0270] All optimized antibody sequences are expressed as full-length antibodies having a κ light chain constant region containing the sequence listed in SEQ ID NO:34 and a modified IgG1 heavy chain constant region containing the sequence listed in SEQ ID NO:35. Table 7 lists the SEQ ID NOs of VL, VH, CDR-L1, CDR-L2, and CDR-H2 of exemplary optimized PD1-blocking antibodies and a reference antibody (P-0734) containing the VL sequence and VH sequence listed in SEQ ID NO:2 and SEQ ID NO:6, respectively. All antibodies of the present invention contain the same CDR-L3 (SEQ ID NO:25), CDR-H1 (SEQ ID NO:26), and CDR-H3 (SEQ ID NO:33).

[0271] Table 7

[0272] Exemplary PD1-blocking antibodies generated by CDR and FR germlining

[0273]

[0274] Example 2

[0275] Construction, Production, and Purification of Optimized PD1 Blocking Antibodies

[0276] All genes were codon-optimized for expression in mammalian cells and were synthesized by GenScript's services and subsequently subcloned into recipient mammalian expression vectors. Protein expression was driven by the CMV promoter, and the synthetic SV40 polyA signal sequence was located at the 3' end of the coding sequence. A leader sequence was engineered at the N-terminus of the construct to ensure proper trafficking and processing for secretion.

[0277] Antibodies were produced by co-transfecting vectors containing the light and heavy chains into ExpiCHO cells (ThermoFisher) at a 1:1 ratio according to the manufacturer's instructions. On the day of transfection, ExpiCHO cells were diluted to 6 x 10 TM cells / mL in ExpiCHO 6 Expression Medium (ThermoFisher). A total of 0.8 μg DNA / mL of culture volume of the expression vectors was mixed with cold OptiPRO TM Medium (40 μL / mL of cell culture). After adding 3.2 μL / mL of cell culture of ExpiFectamine TM CHO reagent, the solution was gently mixed and then incubated at room temperature for 5 minutes. The ExpiFectamine TM CHO / plasmid DNA complex was then slowly transferred to the cells and incubated at 37°C in a shaking incubator with an 8% CO 2 atmosphere at 130 rpm. 18 - 22 hours post-transfection, ExpiFectamine TM CHO Enhancer (6 μL / mL of cell culture) and ExpiCHO TM Feed (240 μL / mL of cell culture) were added to the gently rotating flask. After 8 days of culture, the supernatant was harvested, centrifuged at 2200 rpm for 20 min for purification, and then sterile filtered using a 0.22 μm filter (Corning).

[0278] The secreted antibody was purified from the cell culture supernatant using protein A affinity chromatography. The cell culture supernatant was loaded onto a MabSelect SuRe 5 mL column (Cytiva) equilibrated with 5 column volumes (CV) of phosphate-buffered saline pH 7.2 (ThermoFisher). Unbound proteins were removed by washing with 5 CV of PBS pH 7.2, and the target protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride buffer pH 3.2. The antibody solution was neutralized by adding 3% of 1 M Tris buffer (pH 10.2), and then concentrated and buffer-exchanged to PBS (pH 7.2) using an Ultra-15 Ultracel (MerckMillipore) with a 10 KDa MWCO.

[0279] The purity and molecular weight of the purified antibody were analyzed by SDS-PAGE with and without a reducing agent, and then stained with Coomassie (Imperial TM Protein Stain, ThermoFisher). The SurePAGE TM precast gel system (8-16% Bis-Tris, GenScript) was used according to the manufacturer's instructions. The aggregate content of the antibody was analyzed on an Agilent 1200 high-performance liquid chromatography (HPLC) system. The sample was injected into an AdvanceBio size-exclusion column ( 4.6 x 150 mm, 2.7 μm, LC column, Agilent) and eluted at 25 °C using 150 mM sodium phosphate buffer pH 7.0 as the mobile phase.

[0280] The antibody concentration of the purified protein sample was determined by measuring the absorbance at 280 nm using a Nanodrop spectrophotometer (ThermoFisher) and dividing it by the molar extinction coefficient calculated based on its amino acid sequence. The endotoxin level of the purified protein sample was measured using Endosafe nexgen-PTS (Charles River) according to the manufacturer's instructions.

[0281] Example 3

[0282] Assays for evaluating the biological activity of the optimized PD1-blocking antibody

[0283] The antigen-binding activity of the antibodies of the present invention was tested by well-known methods such as enzyme-linked immunosorbent assay (ELISA). Briefly, Nunc Maxisorp plates (ThermoFisher) were coated overnight at 4 °C with recombinant human PD1 protein (ThermoFisher) in bicarbonate buffer pH 9.4, using 1 μg of antigen per well (100 μL / well). After washing three times with PBS / 0.05% Tween 20, the plates were incubated with SuperBlock (ThermoFisher) for two hours at room temperature to block non-specific binding. Serial three-fold dilutions of the PD1 antibodies in blocking buffer (PBS containing 1% bovine serum albumin) were added to the washed plates (100 μL / well) and incubated for one hour at room temperature. After another wash, the antibodies were detected by incubation with goat anti-human IgG Fc antibody conjugated to horseradish peroxidase (HRP) (ThermoFisher) diluted 1:5000 in blocking buffer (100 μL / well) for 1 hour at room temperature. After the final wash, 100 μL / well of TMB substrate (ThermoFisher) was added. The plates were sealed and incubated in the dark for 5 - 20 minutes. The reaction was stopped by adding 2N sulfuric acid (Ricca Chemical) (50 μL / well), and the absorbance at 450 nm was measured using a plate reader. Curves were plotted and the half maximal effective concentration (EC 50 ) value was calculated using GraphPad Prism software.

[0284] In addition, the cell-based binding strength of the optimized PD1 antibodies was determined by flow cytometry using HEK 293T cells (Crown Bioscience) stably expressing the human PD1 gene. After harvesting, HEK293-hPD1 cells were seeded at 1 x 10 5 cells / well (100 μL) into 96-well U-bottom plates, incubated with Fc block (1:50) for 20 minutes at 4 °C, and subsequently washed with FACS buffer (PBS, 1% FBS). The cells were then treated with serial three-fold dilutions of each antibody in FACS buffer at concentrations ranging from 0.01 - 100 nM for 30 minutes at 4 °C. Subsequently, the cells were washed twice with FACS buffer to remove unbound molecules, and 40 μL of a 1:100 dilution of PE-labeled goat anti-human Fc secondary antibody (eBiosciences) was added to the cells. After incubation for 30 minutes at 4 °C and two more washes with FACS buffer, the antibodies bound to the cells were detected by flow cytometry (BD ACCURI-C6) using the PE-labeled secondary antibody, and the EC 50 value was calculated using GraphPad Prism software.

[0285] In addition, a thaw-and-use form of the Promega luciferase reporter assay, a biologically relevant assay, was used to measure the potency of blocking PD1 interaction. Cell thawing and plating procedures were carried out exactly as described in the manufacturer's protocol.

[0286] Briefly, a vial (0.5 mL) of PD-L1 aAPC / CHO-K1 cells was thawed and mixed with 14.5 mL of cell recovery medium (90% Ham’s F12 / 10% FBS). Next, 100 μL of this cell suspension was added to 60 wells inside two 96-well flat-bottom assay plates, while the outer wells received 100 μL of cell recovery medium. After incubation overnight at 37 °C and 5% CO 2 After incubation overnight, the medium was discarded. The inner wells received 40 μL of 3-fold serial dilutions of the compound, while the outer wells received 80 μL of assay buffer (99% RPMI 1640 / 1% FBS). Subsequently, a vial (0.5 mL) of PD1 effector cells was thawed and mixed with 5.9 mL of assay buffer, and 40 μL of this mixture was added to the inner wells. After incubation at 37 °C, 5% CO 2 After incubation for 6 hours and equilibration for 7 minutes at room temperature, 80 μL of Bio-Glo TM reagent was added to all wells. The plate was then incubated with shaking at room temperature for 10 minutes. The resulting luminescence was measured using a luminescence plate reader (BioTek synergy h1).

[0287] The background was calculated by averaging the relative light units (RLU) of the outer wells. The fold induction was determined as the RLU of the antibody sample minus the background, divided by the RLU of the control sample (no antibody) minus the background, or fold induction = (RLU (antibody - background)) / (RLU (no antibody control - background)). Finally, the EC 50 value was determined using a curve fitted with GraphPad Prism software.

[0288] Example 4

[0289] Evaluate PD1 antibodies containing germline-modified forms based on the closest human germline sequences

[0290] First, the efficacy of P-0734 in inhibiting PD1 / PD-L1 interaction was compared with a pembrolizumab (PBL) biosimilar. Although P-0734 and the PBL biosimilar share the same variable domains, their heavy chain constant regions are different. PBL contains an IgG4 constant chain with an S228P mutation (SEQ ID NO:36), while P-0734 has an IgG1 constant chain with L234A / L235A / G237A mutations (SEQ ID NO:35) to abrogate Fc effector functions. As shown in Figure 4, P-0734 and the PBL biosimilar are equally effective in blocking the interaction between PD1 and PD-L1. This result was expected and confirmed that the ability to block PD1 is determined by the variable domain sequence rather than the immunoglobulin class. P-0734 faithfully reproduced the potency of the PBL biosimilar in blocking PD1 / PD-L1 interaction and is referred to herein as the reference antibody.

[0291] The effect of CDR germline substitutions in CDR-L1 and L H34A in CDR-H2 was evaluated using antibodies with slightly different mutation backgrounds. Antibodies P-1148, P-1150, P-1151, and P-1153 all contain germline substitutions: H K27Q in CDR-L1, L L54R, L E55A in CDR-L2, and L N60A, H E61Q, H K64Q, H N65G in CDR-H2. P-1150 contains an additional H H34A substitution in CDR-L1, P-1151 has an additional L F59Y substitution in CDR-H2, and P-1153 additionally contains H both L H34A and H F59Y changes. Table 8 provides a list of the CDR germline substitutions for these exemplary PD1-blocking antibodies.

[0292] Table 8

[0293] CDR Germline Substitutions of Exemplary PD1-Blocking Antibodies

[0294] PD1 Ab CDR-L1 CDR-L2 CHR-H2 P-1148 <![CDATA L K27Q]]> <![CDATA L L54R, L E55A]]> <![CDATA H N60A, H E61Q, H K64Q, H N65G]]> P-1150 <![CDATA L K27Q, L H34A]]> <![CDATA L L54R, L E55A]]> <![CDATA H N60A, H E61Q, H K64Q, H N65G]]> P-1151 <![CDATA L K27Q]]> <![CDATA L L54R, L E55A]]> <![CDATA H F59Y, H N60A, H E61Q, H K64Q, H N65G]]> P-1153 <![CDATA L K27Q, L H34A]]> <![CDATA L L54R, L E55A]]> <![CDATA H F59Y, H N60A, H E61Q, H K64Q, H N65G]]>

[0295] As illustrated in Figures 5B and 5C and summarized in Table 9, the CDR-L1 germline substitution LH34A consistently results in a ~3.5-fold decrease in PD1 blocking potency (EC 50 ), while both E max (maximal effect / luminescence signal) and fold induction are decreased by 25%, regardless of the H presence (P-1151 and P-1153) or absence (P-1148 and P-1150) of the F59Y substitution. The effect of the H F59Y germline substitution was similarly evaluated, and the data are shown in FIGS. 5B and 5C and summarized in Table 9. Regardless of the L presence (P-1150 and P-1153) or absence (P-1148 and P-1151) of the H34A substitution, H the F59Y CDR germline substitution results in a consistent but modest decrease in both potency (EC 50 ; ~1.8-fold decrease) and signal (E max and fold induction both decreased by 10%-15%). Compared to P-0734, the cumulative CDR germline substitutions in P-1153 ultimately result in a nearly 20-fold decrease in PD1 blocking potency (EC 50 ), and a 50% decrease in E max . Thus, L both the H34A and H F59Y substitutions are considered detrimental in this particular framework, and the original CDR residues L His34 and H Phe59 will be retained.

[0296] However, despite significant differences in the potency of blocking the PD1 / PD-L1 interaction, all four optimized PD1 antibodies and the reference antibody P-0734 exhibit nearly identical binding strengths, with EC 50 values close to 100 pM (FIG. 5A and Table 9).

[0297] Table 9 ELISA binding and PD1 blocking activity of exemplary PD1 blocking antibodies

[0298]

[0299] This portion of the data indicates that mechanism-based functional assays can discern subtle activity changes that are undetectable by ELISA binding assays. Thus, the luciferase PD1 / PD-L1 reporter assay is used herein as the primary tool for characterizing and ranking PD1 blocking antibodies derived from pembrolizumab via germline substitutions. Derived antibodies that are expected to retain full functional activity will exhibit the same in vivo efficacy as pembrolizumab.

[0300] CDR-L2 germline substitution LThe potential negative impact of E55A was further evaluated by comparing P-1127 and P-1129. Both of these molecules contain the L K27Q and L K54E germline substitutions, and the only sequence difference is the additional CDR-L2 substitution in P-1129 L E55A. As shown in Figure 6A, P-1129 exhibited a slight but detectable decrease in potency (EC 50 values for P-1127 and P-1129 were 0.39 nM and 0.58 nM, respectively), and there was a max slight 10% decrease. Therefore, L the E55A amino acid substitution was considered unfavorable, and the original residue L Glu55 would be preserved.

[0301] P-1174, which contains a total of 6 CDR germline substitutions: L K27Q, L L54R, H N60A, H E61Q, H K64Q, and H N65G, exhibited the same PD1 blocking activity as P-1127 and P-0734, with EC 50 values for P-0734, P-1127, and P-1174 of 0.64 nM, 0.54 nM, and 0.67 nM, respectively (Figure 6B). Additionally, P-1148 was derived from P-1174 by eliminating one CDR germline substitution L E55A. When compared to P-0734, P-1174 exhibited higher potency than P-1148 (see Figures 5B and 6B). This portion of the data also cooperatively led to the conclusion that the original CDR residue L Glu55 should not be altered.

[0302] In addition to the low conservation between murine CDRs and their human germline counterparts, multiple non-germline residues in the VH framework of pembrolizumab also contribute to a low sequence similarity score with the germline. These non-germline residues, resulting from unique somatic mutations retained in the receptor framework sequence, include Val9 in FR-1, H Thr76 in FR-3, H Lys82a, H Gln83, H Phe84, and H in FR-4 HThr108, which is considered structurally unimportant. To further enhance the sequence similarity score to the germline or the degree of human origin, these non-germline residues in the P-1174 framework were replaced with their respective germline equivalents H V9A, H T76S, H K82aS, H Q83R, H F84S, H T108L, resulting in P-1271. As expected, P-1271 exhibited the same PD1 blocking activity as the reference antibody P-0734 (Figure 6C), and the EC 50 values of P-1271 and P-0734 were 0.66 nM and 0.70 nM, respectively.

[0303] In summary, the CDR germline substitutions in P-1174 and P-1271 L K27Q, L L54R, H N60A, H E61Q, H K64Q and H N65G enhanced the degree of human origin of the antibody sequence without compromising the potency of blocking the PD1 / PD-L1 interaction. The additional six framework germline substitutions in P-1271 also increased the similarity score to the closest human germline sequence. Table 10 lists the germline substitutions of P-1174 and P-1271 and the similarity scores to the closest human germline sequence compared to the reference antibody P-0734.

[0304] Table 10

[0305] Germline substitutions and similarity scores to the closest human germline sequence of exemplary optimized PD1 antibodies P-1174 and P-1271

[0306]

[0307] Example 5

[0308] Evaluating PD1 antibodies containing germline modifications based on the more common human germline family (VH3)

[0309] The adoption of framework germline substitutions based on the human antibody heavy chain germline IGHV3-23 (SEQ ID NO:38) was investigated to examine whether an antibody framework with significantly lower sequence homology but superior biophysical properties could enhance the drug-like properties of the resulting antibody while fully retaining its functional activity. Among the 33 framework germline substitutions (Table 11A), 5 Vernier region residues HV48, H S49, H I69, H R71 and H the importance of N73 was experimentally evaluated by reverting the mutations singly or in combination to their respective pembrolizumab equivalents H V48M, H S49G, H I69L, H R71T and H N73S. Additionally, six CDR-H2 residues H F59Y, H N60A, H E61D, H K62S, H F63V and H N65G were selected for CDR germline substitution with their corresponding residues in IGHV3-23. Table 11B provides a summary of VH germline substitutions in exemplary antibodies.

[0310] Table 11A

[0311] VH framework germline substitutions based on IGHV3-23

[0312]

[0313]

[0314] Residues in bold and underlined represent a total of 33 framework germline substitutions.

[0315] Table 11B

[0316] VH CDR germlining and FR reversion mutations based on IGHV3-23

[0317]

[0318] Figure 7 depicts the PD1 blocking activity of P-1175 and P-1181, which differ only in their CDR-H2 germline substitutions (as shown in Table 11B). Compared to P-0734, both P-1175 and P-1181 exhibited significantly reduced potency in blocking the PD1 interaction. Specifically, P-1175 showed a 10-fold decrease in potency (EC 50 ), and both the E max and fold induction decreased by 25%. This was compared to a 15-fold decrease in potency for P-1181 and an E maxIn contrast, the induction fold decreased by 40%-50% (illustrated in FIGS. 7A and 7B and summarized in Table 12). Since P-1181 exhibited a more drastic decrease in activity, two different CDR germline substitutions H K62S, H F63V were considered detrimental, and thus the original CDR residues, H Lys62 and H Phe63 would be retained. These findings suggest that the importance of individual CDR residues requires experimental evaluation; even CDR residues close to the boundary or not adjacent to antigen-contact residues can have a negative impact on activity.

[0319] Two to five framework residue revertant mutations were introduced into P-1175 to generate P-1176, P-1177, and P-1178, as detailed in Table 11. As shown by the data in FIG. 8, the combined revertant mutations in P-1176 H I69L, H R71T, and H N73S effectively restored PD1-blocking activity, almost comparable to the level of P-0734. Similarly, due to H V48M and H S49G in the combined revertant mutations, the activity of P-1177 was significantly restored, although not as effective as P-1176. However, incorporation of these two revertant mutations ( H V48M and H S49G) into P-1176 did not result in further enhancement of the activity of the resulting antibody P-1178 (P-1178 compared to P-1176 in FIGS. 8 and 12).

[0320] Table 12 PD1-blocking activity of exemplary PD1-blocking antibodies

[0321]

[0322] The PD1-blocking activity of P-1198 ( H N73S), P-1199 ( H R71T, H N73S), and P-1201 ( H I69L, H R71T, H N73S) was further evaluated by comparing H I69L, H R71T, and HThe importance of each of the three FR revertant mutations, I69L, R71T, and N73S. As shown in Figure 9, each additional revertant mutation led to a slight but significant cumulative increase in PD1 blockade activity. Only the combination of all three revertant mutations in P-1201 led to an almost fully restored functional activity (the EC 50 values of P-1201 and P-0734 were 1.28 nM and 0.78 nM, respectively). Thus, all three revertant mutations H I69L, H R71T, H and N73S were considered essential and were incorporated.

[0323] In addition, the PD1 inhibitory activities of P-1194, P-1201, and P-1238 were compared and are illustrated in Figures 10A and 10B. P-1194 and P-1201, which differ only by an additional CDR germline substitution H F59Y, exhibited the same PD1 blockade potency. This indicates that this particular substitution did not have a negative impact on activity, which contradicts the previous observation that the F59Y germline substitution was detrimental when the IGHV1-2 germline sequence was used H . Thus, it is assumed that the impact of a single CDR germline substitution depends on the context of the surrounding framework sequence. P-1238 had the same potency as the reference antibody P-0734, with EC 50 values of 0.73 nM and 0.70 nM, respectively. Compared to P-1194, two additional framework revertant mutations, H V48M and H S49G in P-1238 led to a slight but discernible improvement in activity.

[0324] In a final assessment, the binding strength of P-1174, P-1193, P-1198, P-1199, and P-1201 to PD1 + cells was evaluated (Figure 11). As expected, P-1174, which fully retained PD1 blockade potency (Figures 6C and 6D), exhibited the same binding affinity for PD1-expressing cells as the reference antibody P-0734 (Figures 11A and 11B). P-1198, P-1199, and P-1201, which contained 1 - 3 framework revertant mutations, showed minor but significant differences in potency in blocking the PD1 interaction (Figure 9), but no such changes in activity were detected in the cell-based binding assay. All three compounds showed the same binding to PD1 +Cell-binding ability (Figure 11C and Figure 11D and Table 13). However, the cell-based binding assay was able to distinguish P-1193 without framework revertant mutations from other compounds, as shown in Figure 11C and Figure 11D and Table 13. However, the degree of reduction was not as obvious as that observed in the blocking assay. The data also confirmed our previous observation that the mechanism-based PD1 / PD-L1 blocking assay was more sensitive than the binding assay in identifying subtle activity differences.

[0325] Table 13

[0326] Exemplary optimized PD1-blocking antibodies bind to PD1 + with cell-binding strength

[0327]

[0328] In summary, the optimized PD1-blocking antibodies P-1194, P-1201, and P-1238, which are based on the VH framework (IGHV3-23), have significantly lower sequence homology but superior biophysical properties, can fully or almost fully retain the functional activity of the antibody, and exhibit improved similarity scores to the closest human germline sequence (IGHV3-23). The mutation details and similarity scores for each antibody are summarized in Table 14.

[0329] Table 14

[0330] Germline substitutions, FR revertant mutations, and similarity scores to the closest human germline sequence of exemplary optimized PD1 antibodies

[0331]

[0332] Example 6

[0333] Germline substitutions result in reduced hydrophobicity of the optimized PD1-blocking antibodies

[0334] Among the 23 FDA- and EMA-approved therapeutic mAbs, pembrolizumab is the most hydrophobic one and thus has the highest aggregation propensity (Goyon et al., J. Chromatogr. B 1065-1066:35-43, 2017). Consistent with the experimentally determined apparent hydrophobic interaction chromatography (HIC) retention factor (k), the SSH2.0 hydrophobicity prediction tool (http: / / i.uestc.edu.cn / SSH2 / ; Zhou et al., Front. Genet. 13:842127, 2022) indicates that both variable chains of pembrolizumab carry a significant risk of hydrophobic interaction. The probabilities of hydrophobic interaction for its VH and VL are 0.97 and 0.61, respectively. If the probability is 0.5 or greater (1 being the maximum possible value), the antibody is predicted to have a high risk of hydrophobic interaction.

[0335] While the focus of germline substitution is to enhance the degree of "humanity" of the antibody sequence, this process also results in a significant reduction in the probability of hydrophobic interaction for a variety of optimized antibody sequences. Table 15 provides a summary of the predicted probabilities of hydrophobic interaction for the variable domains of exemplary optimized PD1-blocking antibodies as estimated by SSH2.0.

[0336] Table 15

[0337] Summary of exemplary antibodies with improved similarity scores and reduced probabilities of hydrophobic interaction while retaining PD1-blocking activity

[0338]

[0339] As shown in Table 15, two light-chain CDR germline substitutions L K27Q and L L54R significantly reduced the hydrophobicity probability of VL from 0.607 of P-0734 to 0.131. These two amino acid changes were applied to the VL in all the optimized PD1-blocking antibodies listed in Table 15. The CDR germline substitutions in the heavy chain only resulted in a minor reduction in hydrophobicity, with the hydrophobicity probability changing from 0.971 of (P-0734) to 0.848 of (P-1174), and the antibody with VH based on the VH-3 family framework changing to approximately 0.8. However, when the germline substitutions ( H V9A, H T76S, H K82aS, H Q83R, H F84S, HWhen the hydrophobicity probability of the resulting construct P-1271 was 0.185 at T108L, it was much lower than that of P-1174.

[0340] Hydrophobic patches on the surface of antibodies typically contribute significantly to their tendency to aggregate. In addition, these hydrophobic patches can lead to high viscosity. Therefore, exemplary PD1-blocking antibodies with a significantly reduced hydrophobic potential are expected to exhibit improved biophysical properties. It is anticipated that PD1-targeted IL-2 immunocytokines and VitoKine fusions constructed using these optimized PD1-blocking antibodies will also have an enhanced developability profile.

[0341] Example 7

[0342] Identifying the optimal IL-2Rα Sushi variants as the masking domain of IL-2 VitoKine

[0343] A representative PD1 Ab IL-2 VitoKine construct is shown in Figure 3. Monomeric IL-2 or an IL-2 variant as the active domain (D2) is fused between the PD1 antibody (D1) and the IL-2Rα Sushi domain as the masking domain (D3). The linker 2 (L2) connecting IL-2 and IL-2Rα is protease-cleavable. The IL-2 within the VitoKine construct will remain inert until it is locally activated by proteases uniquely present or upregulated at the tumor site or within the tumor microenvironment (TME). After cleavage of the L2 linker, the masking α-subunit ideally dissociates, as Figure 2 illustrated. Therefore, it is desirable to identify IL-2Rα variants with reduced binding to IL-2 to ensure that the masking α-subunit (D3) can diffuse away easily after proteolysis but still effectively masks the activity of the IL-2 domain before the linker is cleaved.

[0344] IL-2Rα Sushi variants were designed to weaken binding to IL-2 by incorporating mutations at residues that interact with IL-2. As listed in Table 16, exemplary IL-2Rα Sushi variants P-0751, P-0752, and P-0753 contain Y43A, L42G, and R36A mutations, respectively. They were expressed as monomeric Fc fusion proteins by fusing to the knob Fc chain of a knob-into-hole heterodimeric Fc chain pair (SEQ ID NOs: 187 and 188). P-0757 is an Fc fusion of monomeric wild-type IL-2Rα Sushi. The binding ability of these three IL-2Rα Sushi variants to IL-2 was evaluated using ELISA.

[0345] Briefly, the IL-2Rα Sushi variant Fc fusion protein was coated onto the wells of a Nunc Maxisorp 96-well microplate at 1 μg / well. After incubation overnight at 4°C and blocking with 1% BSA, serial dilutions of P-0689 (a monomeric wild-type IL-2 equivalent (containing the active neutral C125I mutation; SEQ ID NO: 117) Fc fusion) were added to each well at 100 μL / well. After incubation for one hour at room temperature, 100 μL / well of biotinylated anti-IL-2 antibody clone B33-2 (BD biosciences) was added and incubated for 1 hour at room temperature. Subsequently, 100 μL / well of streptavidin-HRP (BioLegend) was added at a dilution of 1:5000. After incubation for 60 minutes and washing, 100 μL / well of TMB substrate (ThermoFisher) was added. The plate was sealed and incubated at room temperature in the dark. The reaction was stopped by adding 2N sulfuric acid (Ricca Chemical). Absorbance at 450 nm was determined and curves were fitted using GraphPad Prism software.

[0346] As summarized in Table 16 and Figure 12 as illustrated therein, the amino acid substitutions Y43A, L42G, and R36A each affect the interaction with IL-2. The Y43A change results in a moderate decrease in IL-2 binding (8.1-fold), the R36A substitution results in a 50 significant 346-fold decrease in binding, while the L42G alteration results in a moderate or 35-fold decrease in its binding to IL-2.

[0347] Table 16

[0348] Effect of IL-2Rα Amino Acid Changes on Binding to IL-2

[0349]

[0350]

[0351] The above three IL-2Rα Sushi variants, together with their wild-type counterparts, were used as masking domains to construct four Fc IL-2 VitoKine molecules. Each of these molecules contains the monomeric IL-2 C125I variant (equivalent to wild-type; SEQ ID NO:117) as the active domain (D2) and the MMP2 / 9-cleavable L2 linker (SEQ ID NO:84) of 15 amino acids that connects IL-2 to IL-2Rα Sushi (D3). The heterodimeric Fc chains (SEQ ID NO:187 and 188) were used as the D1 domain. Subsequently, the masking efficiency of these variants was evaluated by assessing their potency to induce the expression of Ki67, a marker of cell proliferation, in CD8+ T cells and NK cells via human PBMC assays. P-0704, an IL-2 P65R variant (SEQ ID NO:118) Fc fusion that retains its wild-type IL-2 potency towards the dimeric IL-2Rβγ receptor, was included as a fully active IL-2 control for this group of Fc IL-2 VitoKine.

[0352] Briefly, human PBMC were isolated from buffy coats purchased from the Blood Oklahoma Institute by Ficoll-Hypaque centrifugation. The purified human PBMC were treated with serial dilutions of the test compounds and incubated at 37 °C for 5 days. On the fifth day, the cells were washed once with FACS buffer (1% FBS / PBS) and first stained with an Fc blocker (BioLegend) and surface marker antibodies including anti-human CD56-FITC and anti-human CD8-APC (BioLegend) diluted 1:50. After a 30-minute incubation and wash, the cell pellet was resuspended thoroughly with 200 μL / well of 1x fixation and permeabilization working solution (Invitrogen) and incubated at room temperature in the dark for 30 minutes. After centrifugation, 200 μL of 1x permeabilization buffer (Invitrogen) was added to each well for another wash. The cell pellet was resuspended at a 1:25 dilution in permeabilization buffer containing anti-human Ki67-PE (BD Life Sciences). After an additional 30-minute incubation at room temperature, the cells were collected, washed, and resuspended in FACS buffer and analyzed by flow cytometry. Data are represented as the percentage of Ki67-positive cells within the gated population. The dose-response Ki67 proliferation profiles are shown in Figures 13A and 13B. Additionally, CD8+ T cell-specific data are summarized in Table 17.

[0353] Table 17

[0354] Comparison of the activities of various IL-2 VitoKine constructs

[0355]

[0356]

[0357] Figure 13 shows that, compared to P-0704 (the fully active IL-2Fc fusion counterpart of P-0701), P-0701, which has wild-type IL-2Rα Sushi as the shielding partial domain (D3), shows a significant 3-log decrease in inducing CD8+ T cell and NK cell proliferation. We hypothesized that integrating IL-2 binding disrupting mutations into the D3 domain might weaken the shielding ability of D3, thereby reducing the activity inertness of VitoKine. It was also predicted that the degree of this reduction would be consistent with the level of decrease in the binding strength between IL-2 and the IL-2Rα Sushi variant.

[0358] In Figure 13A and Table 17, compared to P-0701, the Fc VitoKines P-0754 and P-0756, which contain Y43A and R36A in the IL-2Rα Sushi as the D3 domain, exhibit weakened shielding ability. This results in a higher VitoKine intrinsic basal level in stimulating CD8+ T cell proliferation, and this trend is consistent in NK cells, as shown in Figure 13B. However, the decrease in shielding efficiency is not consistently correlated with the magnitude of the decrease in binding strength. For example, the Y43A mutation has a small effect on binding, showing only an 8.1-fold decrease, while the R36A mutation results in a significant ~200-fold decrease in binding. In addition, although the L42G variant has 35-fold weaker binding to IL-2, it still maintains its shielding effect almost the same as its wild-type counterpart, as seen in the activity inertness of its corresponding VitoKine P-0755 (Figure 13A and Figure 13B). Although this was unexpected based on previous knowledge, multiple experiments confirmed that changes in binding strength caused by mutations in IL-2Rα Sushi are not consistently correlated with alterations in its shielding ability. This inconsistency may be attributed to unique spatial interactions in the VitoKine form.

[0359] Therefore, the IL-2Rα Sushi L42G variant was selected as the preferred shielding partial domain (D3) of IL-2 VitoKine because of its retained shielding ability to maintain the activity inertness of the corresponding VitoKine, and in view of its weakened binding to IL-2, its potential to diffuse away easily upon proteolysis in vivo to achieve full activity. Meanwhile, when aiming to regulate the inherent basal activity of IL-2 VitoKine and optimize the balance between the desired anti-tumor efficacy and potential systemic toxicity, R36A or Y43A can be used as the shielding partial domain. Additionally, following the same basic principle, other IL-2Rα Sushi variants that differ in the degree of reduced binding to IL-2, such as K38E, can be used as the D3 domain of IL-2 VitoKine.

[0360] Example 8

[0361] Replacing the amino acid at P65 surprisingly had different effects on binding to IL-2Rα

[0362] Due to the high and constitutive expression of IL-2Rα on regulatory T cells (Tregs), the preferential expansion of Tregs by IL-2 represents an undesirable effect of IL-2 for cancer immunotherapy. IL-2 variants designed to attenuate or eliminate binding to IL-2Rα will reduce their responsiveness to Tregs. IL-2 variants that no longer bind to IL-2Rα are expected not to preferentially activate Tregs, but only to activate Tregs at concentrations when CD8+ T cells and NK cells are also activated.

[0363] The P65 residue of the IL-2 molecule makes van der Waals interactions with key residues at the IL-2Rα interface, especially R36 and L42. However, it does not form salt bridges or hydrogen bonds with IL-2Rα (Xinquan Wang, et al., Science (2005), 310:1159-1163). Given this, it can be hypothesized that changes in P65 would only slightly alter its interaction with the IL-2Rα subunit and may only have a minimal impact on binding. However, the actual effects of P65 modification on its interaction with IL-2Rα are surprisingly diverse, ranging from fully maintaining binding or even improving binding to weakening binding or completely eliminating binding.

[0364] A set of IL-2 variants with different P65 substitutions are fused to Fc in dimer or monomer form via a flexible GS-based linker (SEQ ID NO:103). All of these variants contain the active neutral C125I mutation, intended to enhance their developability. Then their binding affinities to IL-2Rα (CD25) were evaluated using ELISA. Briefly, IL-2Rα-ECD was coated onto wells at 0.1 μg / well. After incubation overnight at 4 °C and blocking, serially diluted IL-2Fc fusion proteins were added to each well at 100 μL / well. After incubation for 1 hour at room temperature, 100 μL / well of goat anti-human IgG Fc-HRP (diluted 1:5000 in diluent) was added to each well and incubated for 1 hour at room temperature. After adding 100 μL of TMB substrate, the plate was developed at room temperature in the dark for 10 minutes, and 100 μL / well of stop solution was added. The absorbance at 450 nm was determined, and the curve was fitted using Prism software (GraphPad).

[0365] The ELISA binding curves are shown in Figure 14. In addition, the ELISA binding EC 50 values of the IL-2 variants were normalized against the ELISA binding EC 50 values of the wild type (P-0531 or P-0689, depending on the valency of each construct), as detailed in Table 18.

[0366] Table 18

[0367] The binding of IL-2 variants with P65 mutations to IL-2Rα was evaluated by ELISA

[0368]

[0369] As illustrated in Figures 14A and 14B, the P65G mutation in P-0608, the P65E mutation in P-0633, and the P65A mutation in P-0706 do not seem to impair the interaction with the IL-2Rα subunit. Instead, when compared to their respective wild-type IL-2 controls, these mutations enhance the binding affinity to IL-2Rα by 18-fold, 10-fold, and 10-fold, respectively.

[0370] In another group, the IL-2 variant Fc fusions, namely P-0634, P-0708, and P-0709, carry P65 alterations that result in different levels of interference with binding to the IL-2Rα subunit. As shown in Figure 14C and detailed in Table 10, the P65N mutation in P-0708 leads to a moderate 8.6-fold decrease in IL-2Rα binding. On the other hand, the P65H (P-0634) and P65Q (P-0709) alterations have a more pronounced effect, resulting in 23-fold and 43-fold decreases in binding, respectively.

[0371] Another set of P65 substitutions, particularly P65R and P65K, manifested as causing a drastic disruption in the IL-2 and IL-2Rα interaction, completely abolishing the binding of P-0635, P-0704, and P-0707 to IL-2Rα. Here, P-0635 and P-0704 are the dimeric and monomeric forms of the IL-2 P65R variant Fc fusion, and P-0707 has the P65K amino acid change. Figure 14D reveals that even at IL-2Rα concentrations up to 100 nM, there is little to no detectable binding signal for these three IL-2 mutant protein Fc fusions. This is comparable to a benchmark molecule carrying three CD25 disrupting mutations F42A / Y45A / L72G known to abolish binding, as documented by Christian Klein et al., OncoImmunology (2017), 6:3, e1277306.

[0372] In summary, alterations in the P65 residue result in a diverse range of effects on IL-2Rα binding, including increased, maintained, decreased, or complete elimination of the binding of the resulting IL-2 variants to IL-2Rα. Such a broad spectrum of outcomes caused by the modification of seemingly non-critical amino acids cannot be predicted by structure-based mutagenesis approaches. The complete loss of IL-2Rα binding was unforeseen and not predictable by the prior art. This is particularly surprising given that the P65 mutation modifies only a small portion of the van der Waals interaction surface.

[0373] Changes in IL-2Rα binding strength were expected to correlate with the IL-2 potency in activating Treg cells. To verify this, the ability of IL-2 variant Fc fusion proteins with enhanced (P-0608), reduced (P-0634 and P-0709), or abolished (P-0635 and P-0704) binding to IL-2Rα to differentially stimulate STAT5 phosphorylation in CD4+ Treg cells was examined. STAT5 is known to be involved in the downstream signaling cascade after IL-2 binds to the transmembrane IL-2 receptor. The wild-type IL-2 fusion P-0531 and the benchmark molecule were included for comparison.

[0374] Measure phosphorylation of STAT5 in lymphocyte subsets in fresh human PBMCs using the transcription factor FOXP3 to identify the Treg population in FACS analysis. Specifically, purified PBMCs are serum-starved in MACS buffer (Miltenyi Biotech) at 4 °C for 1 hour and subsequently treated with serial dilutions of the test compound at 37 °C for 30 minutes. The cells are then fixed, permeabilized, stained with specific antibodies, and further analyzed by flow cytometry according to a similar procedure detailed in Example 7. Staining is achieved using a mixture of anti-CD25-PE, anti-FOXP3-APC, anti-pSTAT5-FITC, and anti-CD4-PerCP-Cy5.5 antibodies (purchased from BioLegend or BD Life Sciences). Flow cytometry data for the Treg cell subset are gated as CD4+ / Foxp3+ / CD25 高 group. Data are represented as the percentage of pStat5-positive cells in the gated population.

[0375] As illustrated in Figure 15, there is a clear correlation between IL-2Rα binding strength and the potency to stimulate STAT5 phosphorylation in CD4+ Treg cells. The compounds in Figure 15A are all characterized by bivalent IL-2 variants, while the compounds in Figure 15B all have monomeric IL-2 variants. Compared to P-0531, P-0608 with enhanced IL-2Rα binding exhibits significantly higher potency. Compared to P-0531 / P-0689, P-0626 (Figure 15A) and P-0709 (Figure 15B), consistent with their reduced IL-2Rα binding, show reduced pSTAT5 potency. However, their remaining (albeit reduced) binding to IL-2Rα ensures more effective activation of Tregs than both P-0635 / P-0689 and the benchmark molecule (valence-matched) that have completely lost IL-2Rα binding. Similarly, complete loss of IL-2Rα binding results in a significant shift of Treg potency to the right by approximately 5 logs. This remaining Treg signaling is caused by the activation of IL-Rβγ found on Treg cells.

[0376] In addition, exemplary IL-2 variant Fc fusions that contain mutations that enhance, reduce, or eliminate binding to IL-2Rα all show unaltered binding to IL-2Rβγ (Figure 16A). They also show almost equal potency in inducing Ki-67 expression in CD8+ T cells (Figure 16B). The data highlight the fact that IL-2 mutations at the IL-2Rα interface, regardless of their effect on IL-2Rα binding, do not alter the interaction with IL-2Rβγ.

[0377] Example 9

[0378] Identification of IL-2 variants with optimal IL-2Rα binding as the active domain structure of VitoKine

[0379] The active domain (D2) of the IL-2 VitoKine construct is selected from a group of IL-2 variants identified in Example 8 with different levels of binding strength to IL-2Rα. Incorporating IL-2 variants with reduced or eliminated IL-2Rα binding in VitoKine can reduce the reactivity to Tregs after proteolytic activation near the tumor. However, since the binding between D2 and D3 is considered necessary for the shielding ability of VitoKine, a balance must be achieved between the reduced degree of IL-2Rα binding and the shielding efficiency.

[0380] Four exemplary IL-2 VitoKines, namely P-0800, P-0830, P-0831, and P-0802, all contain the anti-mouse PD1 antibody P-0722 (SEQ ID NO:52, 189, and 190) as the D1 domain, the IL-2Rα Sushi L42G variant (SEQ ID NO:184) as the shielding domain (D3), the non-cleavable linker (SEQ ID NO:103) as the L1 linker, and the MMP-2 / 9 cleavable linker (SEQ ID NO:84) as the L2 linker. As detailed in Table 19, the active domain (D2) contains the P65R mutation in P-0800, the P65N mutation in P-0830, and the P65Q mutation in P-0831. P-0802 has the IL-2 wild-type equivalent as the D2 domain.

[0381] The ability of the exemplary VitoKines to induce Ki67 expression on CD8+ T cells (Figure 17A) and NK cells (Figure 17B) was evaluated using fresh human PBMCs. P-0782 is the non-VitoKine immunocytokine counterpart of P-0800 and contains the monomeric IL-2 P65R variant and was included as a fully active IL-2 reference. The EC 50 values of these compounds in stimulating Ki67 expression in NK cells, as well as the fold changes compared to P-0782, are summarized in Table 19.

[0382] Data revealed that when the D2 domain was wild-type IL-2, the D3 domain of the VitoKine construct (P-0802) caused a roughly 3-log decrease in activity, indicating a strong shielding ability of the D3 domain. In contrast, for P-0800 incorporating the IL-2 variant (P65R) with IL-2Rα binding elimination, the activity decreased only 10- to 20-fold, indicating a significant attenuation of the shielding ability of D3 due to the absence of binding between the D2 and D3 domains. Intriguingly, when IL-2 variants with moderately reduced IL-2Rα binding (such as P65N and P65Q) were used in P-0830 and P-0831, the D3 domain provided a shielding efficiency equivalent to or only slightly lower than that promoting wild-type IL-2.

[0383] Table 19

[0384] Activity comparison of IL-2 VitoKine containing IL-2 variants with different IL-2Rα binding strengths

[0385]

[0386] It is hypothesized that the binding affinity threshold between the D2 and D3 domains and the ideal spatial configuration of the binding interface are crucial in determining the shielding efficiency of IL-2 VitoKine. Given that the IL-2 P65Q variant exhibits a significantly reduced binding strength to IL-2Rα (as depicted in Figure 14C and Table 18), and is still effectively shielded by IL-2Rα Sushi L42G to remain inert as a VitoKine, the IL-2 P65Q variant was selected as the preferred D2 domain for IL-2 VitoKine design. Due to the reduced binding between the D2 and D3 domains, the D3 domain is expected to diffuse away easily after protease cleavage. It is also expected that once the biological activity is fully restored after proteolytic activation, this variant will have a significantly reduced ability to stimulate Treg cells compared to wild-type, as illustrated in Figure 15B. However, other IL-2 variants with reduced IL-2Rα binding, such as P65H and P65N, can also be considered to achieve the right balance between the desired anti-tumor efficacy and minimized potential systemic toxicity.

[0387] In addition to the P65 mutation to achieve a balance between reduced binding and effective shielding by IL-2Rα Sushi, additional mutations that alter the binding affinity of IL-2 for IL-2Rβγ can be incorporated into the D2 domain of VitoKine. These mutations regulate the overall response of IL-2 in cells that predominantly express the β and γ receptor subunits, such as CD8+ T cells and NK cells. By this strategy, the intrinsic basal activity of VitoKine as well as their activity after proteolytic activation can be fine-tuned.

[0388] Example 10

[0389] IL-2 variants with IL-2Rβγ disrupting substitutions for overall potency attenuation

[0390] Information on the selection of mutations that disrupt the IL-2Rβ and common γ chain (γc) was obtained by examining the IL-2 / IL-2R co-crystal structure (PDB code 2B51). Substituting energy hot spot residues that directly interact with IL-2Rβ, such as D20 and N88, may result in a significant reduction in activity, making the potency poor. Therefore, substitutions were introduced at non-critical residues such as L19 at the IL-2 / IL-2Rβ interface. The L19 residue only makes van der Waals interactions with IL-2Rβ, and the resulting mutant is expected to only slightly modulate rather than significantly reduce the functional activity of IL-2. In addition, replacing L19 with a non-aliphatic residue eliminates the proposed " 19 LDL" motif (Baluna R, Rizo et al., Proc Natl Acad Sci 1999; 96:3957-62) that may contribute to the vascular toxicity of IL-2.

[0391] Exemplary IL-2Rβ disrupting mutations L19H, L19Q, L19Y were introduced into IL-2 together with the mutation backgrounds of P65R and C125I in P-0704 to construct monomeric IL-2Fc fusions. The P65R mutation results in complete loss of IL-2Rα binding, and the C125I modification was made for developability purposes, and neither of these changes affects the functional activity of IL-2 on IL-2Rβγ. The resulting fusion proteins, namely P-0731, P-0759, and P-0761, were evaluated by flow cytometry for their potency to stimulate Ki67 expression on human CD8+ T cells and NK cells. The results are depicted in FIGS. 18A and 18B and detailed in Table 20A. When compared to P-0704, all variants showed a reduced ability to promote the proliferation of human CD8+ T cells and NK cells. Specifically, P-0759 (L19Q) and P-0761 (L19Y) exhibited a moderate 3-fold decrease in potency, while the L19H mutation in P-0731 resulted in a more significant 18- to 25-fold decrease in potency. A decrease in potency could also be achieved by incorporating other L19 mutations such as L19D, L19R, and L19S.

[0392] Table 20A

[0393] Exemplary Fc fusions of IL-2 variants containing L19 mutations and their ex vivo activities

[0394]

[0395] Similarly, amino acid substitutions were made at Q126 (a residue indispensable for interaction with γc) to attenuate the interaction of IL-2 with γc. All of these mutations were also introduced into IL-2 in the mutation background of P65R and C125I. Fc fusions of monomeric IL-2 variants containing Q126 mutations are listed in Table 20B.

[0396] Compared to P-0704, the increased Ki67 expression of human CD8+ T cells and NK cells in response to these IL-2 variants is depicted in FIGS. 19A to 19F and also summarized in Table 20B. The IL-2 Q126 mutations have varying effects on CD8+ T cell and NK cell proliferation. For CD8+ T cells (FIGS. 19A, 19C, and 19E), Q126N, Q126H, Q126M, Q126F, Q126W, and Q126Y showed a slight 1.5- to 5-fold decrease; a moderate 5- to 20-fold decrease was observed for Q126R, Q126G, Q126S; a significant 20- to 50-fold decrease was noted for mutations including Q126A, Q126V, Q126E, Q126E, Q126L, and Q126T; a more drastic decrease (>50-fold) was observed for Q126P and Q126I; and Q126D completely abolished activity. Due to the Q126 mutations, a comparable trend in potency change was observed in NK cells (FIGS. 19B, 19D, and 19F).

[0397] Table 20B Fc fusions of IL-2 variants containing Q126 mutations and their ex vivo activities

[0398]

[0399] * Fold change is compared to the EC 50 value of P-0704 in each individual experiment.

[0400] IL-2 potency can be further fine-tuned by combining IL-2Rβ and γc disrupting mutations, as shown for P-1247 (IL-2 domain SEQ ID NO: 173) compared to P-1158 and P-0704. In addition to the P65R and C125I mutations in P-0704, P-1158 contains the Q126N mutation, and P-1247 contains the L19Y and Q126N mutations. As shown in FIG. 20A, for stimulating Ki67 expression in human CD8+ T cells, incorporation of the L19Y mutation in P-1247 resulted in an additive 2.6-fold decrease in potency compared to P-1158 (9.2 nM vs. 3.6 nM), and a 4-fold decrease in combined potency compared to P-0704 (9.2 nM vs. 2.3 nM). A similar trend was observed with NK cells (FIG. 20B). As will be understood by those skilled in the art, combining different mutations at positions L19 and Q126 can result in varying degrees of activity modulation and is within the spirit and scope of the present invention.

[0401] In summary, in addition to using IL-2Rα disruption substitution in IL-2 to limit the undesired expansion of immunosuppressive Tregs, integrating IL-2Rβγ disruption substitution provides a way to attenuate the overall potency to achieve optimal activity. Different degrees of potency can be achieved by introducing specific mutations at L19 or Q126. The desired potency of IL-2 can be finely tuned by combining mutations at the L19 and Q126 positions. The reduced potency helps avoid over-activation of this pathway and minimizes undesired target sinking. Therefore, this strategy can potentially reduce the toxicity associated with IL-2 treatment and improve pharmacokinetics and pharmacodynamics. Incorporating potency-reduced IL-2 into VitoKine helps fine-tune its inherent basal activity as well as its activity after proteolytic activation.

[0402] Example 11

[0403] Construct PD1 Ab-IL-2 VitoKine using an optimized PD1-blocking antibody and a preferred IL-2 and IL-2Rα Sushi domain

[0404] Antibodies that block PD1 and thus bypass the immunosuppressive effects in the tumor microenvironment can enhance the IL-2 response and further potentiate immunity against tumors. The PD1 antibody used as the D1 domain to construct PD1 Ab-IL-2 VitoKine is selected from optimized human PD1-blocking antibodies comprising the light chain sequences listed in SEQ ID NO:44 and the heavy chain sequences listed in SEQ ID NO:45-49. These optimized PD1-blocking antibodies have high affinity for the human PD1 protein and exhibit potency equal to or comparable to pembrolizumab in blocking PD1. They also have a higher sequence similarity score to their closest human germline sequences, resulting in a higher degree of human origin compared to pembrolizumab. In addition, they are predicted to have lower hydrophobicity, which in turn may reduce their tendency to aggregate compared to pembrolizumab. The PD1-targeted IL-2 VitoKine constructed using these optimized PD1-blocking antibodies is also expected to have an enhanced developability profile.

[0405] Table 21 lists exemplary PD1 Ab-IL-2 VitoKines, the structures of which are depicted in Figure 3A. All exemplary VitoKines contain an IL-2 P65Q variant with or without mutations that modulate activity towards IL-2Rβγ as the active partial domain (D2), an IL-2Rα Sushi L42G variant as the shielding partial domain (D3), and a cleavable L2 linker (SEQ ID NO:84) that connects the D2 domain to the D3 domain. However, when it is desired to modulate the intrinsic basal activity of the IL-2 VitoKine, other IL-2Rα Sushi variants, such as R36A, can be used as the shielding partial domain. Additionally, the L1 linker that connects the PD1 Ab and IL-2 can also be cleavable. The composition of the cleavable linker can be further optimized by using the various sequences listed in SEQ ID NOs:78-94 to better suit different disease indications and / or stages.

[0406] Table 21A

[0407] Exemplary human PD1 Ab-IL-2 VitoKine

[0408]

[0409] All genes were codon-optimized for expression in mammalian cells, synthesized via GenScript's services, and subcloned into recipient mammalian expression vectors. VitoKine constructs were generated by co-transfecting Expi293 cells (ThermoFisher) with the mammalian expression vector according to the manufacturer's instructions. Protein purification and characterization were performed according to the same procedures detailed in Example 2.

[0410] PD1 Ab-IL-2 VitoKines containing optimized antibody sequences (including P-1197, P-1239, and P-1272) were found to be expressed at significantly higher levels than P-1120 containing the reference antibody P-0734. Under the same transient expression conditions using the same batch of Expi293 cells, P-1197, P-1239, and P-1272 were expressed at titers of 137-150 mg / L, compared to a titer of 60 mg / L for P-1120. The data suggest that PD1-blocking antibodies with optimized sequences that eliminate potential sequence defects may lead to improved developability characteristics for the corresponding VitoKine constructs.

[0411] Because these optimized PD1 antibodies do not react with murine PD1, surrogate murine PD1-Ab-IL-2 VitoKines and additional controls were similarly generated. These were used for in vivo studies, particularly for pharmacokinetics (PK) / pharmacodynamics (PD) and tumor experiments in immunocompetent mice. Table 21B provides detailed information on these molecules. Each VitoKine construct listed in this table incorporates the same D3 domain (SEQ ID NO:184). Except for P-0871, these VitoKines contain the murine PD1 antibody P-0722 (SEQ ID NO:189, 190, and 52) as the D1 domain. P-0871 is the non-targeting VitoKine counterpart of P-0831 and contains the germline antibody P-1260 (SEQ ID NO:192, 193, and 194) as the D1 domain. P-0877 is a non-cleavable VitoKine, and P-0838 lacks the L2 linker and D3 and its structure is shown in Figure 3B and serves as the non-VitoKine immunocytokine counterpart of P-0831.

[0412] Table 21B

[0413] Exemplary surrogate murine PD1 Ab-IL-2 VitoKines and control molecules

[0414]

[0415] While P-0831 was the main subject for in vivo studies in the following examples, when appropriately dosed, it is predicted that other VitoKines containing IL-2 variants with additional mutations targeting IL-2Rβγ (such as those listed in Table 21B) can achieve similar anti-tumor efficacy. Since the intrinsic basal activity of the IL-2 VitoKine is directly related to the activity of its active domain (D2), the attenuated D2 activity and the proportionally regulated basal activity of the VitoKine enable the administration of higher doses without adverse effects. This helps to fully support the function of the PD1 antibody in reversing T cell anergy or exhaustion, thereby potentially enhancing the synergistic effect with IL-2 immunotherapy and expanding the therapeutic window.

[0416] Example 12

[0417] Ex vivo activity and in vitro proteolytic activation of PD1 Ab-IL-2 VitoKine

[0418] It is important that the PD1 antibody retains its binding and functional activity when incorporated into PD1 Ab-IL-2 VitoKine. PD1 antibodies with superior target binding and PD1 blocking function can enhance the specificity and selectivity of TIL targeting, and further synergize with the IL-2 anti-cancer immune response by efficiently reversing T cell anergy and exhaustion.

[0419] In a comparative analysis using luciferase reporter assays, the PD1 inhibitory capabilities of exemplary VitoKine P-1197, P-1239, and P-1272 were set against their respective PD1 blocking antibodies P-1174, P-1238, and P-1271. As illustrated in FIGS. 21A and 21B, each of the three antibodies, when incorporated into their corresponding VitoKine constructs, not only maintained their blocking potency but also slightly improved it. For example, PD1 antibodies P-1174, P-1238, and P-1271 block the PD1 / PD-L1 interaction, with EC 50 values of 1.29 nM, 1.82 nM, and 1.53 nM, respectively. On the other hand, their corresponding VitoKine P-1197, P-1239, and P-1272 showed a 1.5 - 2-fold subtle enhancement in blocking efficiency, with EC 50 values of 0.92 nM, 1.27 nM, and 1.20 nM, respectively. Additionally, in VitoKine form, both E max and the fold induction increased by 11% - 17%.

[0420] FIG. 22 also confirmed that the activity of IL-2 remained effectively masked by the IL-2Rα Sushi domain, regardless of the specific PD1 antibody composition. Exemplary PD1 Ab IL-2 VitoKine P-1197, P-1272, and P-0872 (which differ only based on their different PD1 blocking antibodies) (see in detail in Table 21A) showed approximately 300-fold reduced ability to induce CD8+ T cell and NK cell proliferation when compared to P-0879 (FIGS. 22A and 22B) or P-1273 (FIGS. 22C and 22D). For background, P-0879 and P-1273 are the non-VitoKine immunocytokine counterparts of P-0872 and P-1272 lacking the masking D3 domain. EC 50 values are shown in Table 22. For CD8+ T cells, the low potency of VitoKine prevented curve fitting, and only approximate EC 50 values were obtained. P-1174 is the PD1 antibody component of P-1197 and was included as a negative control.

[0421] Table 22

[0422] EC of exemplary PD1 Ab-IL-2 VitoKine compared to its non-VitoKine immunocytokine counterpart 50 value

[0423]

[0424] Further evaluate the in vitro proteolytic activation of P-1272. Consistent with other exemplary PD1 Ab-IL-2 VitoKines in the present invention, P1272 contains a single MMP-2 / 9 cleavable L2 linker (SEQ ID NO:84). In the procedure, first, 3.3 μg of latent MMP-2 (BioLegend) was activated with APMA (Millipore Sigma) according to the manufacturer's instructions, then its exchange buffer was changed, and it was added to 120 μg of P-1272 in 0.4 ml of the manufacturer-recommended assay buffer (100 mM Tris, 20 mM CaCl 2 , 300 mM NaCl, 0.1% (w / v) Brij 35, pH 7.5). After incubation at 37 °C for 3 hours, the treated sample was then purified in a binding and elution mode using protein A resin (MabSelect SuRe; Cytiva). The eluted sample was analyzed on a reducing SDS-PAGE gel, and its biological function was evaluated in an ex vivo functional assay.

[0425] Figure 23A shows that the masked domain structure of P-1272 was effectively and completely cleaved, generating P-1272-activated, which corresponds to Figure 2 active form 2 depicted in. Effective in vitro proteolysis led to a complete restoration of IL-2 activity, exemplified by the indistinguishable activities of P-1272-activated and P-1273 in inducing the dose-dependent expression of Ki67 in CD8+ T cells of fresh human PBMCs (Figure 23B). Comparable findings were observed with other PD1 Ab-IL-2 VitoKines, as exemplified by P-0831 and depicted in Figure 23C.

[0426] In addition, P-1345 (a different form of PD1 Ab-IL-2 VitoKine) differs from other VitoKines in that it contains a cleavable L1 linker (SEQ ID NO:84) and an uncleavable L2 linker (SEQ ID NO:115). P-1345 was similarly activated by in vitro protease cleavage, and its only activated form was isolated, in Figure 2is referred to as active form 1. Then, the potency of this form to induce Ki67 expression in CD8+ T cells of human PBMCs was evaluated. As shown in Figure 23D, proteolytic activation led to approximately 50-fold increase in activity compared to its intact VitoKine form. However, the activation did not fully restore the activity of the IL-2 domain, showing its activity to be 6-fold lower than that of its non-VitoKine counterpart P-0838, with corresponding EC 50 values of 8.29 nM and 1.34 nM.

[0427] These results imply that using a cleavable L2 linker is more advantageous than a cleavable L1 linker. This is because active form 2 derived from the cleavage of the L2 linker is a fully functional IL-2 domain fused to a PD1 Ab. This form can activate IL-2R signaling in PD1-expressing T cells near the disease site, enhancing both pathways and synergistically anti-cancer immune responses while reducing systemic toxicity. On the other hand, active form 1 exhibits reduced potency, shorter half-life, and lack of TIL targeting ability.

[0428] These observations also suggest that the masking domain alone is not sufficient to effectively mask the active domain, resulting in a moderate masking efficiency of approximately 6-fold. To effectively mask IL-2 activity, the structure must be in the form of the VitoKine platform disclosed herein and by the present inventors in WO2019246392 and WO2021119516, which includes the conjugation of both a targeting domain (D1) and a masking domain (D3).

[0429] Example 13

[0430] Prolonged in vivo half-life of PD1-Ab-IL-2 VitoKine in non-tumor-bearing mice

[0431] The IL-2 domain of VitoKine was designed to remain inert until locally activated by proteases upregulated in diseased tissues. As a result, it was expected that the binding of IL-2 VitoKine to IL-2 receptors on the cell surface in peripheral and non-lesional tissues would be significantly reduced. This would alleviate potential antigen sink and / or target-mediated deposition, thus prolonging the in vivo half-life. A study was conducted to compare the pharmacokinetics of murine PD1 Ab IL-2 VitoKine P-0831 with its non-VitoKine immunocytokine counterpart P-0838 in non-tumor-bearing C57BL / 6 mice.

[0432] Initial female C57BL / 6 mice at 7 weeks of age were received from Charles River Laboratories. Before the start of the study, they were allowed to acclimatize within the institution for 7 days. At the start, P-0831 and P-0838 were each administered at a dose of 1 mg / kg by intravenous injection. Vehicle (PBS) was included as a negative control. Blood samples were drawn by cheek bleeding at 10 minutes, 2 hours, 6 hours, 24 hours, 48 hours, 72 hours, 120 hours, 168 hours, 240 hours, and 360 hours post-injection. Each group consisted of 3 mice, and blood was drawn once a week or every three days, with a maximum frequency of twice per group.

[0433] Serum concentrations of the compounds were determined using ELISA assays. Three different ELISA methods were developed for P-0831 to measure: 1) total VitoKine concentration (including both the activated and intact forms); 2) concentration of intact VitoKine; and 3) concentration of activated VitoKine. For all three methods, maxisorp plates were coated overnight at 4 °C with mouse PD1 protein (R&D systems). Subsequently, the plates were blocked with Superblock (ThermoFisher). Blood samples at various dilutions were added to the plates and incubated for 1 hour at room temperature.

[0434] For total VitoKine detection, anti-IL-2 goat polyclonal antibody (R&D Systems) was added, followed by a second HRP-conjugated donkey anti-goat IgG (ThermoFisher). To detect intact VitoKine, a polyclonal anti-CD25 antibody (R&D Systems) was used, which was probed with HRP-conjugated streptavidin protein (ThermoFisher). For activated VitoKine detection, a biotinylated monoclonal anti-IL-2 antibody (BD Pharmingen) was applied, paired with HRP-conjugated streptavidin. For P-0838 detection, the same anti-IL-2 goat polyclonal antibody (R&D Systems) used for detecting total VitoKine concentration was used, followed by donkey anti-goat IgG-HRP. The resulting signals were developed using Ultra TMB substrate solution, and values were inferred from non-linear regression curve fitting in GraphPad Prism.

[0435] As Figure 24 shown, the concentration profiles of intact P-0831 and total (including both intact and activated forms) P-0831 aligned closely, indicating that P-0831 circulates primarily in its intact state. Additionally, no evidence of activated P-0831 was detected at any time point post-administration, corroborating the view that P-0831 remains intact peripherally.

[0436] In contrast to the concentration profile of P-0831, which was still measurable 360 hours after a 1 mg / kg dose, the serum concentration of P-0838 declined rapidly. At 72 hours after dosing, P-0838 was significantly reduced and undetectable at 120 hours after dosing. Figure 24 The grey horizontal dotted line in [reference] indicates the lower limit of quantification (LLOQ) of serum P-0838 levels. For measurements below the LLOQ, the value was assigned as 10 - 3 nM.

[0437] These findings strongly support the view that the VitoKine format is superior in prolonging the in vivo half-life of the active domain. The remarkable prolonged in vivo half-life of VitoKine is thought to be caused by the inertness of the IL-2 domain in peripheral blood. This inactivity may reduce the interaction with IL-2 receptors on the cell surface in both peripheral and non-diseased tissues, significantly reducing cell activation and expansion, and thus alleviating potential antigen sink and / or target-mediated deposition.

[0438] Example 14

[0439] Minimized systemic pharmacodynamic effects of PD1-Ab-IL-2 VitoKine in non-tumor-bearing mice

[0440] The VitoKine platform is designed to mitigate target toxicity in the whole body and expand the therapeutic window for cytokine therapy. This is achieved by rendering the active cytokine inert within the construct, which prevents its interaction with receptors in peripheral blood or on the surface of non-diseased cells. This design helps to limit the over-activation of cytokine pathways and reduce the risk of unwanted "on-target" effects at "extra-tissue" locations. To evaluate this hypothesis, murine PD1 Ab-IL-2 VitoKine P-0831 was administered to non-tumor-bearing C57BL / 6 mice to assess its systemic effects compared to its non-VitoKine immunocytokine counterpart P-0838 by monitoring the proliferation and expansion of peripheral blood lymphocytes over a given time period.

[0441] Initial C57BL / 6 mice between 7-9 weeks of age were administered 2 mg / kg and 10 mg / kg of P-0831 and 0.3 mg / kg, 1 mg / kg and 2 mg / kg of P-0838 (n = 4 per group) via a single intraperitoneal injection. Vehicle (PBS) was included as a negative control. Blood samples were collected into heparin tubes on days 0, 3, 5, 7 and 10 after dosing for immunophenotyping.

[0442] Subsequently, heparin-treated blood samples were stained with a panel of antibodies targeting common surface immune cell markers. After lysing red blood cells using BD Pharmingen lysis buffer, the total number of viable mononuclear blood cells was determined by excluding dead cells with trypan blue. The lysed immune cells were then fixed and permeabilized for 30 minutes at room temperature in the dark using fixation / permeabilization buffer (eBioscience). After washing, the cells were intracellularly stained with an antibody against the Ki67 proliferation marker. Different immune cell subsets were identified and their absolute counts in the circulation were quantified using a flow cytometer (Beckton Dickinson). This was done using commercially available antibodies, namely CD3-APC.Cy7, CD8-Percp-cy5.5, CD335-APC, CD45-AF700, CD4-BV421, CD25-BV510, Foxp3-FITC, Ki67-PE, and granzyme B-BV421. Flow cytometry analysis was performed using FlowJo software and the results were plotted using GraphPad Prism.

[0443] In Figure 25, the data revealed that P-0838 significantly amplified peripheral blood CD8+ T cells (Figure 25A) and granzyme B+ CD8+ T cells (Figure 25B) at doses of 1 mg / kg and 2 mg / kg, showing a dose-dependent response. Specifically, with a 2 mg / kg dose of P-0838, CD8+ T cells expanded from a baseline level of 900 cells / μL to 2400 cells / μL on day 3 (a 2.7-fold increase). The expansion peaked on day 5 at 4200 cells / μL (a 4.7-fold increase), and then decreased to near baseline levels on day 7. With a 1 mg / kg dose of P-0838, a peak expansion of CD8+ T cells was observed on day 3, a 2.3-fold increase, and returned to near baseline levels on day 7. At a lower dose of 0.3 mg / kg of P-0838, CD8+ T cell expansion was only slightly evident. A similar trend was observed in the expansion of cytotoxic granzyme B+ CD8+ T cells (Figure 25B). In stark contrast, even at higher doses of 2 mg / kg and 10 mg / kg, VitoKine P-0831 did not show significant expansion of CD8+ T cells or granzyme B+ cells over the entire 10-day period (illustrated in Figures 25A and 25B).

[0444] Consistent with the in vitro assay results, for IL-2 treatment, NK cells demonstrated higher reactivity than CD8+ T cells. This was evident from the significant NK cell expansion observed with a 0.3 mg / kg dose of P-0838 (Figure 25C). NK cell expansion showed a dose-dependence between 0.3 mg / kg and 1 mg / kg doses, with no significant difference between 1 mg / kg and 2 mg / kg doses. Across all three doses of P-0838, the peak NK cell expansion occurred on day 3 (Figure 25C). In the cytotoxic granzyme B + A similar pattern was observed in the expansion of NK cells (Figure 25D). In contrast, even when administered at a relatively high dose of 10 mg / kg, P-0831 treatment only resulted in a slight and delayed increase in the numbers of both NK cells and granzyme B+ NK cells (Figure 25C and Figure 25D).

[0445] In summary, when compared to the active IL-2 fusion molecule P-0838, P-0831 exhibited a significantly reduced systemic proliferation and expansion of specific lymphocytes. This highlights the effectiveness of the VitoKine format in masking IL-2 activity, thereby preventing unwanted activation of the IL-2 pathway and reducing the risk of unwanted "off-target" effects in the "extratissue".

[0446] Example 15

[0447] Reducing cytokine-related toxicity of PD1 Ab-IL-2 VitoKine in mice

[0448] Cytokine-related toxicity, also known as cytokine release syndrome (CRS), is one of the major risks associated with cancer immunotherapy. CRS results from a strong immune response and is typically associated with elevated circulating levels of several cytokines, including interleukin-6 and interferon gamma (INFγ). As the potency of immune-based therapies increases, the magnitude of immune activation may exceed levels that occur in a more natural setting, potentially escalating CRS to life-threatening levels. Given that the VitoKine platform is designed to limit the overactivation of cytokine pathways and considering the demonstrated ability of the exemplary IL-2 VitoKine P-0831 to minimize the systemic activation and expansion of targeted lymphocyte populations (see Example 14), VitoKine holds promise for significantly reducing cytokine-related toxicity.

[0449] To investigate the potential for reducing cytokine-related toxicity, murine PD1 Ab-IL-2 VitoKine P-0831 and its non-VitoKine immunocytokine counterpart P-0838 were administered at different doses to naive C57BL / 6 mice without tumors. Subsequently, the circulating levels of INFγ, one of the key serum inflammatory cytokines, were determined.

[0450] Naive C57BL / 6 mice, 7 - 9 weeks old, were grouped, with 3 mice per group (n = 3), and given a single intraperitoneal injection of P-0831 at doses of 1 mg / kg, 3 mg / kg, 6 mg / kg, 10 mg / kg, and 20 mg / kg and P-0838 at doses of 1 mg / kg, 3 mg / kg, and 6 mg / kg. A vehicle (PBS) and murine PD1 antibody P-0722 (containing a homodimeric Fc with SEQ ID NO:52 and 53) were included as negative controls. Serum samples were collected and isolated from the mice 48 hours after treatment. Serum IFNγ concentrations were determined using a murine INFγ DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions.

[0451] Figure 26A and the accompanying Table 23 reveal that both P-0831 and P-0838 treatments resulted in a dose-dependent increase in serum IFNγ levels. However, compared to P-0838, VitoKine P-0831 exhibited significantly reduced serum IFNγ levels. At the 1 mg / kg dose, P-0838 treatment resulted in an INFγ concentration of 253 pg / mL, while P-0831 resulted in only 10.6 pg / mL of INFγ concentration. More strikingly, at the 3 mg / kg dose of P-0838, serum INFγ rose to 12482 pg / mL, which is 50 times the level seen at the 1 mg / kg dose. In contrast, when administered at 3 mg / kg, P-0831 showed only a 2.5-fold increase in IFNγ levels compared to its 1 mg / kg dose. Surprisingly, even when administered at 20 mg / kg dose, P-0831 resulted in only a modest INFγ level of 182 pg / mL, which is still lower than the level seen with P-0838 at the 1 mg / kg dose. As expected, neither vehicle nor antibody treatment induced any discernible release of this inflammatory cytokine, as illustrated in Figure 26A.

[0452] Table 23

[0453] Serum INFγ concentration 48 hours after treatment

[0454]

[0455] A sharp increase in circulating INFγ levels was observed in the P-0838 treatment groups at doses of 3 mg / kg and 6 mg / kg, which was caused by high levels of systemic immune activation and could lead to severe toxicity. In a concurrent experiment, P-0831 at doses of 3 mg / kg, 6 mg / kg, and 20 mg / kg and P-0838 at doses of 1 mg / kg, 3 mg / kg, and 6 mg / kg were administered via intraperitoneal injection to naïve C57BL / 6 mice (7-9 weeks old, n = 4 mice / group). In mice treated with 3 mg / kg and 6 mg / kg of P-0838, such cytokine-related toxic effects were accompanied by significant weight loss (Figure 26B) and other signs of stress. Given that any mouse experiencing more than a 10% weight loss must be sacrificed according to the established protocol, all mice in the P-0838 6 mg / kg treatment group did not survive beyond 4 days, and 3 out of 4 mice in the 3 mg / kg P-0838 group had to be sacrificed on the 4th day after a single injection. In contrast, mice treated with P-0831 across all tested doses (even up to 20 mg / kg) remained alive after 3 doses on the Q2W dosing schedule and did not show significant weight loss (Figure 26B). This indicates that the VitoKine platform presents a significantly lower toxicity profile.

[0456] In summary, PD1 Ab IL-2 VitoKine significantly alleviated cytokine-related toxicity in mice, as demonstrated by a marked reduction in the circulating levels of inflammatory cytokines exemplified by INFγ, and showed minimal weight changes even at much higher doses compared to non-VitoKine immunocytokine counterparts. The PD1 Ab-IL-2 VitoKine platform effectively minimized off-target toxicity, thus providing a wider therapeutic window.

[0457] Furthermore, the ability of PD1 Ab IL-2 VitoKine to be tolerated at much elevated doses provides greater flexibility in optimizing dosing regimens. At higher doses, the function of the PD1 antibody to reverse T cell anergy or exhaustion can be fully realized as the dose levels are within its effective range, potentially enhancing the synergy with IL-2 immunotherapy. Additionally, incorporation of IL-2 with reduced potency achieved by introducing mutations that disrupt the IL-2Rβ or γc interaction will result in a VitoKine with lower intrinsic basal activity and may also potentially broaden the therapeutic margin.

[0458] Example 16

[0459] Inhibition of the growth of established tumors in mice by PD1 Ab-IL-2 VitoKine

[0460] In a syngeneic MC38 murine colon cancer model, the anti-tumor efficacy of PD1 Ab IL-2 VitoKine P-0831 was studied compared to its non-VitoKine immunocytokine counterpart P-0838. In these experiments, 5×10 5 MC38 colon cancer cells were implanted subcutaneously in the right flank of female C57BL / 6 mice between 7 and 9 weeks of age. Approximately 2 weeks later, once the tumors had grown to an average volume of approximately ~75 mm 3 , on study day 0, the mice were randomly divided into groups of 8 mice. On study day 1, the following treatments were given: murine PD1 antibody P-0722 at 9 mg / kg, P-0831 at different dosing levels (3 mg / kg, 6 mg / kg, and 9 mg / kg), and P-0838 at 1 mg / kg. These treatments were administered intraperitoneally every 10 days (Q10D) for a total of 2 doses. Vehicle (PBS) was used as a control. Both tumor growth and the body weight of the mice were monitored twice a week. Tumor volume (TV) was determined using calipers and calculated as: volume = 0.5 x (width) 2 x (length). Tumor growth inhibition (TGI, %) was calculated using the following formula: TGI (%) = [1 - (TV of the treatment group) / (TV of the control group)] × 100 (%). Based on established criteria, if the tumor grew to or exceeded 1500 mm 3 or the tumor became necrotic, the mice were euthanized.

[0461] Figures 27A - 27D show the tumor growth curves of individual mice in four different treatment groups containing the IL-2 moiety. Each line in the figure represents one mouse, and the average tumor growth of the vehicle group is represented by the dashed line. The small arrows below the X-axis indicate each dose. Treatment with 6 mg / kg of P-0831 showed the most significant and sustained effect, with complete eradication of tumor growth in all 8 mice in this group on day 45, which was 34 days after the second and last treatment (Figure 27B). Similarly, in the group treated with 9 mg / kg of P-0831, 7 out of 8 mice remained tumor-free at the end of the study (Figure 27C). On the other hand, P-0831 administered at 3 mg / kg had a slightly lower efficacy, with 5 out of 8 mice remaining tumor-free, while 3 mice showed tumor growth after an initial phase of delayed tumor growth (Figure 27A). For mice treated with 1 mg / kg of P-0838, according to the study conclusion, 6 out of 8 mice were tumor-free (Figure 27D).

[0462] Also illustrated in Figure 27E are the changes in the average tumor volume of each group and the standard error of the mean (SEM) over time. Mice treated with the vehicle rapidly developed large subcutaneous tumors. Treatment with the PD1 antibody showed limited efficacy, resulting in 27% tumor growth inhibition (TGI) when compared to the vehicle group. In contrast, all other treatment groups showed high efficacy in inhibiting tumor growth, with 100% TGI on day 45 after the start of treatment.

[0463] Figure 27F shows that P-0831 was well tolerated even at doses significantly higher than P-0838, with little or no weight loss. P-0838 was previously shown to be intolerant at doses of 3 mg / kg or higher (as shown in Figure 26B). The in vivo findings of the combination indicate that a 6 mg / kg dose of P-0831 resulted in a more pronounced and prolonged response. Notably, when compared to the effect of a 1 mg / kg dose of P-0838, this anti-tumor efficacy was achieved with much lower peripheral lymphocyte proliferation and expansion (as shown in Figure 25) and a significant reduction in the production of circulating INFγ (Figure 26). This efficacy is partly attributed to the high-dose tolerance provided by the VitoKine format. Thus, the PD1 Ab-IL-2 VitoKine platform provides a wider therapeutic window, enabling the antibody component to fully realize its potential to reverse T cell anergy and exhaustion.

[0464] In a parallel study, immunohistochemistry (IHC) was utilized to evaluate the effect of PD1 Ab-IL-2 VitoKine on tumor tissues obtained 5 days after treatment. In this setting, MC38 subcutaneous tumors were similarly established. After randomization (5 mice / group), mice were treated with a single dose of vehicle, P-0722 (6 mg / kg), P-0831 (6 mg / kg), or P-0838 (1 mg / kg). Five days after treatment, the mice were euthanized, tumors were excised, and tissue samples were prepared. Tissue sections were fixed in 10% formalin, paraffin-embedded, processed, and stained with antibodies by HistoWiz according to the manufacturer's instructions to evaluate immune cells in the tumor tissue. Representative IHC images of each group are illustrated in Figure 28 In.

[0465] Figure 28P-0831 treatment was shown to cause extensive infiltration of CD3+ T cells and CD8+ T cells into tumor tissues. Additionally, the infiltrating CD8+ T cells were characterized by strong cytotoxic capacity, as demonstrated by strong granzyme B expression. These observations highlight the expanded numbers and activity of cytotoxic CD8+ T cells in P-0831-treated murine tumors and also validate the anti-tumor efficacy data. In sharp contrast, the murine PD1 antibody P-0722 at 6 mg / kg induced only minimal tumor-infiltrating lymphocytes (TILs). Although P-0838 treatment led to limited T cell infiltration, it did show high granzyme B expression. Importantly, no treatment significantly led to the emergence of inhibitory FOXP3+ cells.

[0466] In summary, the PD1 Ab IL-2 VitoKine exemplified by the surrogate molecule P-0831 effectively inhibits tumor growth by promoting extensive infiltration of cytotoxic T cells into tumor tissues while minimizing the proliferation and expansion of peripheral lymphocytes. Thus, the use of the VitoKine format can mitigate problems commonly associated with fully active cytokines, such as overstimulation of immune pathways, unwanted "off-tissue" "on-target" toxicities, and unwanted target sink, while still showing strong anti-tumor efficacy. Importantly, the compatibility of the PD1 Ab-IL-2 VitoKine with higher doses ensures that the antibody arm can optimally target and reverse T cell anergy and exhaustion, enhancing existing immune responses. This will lead to a further enhancement of the anti-tumor activity of the immune system. Additionally, the incorporation of a potency-reduced IL-2 achieved by introducing mutations that disrupt the IL-2Rβ or γc interaction results in a VitoKine with lower intrinsic basal activity and post-activation activity. This may also potentially widen the therapeutic window.

[0467] Example 17

[0468] The in vivo activity of PD1 Ab-IL-2 VitoKine depends on proteolytic activation and is PD1-targeting dependent

[0469] The key role of VitoKine activation in its anti-tumor efficacy was investigated by comparing P-0831 and its non-cleavable VitoKine counterpart P-0877 using the murine CT26 colon carcinoma tumor model. The only difference between P-0831 and P-0877 lies in the L2 linker connecting the IL-2 (D2) and IL-2Rα (D3) domains (see Table 21B for details). Although P-0877 demonstrated the same activity as P-0831 in inducing Ki67 expression in CD8+ T cells and NK cells in human PBMCs, as depicted in Figures 29A and 29B, the IL-2 domain in P-0877 remained masked and inactive because the D3 domain could not be cleaved for activation.

[0470] For this study, 5 × 10 5 CT26 cells were subcutaneously injected into the right flank of female Balb / C mice at 7 - 9 weeks of age. On day 11, when the mean tumor volume reached approximately 75 mm 3 , the mice were randomly divided into five groups of eight animals each. Starting on the first study day (i.e., the day after randomization), they received either vehicle (PBS), or two intraperitoneal injections of P-0722, P-0831, or P-0877 at 10 mg / kg Q12D. Tumor size and body weight were monitored twice a week. Based on established criteria, mice were euthanized if the tumor grew to or exceeded 1500 mm 3 or the tumor became necrotic.

[0471] Compared to the MC38 model, the CT26 syngeneic tumor model generally has a lower response to PD1 therapy. As depicted in Figure 29C, tumors eventually developed in all mice. When treated with the murine PD1 antibody P-0722, tumor growth was only slightly delayed, resulting in 25% tumor growth inhibition (TGI). On the other hand, administration of P-0831 at the same dose demonstrated significantly improved efficacy, showing 81% TGI. In stark contrast, P-0877 with an inactivated and inert IL-2 domain did not show any improvement in inhibiting tumor growth compared to P-0722. These findings suggest that the enhanced anti-tumor effectiveness of VitoKine molecules depends on enzymatic cleavage of the linker to release the masked moiety, thereby activating the IL-2 domain around the tumor. Notably, all tested compounds were well tolerated at 10 mg / kg, with no evidence of weight loss in the mice, as shown in Figure 29D.

[0472] In studies conducted concurrently using the CT26 tumor model, the importance of PD1 targeting for the anti-tumor efficacy of IL-2 VitoKine was evaluated. This was done by comparing P-0871, a non-targeting IL-2 VitoKine, with P-0831. Both P-0871 and P-0831 share the same D2 and D3 domains, as well as L1 and L2 linkers, but the D1 domain of P-0871 is a non-targeting germline antibody, P-1260, containing heterodimeric heavy and light chains with SEQ ID NOs: 191, 192, and 193.

[0473] Mice bearing subcutaneously implanted CT26 tumors were administered twice-weekly (Q12D) intraperitoneal injections of vehicle (PBS), P-0722, P-0831, or P-0871 at a dose of 10 mg / kg. Figure 30 The mean tumor volume (along with SEM) for each group is depicted over time. The results showed that P-0831 was significantly more effective than its non-targeting counterpart, P-0871, in delaying tumor growth. This indicates that targeting PD1 is crucial for enhancing the anti-tumor potency of P-0831.

[0474] In summary, these findings strongly suggest that the anti-tumor action of PD1 Ab-IL-2 VitoKine depends on an in vivo proteolytic cleavage process, which subsequently leads to the activation of IL-2. Furthermore, the efficacy of this VitoKine was shown to be closely related to its ability to target PD1, indicating that PD1 targeting plays an important role in its therapeutic potential.

[0475] Example 18

[0476] Construction and ex vivo characterization of PD1 Ab-IL-2 immunocytokines

[0477] Tethering an IL-2 variant to a PD1 antibody is intended to preferentially cis-deliver the IL-2 variant to PD1+ cells, such as activated and exhausted CD8+ T cells in the tumor microenvironment, promoting selective signaling. This strategy also reduces systemic exposure to IL-2 and can provide synergy by removing negative regulation and reconstituting T cells both functionally and numerically. In addition to the VitoKine platform, using an IL-2 variant with reduced / abolished binding to IL-2Rα and attenuated IL-2Rβγ activity provides an alternative approach to balance the ratio between the cytokine and antibody arms, which exhibit significantly different potencies and molecular weights in their native forms. This balance allows for an optimal dose and preserves the function of each arm. Reduced cytokine activity is expected to minimize peripheral activation, mitigate in vivo antigen sink and target-mediated deposition, and facilitate tumor targeting via the antibody arm.

[0478] The PD1 antibodies used to construct the PD1 Ab-IL-2 immunocytokine are selected from optimized human PD1 blocking antibodies comprising the light chain sequences listed in SEQ ID NO:44 and the heavy chain sequences listed in SEQ ID NO:45-49. These optimized PD1 blocking antibodies have high affinity for the human PD1 protein and exhibit equal or comparable potency to pembrolizumab in blocking PD1. They also have higher sequence similarity scores to their closest human germline sequences, resulting in a higher degree of human origin compared to pembrolizumab. In addition, they are predicted to have lower hydrophobicity, which in turn may reduce their tendency to aggregate compared to pembrolizumab. It is expected that the PD1-targeted IL-2 immunocytokines constructed using these optimized PD1 blocking antibodies will also have an enhanced developability profile.

[0479] When generating the PD1 Ab monomeric IL-2 immunocytokine fusion, the IL-2 variant is fused via a peptide linker to the C-terminus of the knob-containing heterodimeric heavy chain of the PD1 antibody. The human IgG1 knob-into-hole heavy chain pair also contains L234A, L235A, G237A mutations to eliminate binding to FcγR and C1q, but retains FcRn binding for pharmacokinetics (PK). The structure of the PD1Ab-IL-2 immunocytokine is depicted in Figure 3B, and exemplary immunocytokines are listed in Table 24.

[0480] Table 24

[0481] Exemplary human PD1 Ab-IL-2 immunocytokines

[0482]

[0483] As can be appreciated by those skilled in the art, any of the optimized PD1 antibodies disclosed in the present invention, including those having the sequences listed in SEQ ID NO:44-49, can be used to construct PD1 Ab-IL-2 immunocytokines, which are within the spirit and scope of the present invention. Similarly, any of the IL-2 variants with varying degrees of potency reduction disclosed in the present invention, particularly those having the sequences listed in SEQ ID NO:151-180, can be used as building blocks for constructing PD1-targeted IL-2 immunocytokines. These designs are intended to enhance and / or potentiate PD1 antibody-based therapies for a range of cancers.

[0484] All genes were codon-optimized for expression in mammalian cells, sub-synthesized by GenScript's service and subsequently cloned into a recipient mammalian expression vector. Constructs were generated by co-transfecting ExpiCHO cells (ThermoFisher) with the expression vector according to the manufacturer's instructions. Protein purification and characterization were performed according to the same procedures detailed in Example 2.

[0485] As expected, the potency of these IL-2 variants, initially observed in the form of Fc fusions, was faithfully retained upon integration with the PD1 antibody, as shown in the human PBMC assay measuring Ki67 expression. In addition, these exemplary immunocytokines listed in Table 24 retained the binding and PD1 blocking activity of their component PD1 antibody P-1271 (SEQ ID NO:49 and 44), as confirmed in the Promega PD1 / PD-L1 blocking reporter assay.

[0486] Mouse PD1 Ab-IL-2 immunocytokines were similarly generated for in vivo tumor models in immunocompetent mice. The IL-2 variant was fused to the C-terminus of the anti-mouse PD1 HC chain 2 (SEQ ID NO:190) of a heterodimeric heavy chain pair (SEQ ID NO:189 and 190) via a GS linker (SEQ ID NO:114). The light chain of the mouse PD1 antibody has the sequence listed in SEQ ID NO:52. Exemplary mouse PD1 Ab IL-2 immunocytokines, particularly P-0782, P-0786 and P-0783, are characterized by IL-2 variants having P65R / C125I mutations (SEQ ID NO:118), L19Q / P65R / C125I mutations (SEQ ID NO:152) and L19H / P65R / C125I mutations (SEQ ID NO:151), respectively. P-0837 containing the IL-2 domain of SEQ ID NO:117 was used as a wild-type IL-2 immunocytokine control.

[0487] Subsequently, the activities of P-0782, P-0786 and P-0783 to stimulate Ki67 expression in CD8+ T cells and NK cells were evaluated using human PBMCs. As illustrated in FIGS. 18A and 18B, the degree of potency attenuation of P-0786 and P-0783 compared to P-0782 due to the incorporation of the L19 mutation (shown in FIGS. 31A and 31B) paralleled that of their respective Fc fusion counterparts P-0759 and P-0731 compared to P-0704.

[0488] The proliferative activities of P-0782, P-0786, and P-0783 were further evaluated using CTLL-2 cells, which are cytotoxic T cells derived from C57BL / 6 mice. Briefly, CTLL2 cells were harvested, washed, and resuspended in medium without IL-2 (RPMI1640, 10% FCS, 2 mM glutamine) for a 2-hour starvation period. After starvation, these cells were transferred at 50,000 cells / mL into 96-well U-bottom plates. Then, serial dilutions of the PD1 Ab-IL-2 immunocytokine were added, followed by a 2-day incubation. Cell proliferation was evaluated using CellTiter-Glo (Promega) and measuring the luminescence signal according to the manufacturer's instructions. As depicted in Figure 31C, the potency reduction caused by the L19Q in P-0786 and L19H in P-0783 mutations was consistent between mouse-derived cells and human primary cells when compared to P-0782. This consistency highlights the mouse as a reliable model for analyzing the impact of IL-2 potency changes on in vivo pharmacodynamics and anti-tumor efficacy.

[0489] Example 19

[0490] Pharmacodynamic effects of PD1 Ab IL-2 immunocytokine in mice

[0491] The pharmacodynamic effects of the mouse PD1 Ab-IL-2 immunocytokine were evaluated in C57BL / 6 mice using a single-dose administration. Before starting the study, 7-week-old female C57BL / 6 mice from Charles River Laboratories were allowed a 7-day acclimation period. On day 0, the mice were given an intraperitoneal injection of vehicle or one of the following test compounds: P-0837, P-0782, P-0783, or P-0786. Blood samples were drawn on days 0, 3, 5, and 10 after injection. Each group consisted of 5 mice. Immunoprofiling of heparinized whole blood was performed according to the procedure outlined in Example 14.

[0492] After a single injection at 2 mg / kg, significant differences in CD8 cell and NK cell expansion were observed between the test compounds. P-0782, which contains a mutation that abrogates IL-2Rα binding but does not affect IL-2Rβγ interaction, exhibited robust expansion of CD8+ T cells (Figure 32A) and NK cells (Figure 32B). Expansion of these lymphocyte subsets began on day 3 and peaked on day 7, with a 68-fold increase in CD8+ T cells and a significant 182-fold increase in NK cells. In stark contrast, P-0837, which served as the wild-type control, demonstrated a much milder response. The peak cell expansion for both lymphocyte populations occurred on day 5, with a more modest 3.9-fold increase in CD8+ T cells and a 6.8-fold increase in NK cells, as illustrated in Figure 32A and Figure 32B.

[0493] Mutations that abrogate IL-2Rα binding can minimize the IL-2Rα (CD25) sink effect, subsequently increasing the availability of IL-2Rβγ. As demonstrated in P-0782, an IL-2Rβγ-selective full agonist, this enriched receptor engagement triggers robust expansion of cytotoxic cells. It is hypothesized that IL-2 mutations designed to reduce but not eliminate IL-2Rα binding can fine-tune the response of regulatory T cells (as illustrated in Figure 15B). Appropriate modulation of IL-2Rα binding can establish an immune balance to improve systemic tolerance without compromising tumor-killing efficacy.

[0494] Figure 32A and Figure 32B also show the pharmacodynamics of P-0783 and P-0786 after a 2 mg / kg dose. Compared to P-0782, the maximum responses observed for P-0786 and P-0783 were significantly reduced, consistent with their overall attenuated potency. However, in contrast to P-0837, P-0786 exhibited a significantly prolonged and enhanced dose-response effect on cell expansion. The increase in the number of both CD8+ T cells and NK cells was delayed but sustained and persistent. The peak response was observed on day 7, demonstrating an 8.6-fold increase in CD8+ T cells and a 13-fold increase in NK cells. These numbers did not return to baseline levels by day 10. P-0783, which contains an even weaker IL-2 agonist, showed a similar delayed but persistent effect, resulting in a 5.5-fold expansion of CD8+ T cells and a 14-fold expansion of NK cells in a dose-dependent manner (Figure 32A and Figure 32B).

[0495] In addition, Figure 32C highlights the direct correlation between the potency level in mice, the expansion of cytotoxic lymphocytes, and the resulting weight loss. Specifically, P-0782 is an IL-2Rβγ-selective full agonist that induces a sharp increase in the numbers of both CD8+ T cells and NK cells, resulting in the most significant weight loss among the test compounds. The less potent agonists P-0786 and P-0783 show enhanced in vivo tolerance. Among them, P-0783 appears to be slightly more tolerant than P-0786, consistent with its characterization as the weaker agonist of the two.

[0496] In summary, P-0782 demonstrated robust pharmacodynamic effects by significantly promoting the proliferation and expansion of CD8+ T cells and NK cells. Although P-0786 and P-0783 exhibited weaker effects, their responses were sustained. The in vitro and in vivo potency evaluations of these compounds were generally consistent. Notably, compared with the full-effect P-0782, the attenuated potency of P-0786 and P-0783 led to improved in vivo tolerance. These findings support our design premise that attenuating cytokine potency helps to alleviate pathway overactivation and reduce antigen sink and target-mediated deposition, ultimately reducing toxicity and improving pharmacokinetic and pharmacodynamic outcomes.

[0497] Example 20

[0498] In vivo efficacy of PD1 Ab IL-2 immunocytokine in a syngeneic mouse tumor model

[0499] The anti-tumor efficacy of PD1 Ab-IL-2 immunocytokines (including P-0837, P-0782, P-0783, and P-0786) was investigated in the MC38 murine colon cancer model. Female C57BL / 6 mice, 7-9 weeks old, were subcutaneously implanted with MC38 cells. Once the tumor volume averaged ~75 mm 3 after approximately 2 weeks, the mice were randomized (n = 8) and treated with 2 injections of the immunocytokine at 0.5 mg / kg every 12 days (Q12D). Vehicle (PBS) was included as a control. Tumor size and mouse body weight were monitored twice a week. If the tumor reached / exceeded 1500 mm 3 or became necrotic, the animals were euthanized.

[0500] In Figure 33A, the average tumor volume per group and the standard error of the mean (SEM) are presented as a function of time. The vehicle group (PBS treatment) showed rapid tumor growth. P-0837, a PD1Ab immunocytokine with a wild-type IL-2 equivalent, inhibited tumor growth by only 37% compared to the vehicle group. However, the other PD1 Ab-IL-2 immunocytokines, P-0782, P-0783, and P-0786, all significantly reduced tumor growth by 85%-90% from the time of treatment until day 26. Further analysis of individual tumor volumes (represented by each point) on day 26 (Figure 33B) revealed that although tumor growth inhibition was similar, only two mice were tumor-free with P-0782, while five mice were tumor-free with both P-0783 and P-0786. The latter two both had attenuated IL-2 potency. It is hypothesized that the full IL-2 agonist P-0782 may lead to overactivation of the pathway, target-mediated deposition, activation-induced cell death, and upregulation of inhibitory signals on T cells, ultimately resulting in reduced in vivo anti-tumor efficacy. Therefore, only PD1 Ab-IL-2 immunocytokines with attenuated IL-2 will be further investigated for in vivo anti-tumor efficacy.

[0501] In a similarly conducted experiment depicted in Figure 34, both P-0783 and P-0786 showed significant tumor growth inhibition at a low dose of 0.3 mg / kg (two Q10D doses). On day 41, 30 days after the second and final treatment, 5 out of 8 mice treated with P-0783 and 6 out of 8 mice treated with P-0786 had no tumor growth. At this dose, P-0786 showed slightly better anti-tumor activity compared to P-0783.

[0502] Figures 35A and 35B show the progression of tumor volume and body weight changes over time in mice treated with two Q10D doses of P-0782 or the murine PD1 antibody P-0722. At a dose of 9 mg / kg, P-0722 showed minimal efficacy. In stark contrast, even when the dose of P-0786 was reduced to 1 / 9 (1 mg / kg), a significant and prolonged anti-tumor response was observed. By day 45, 34 days after the last treatment, all mice treated with P-0786 showed no tumor growth. In addition, no significant body weight loss was observed with 1 mg / kg of P-0786 (Figure 35B). These findings highlight the crucial role of the IL-2 component in enhancing the anti-tumor efficacy of PD1Ab-IL-2 immunocytokines.

[0503] In addition, the dose response of P-0786 on MC38 tumor growth inhibition was evaluated. Mice with established MC38 tumors (average tumor volume of ~75 mm 3Mice (n = 8) received two Q14D doses of P-0786 at 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg intraperitoneally. Figure 36A The mean tumor volume ± SEM of each group over time is shown, and FIGS. 36B to 36E illustrate the individual tumor growth curves for each dose group. The mean tumor size ± SEM of the vehicle group (represented by the dashed line) is shown for comparison. Both the 0.03 mg / kg and 0.1 mg / kg doses resulted in a moderate 36% TGI, and no mice showed complete tumor regression by day 21. However, a clear dose response emerged when the dose was increased from 0.1 mg / kg to 1 mg / kg in approximately 3-fold increments. At 0.3 mg / kg, 81% TGI was reached by day 21, and 2 out of 8 mice were tumor-free. Impressively, at the 1 mg / kg dose, all 8 mice showed complete tumor eradication.

[0504] Ten mice that did not show tumor growth (2 from the 0.3 mg / kg dose and 8 from the 1 mg / kg dose) were rechallenged with MC38 cells on day 109 after initial implantation or 94 days after the first P-0786 dose. Figure 37 It was revealed that none of these rechallenged mice had tumor recurrence, unlike the age-matched naive mice that successfully developed tumors and were used as controls. These findings suggest that the PD1 Ab IL-2 immunocytokine successfully induced long-term immunity.

[0505] The efficacy of P-0786 was also evaluated in two other syngeneic tumor models: the murine CT26 colon carcinoma and the B16F10 murine melanoma model. In the CT26 model, female Balb / C mice were subcutaneously implanted with 5 × 10 5 CT26 cells, while the B16F10 model was similarly established by implanting 5 x 10 5 B16F10 cells into female C57BL / 6 mice. Mice bearing CT26 tumors received two doses of P-0786 (0.6 mg / kg and 2 mg / kg) every 12 days, while mice bearing B16F10 tumors received the same doses every 10 days. All mice were routinely monitored with twice-weekly tumor measurements.

[0506] Figure 38 reveals the dose-dependent single-agent anti-tumor effect of P-0786 in both the CT26 model (Figure 38A) and the B16F10 model (Figure 38B). In the CT26 model, both doses had strong tumor growth inhibition (65% TGI at 0.6 mg / kg and 91% TGI at 2 mg / kg by day 21). By day 41 after the first treatment, 1 out of 7 mice in the 0.6 mg / kg group was tumor-free, and 2 out of 7 mice in the 2 mg / kg group were tumor-free. Compared to the MC38 model, the B16F10 model grew aggressively and had a lower response to PD1 therapy, showing an initial tumor growth delay for P-0786 (35% TGI at 0.6 mg / kg and 58% TGI at 2 mg / kg), but tumors eventually developed in all mice (Figure 38B).

[0507] In summary, the PD1 Ab-IL-2 immunocytokine effectively inhibits tumor growth across more than one syngeneic mouse tumor model. When compared to an IL-2Rβγ-selective full agonist, the IL-2 with attenuated potency achieved by disrupting the IL-2Rβ interaction showed improved in vivo tolerance and enhanced single-agent anti-tumor efficacy. It is predicted that the PD1 Ab IL-2 immunocytokine characterized by IL-2 mutations that interfere with γc interaction will have a similar improvement. Given the different expression profiles of γc in peripheral cells, IL-2 variants with attenuated γc activity may provide the additional benefits of reduced target sink and enhanced bioavailability.

[0508] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the invention. It will be apparent to those of skill in the art that all such variations and equivalents, whether now existing or later developed, are considered to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification indicate the level of skill of those of ordinary skill in the art to which this invention pertains. For all purposes, all patents, patent applications, and publications are hereby incorporated by reference in their entirety, and to the extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The invention described herein by way of example can be practiced appropriately in the absence of any one or more of the elements specifically disclosed herein. Accordingly, it is to be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, those of skill in the art may seek alterations and variations of the concepts disclosed herein, and such alterations and variations are considered to be within the scope of the invention as defined by the appended claims.

[0509] Sequence Listing

[0510] The amino acid sequences listed in the attached Sequence Listing are shown using the standard letter abbreviations for nucleotide bases and the single-letter codes for amino acids as defined in 37 C.F.R. 1.822.

[0511] SEQ ID NO:1 is the amino acid sequence of the mature human PD1 polypeptide.

[0512] SEQ ID NOs: 2 - 5 are the amino acid sequences of the light chain variable domains of human PD1 blocking antibodies.

[0513] SEQ ID NOs: 6 - 18 are the amino acid sequences of the heavy chain variable domains of human PD1 blocking antibodies.

[0514] SEQ ID NOs: 19 - 21 are the amino acid sequences of the light chain CDR1 of human PD1 blocking antibodies.

[0515] SEQ ID NOs: 22 - 24 are the amino acid sequences of the light chain CDR2 of human PD1 blocking antibodies.

[0516] SEQ ID NO: 25 is the amino acid sequence of the light chain CDR3 of human PD1 blocking antibodies.

[0517] SEQ ID NO: 26 is the amino acid sequence of the heavy chain CDR1 of human PD1 blocking antibodies.

[0518] SEQ ID NO: 27 - 32 are the amino acid sequences of the heavy chain CDR2 of human PD1 - blocking antibodies.

[0519] SEQ ID NO: 33 is the amino acid sequence of the heavy chain CDR3 of human PD1 - blocking antibodies.

[0520] SEQ ID NO: 34 is the amino acid sequence of the constant domain of human κ light chain.

[0521] SEQ ID NO: 35 is the amino acid sequence of the constant domain of human IgG1 heavy chain containing the L234A / L235A / G237A mutations.

[0522] SEQ ID NO: 36 is the amino acid sequence of the constant domain of human IgG4 heavy chain containing the S228P mutation.

[0523] SEQ ID NO: 37 is the amino acid sequence of human immunoglobulin germline exon HGHV1 - 2 (GenBank accession number: X62106).

[0524] SEQ ID NO: 38 is the amino acid sequence of human immunoglobulin germline exon HGHV3 - 23 (GenBank accession number: M99660).

[0525] SEQ ID NO: 39 is the amino acid sequence of human immunoglobulin germline exon HGKV3D - 11 (GenBank accession number: X17264).

[0526] SEQ ID NO: 40 is the amino acid sequence of the variable domain of human antibody heavy chain, with GenBank accession number: AB063829.

[0527] SEQ ID NO: 41 is the amino acid sequence of the variable domain of human antibody light chain, with GenBank accession number: M29469.

[0528] SEQ ID NO: 42 is the amino acid sequence of the light chain of the reference human PD1 - blocking antibody P - 0734.

[0529] SEQ ID NO: 43 is the amino acid sequence of the heavy chain of the reference human PD1 - blocking antibody P - 0734.

[0530] SEQ ID NO: 44 is the amino acid sequence of the light chain of human PD1 - blocking antibodies.

[0531] SEQ ID NO: 45 is the amino acid sequence of the heavy chain of human PD1 - blocking antibody P - 1174.

[0532] SEQ ID NO:46 is the amino acid sequence of the heavy chain of the human PD1-blocking antibody P-1194.

[0533] SEQ ID NO:47 is the amino acid sequence of the heavy chain of the human PD1-blocking antibody P-1201.

[0534] SEQ ID NO:48 is the amino acid sequence of the heavy chain of the human PD1-blocking antibody P-1238.

[0535] SEQ ID NO:49 is the amino acid sequence of the heavy chain of the PD1 human-blocking antibody P-1271.

[0536] SEQ ID NO:50 is the amino acid sequence of the light chain of the benchmark human PD1-blocking antibody P-0795.

[0537] SEQ ID NO:51 is the amino acid sequence of the heavy chain of the benchmark human PD1-blocking antibody P-0795.

[0538] SEQ ID NO:52 is the amino acid sequence of the light chain of the surrogate mouse PD1-blocking antibody P-0722.

[0539] SEQ ID NO:53 is the amino acid sequence of the heavy chain of the surrogate mouse PD1-blocking antibody P-0722.

[0540] SEQ ID NO:54-77 are the amino acid sequences of various protease substrate peptides.

[0541] SEQ ID NO:78-94 are the amino acid sequences of various protease-cleavable linkers containing various spacer peptides flanking the protease substrate peptide.

[0542] SEQ ID NO:95-115 are the amino acid sequences of various non-cleavable linker sequences.

[0543] SEQ ID NO:116 is the amino acid sequence of the mature form of human IL-2.

[0544] SEQ ID NO:117-180 are the amino acid sequences of human IL-2 variant polypeptides.

[0545] SEQ ID NO:181 is the amino acid sequence of human IL-2Rα.

[0546] SEQ ID NO:182 is the amino acid sequence of the sushi domain of human IL-2Rα.

[0547] SEQ ID NO: 183 - 185 are the amino acid sequences of human IL-2Rα sushi domain variant polypeptides.

[0548] SEQ ID NO: 186 is the amino acid sequence of human IgG1 Fc containing the L234A / L235A / G237A mutation.

[0549] SEQ ID NO: 187 is the amino acid sequence of human IgG1 Knob-Fc containing the L234A / L235A / G237A mutation.

[0550] SEQ ID NO: 188 is the amino acid sequence of human IgG1 Hole-Fc containing the L234A / L235A / G237A mutation.

[0551] SEQ ID NO: 189 and 190 are the amino acid sequences of the heterodimeric heavy chains of the surrogate murine PD1 Ab P-0722.

[0552] SEQ ID NO: 191 and 192 are the amino acid sequences of the heterodimeric heavy chains of the germline antibody P-1260.

[0553] SEQ ID NO: 193 is the amino acid sequence of the light chain of the germline antibody P-1260.

[0554] SEQ ID NO: 194 - 209 are the amino acid sequences of the heavy chains of various human PD1 Abs and / or human PD1 Ab-IL-2 VitoKine.

[0555] SEQ ID NO: 210 - 215 are the amino acid sequences of the heavy chains of various human PD1 Ab-IL-2 immunocytokines.

[0556] Sequence Listing

[0557] The sequence of human PD1 mature protein: FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPS

[0558] PSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL(SEQ ID NO:1)

[0559] Human PD1-blocking antibody light chain variable domain sequence

[0560] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR(SEQ ID NO:2)

[0561] Human PD1-blocking antibody light chain variable domain sequence

[0562] EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR(SEQ ID NO:3)

[0563] Human PD1-blocking antibody light chain variable domain sequence

[0564] EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR(SEQ ID NO:4)

[0565] Human PD1-blocking antibody light chain variable domain sequence

[0566] EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLAWYQQKPGQAPRLLIYLASYRASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR(SEQ ID NO:5)

[0567] Human PD1-blocking antibody heavy chain variable domain sequence

[0568] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS(SEQ ID NO:6)

[0569] Human PD1-blocking antibody heavy chain variable domain sequence

[0570] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS(SEQ ID NO:7)

[0571] Human PD1-blocking antibody heavy chain variable domain sequence

[0572] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNYAQK FQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS(SEQ ID NO:8)

[0573] Human PD1-blocking antibody heavy chain variable domain sequence

[0574] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKF QGRVTLTTDSSTSTAYMELSSLRSDDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:9) Human PD1-blocking antibody heavy chain variable domain sequence

[0575] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:10)

[0576] Human PD1-blocking antibody heavy chain variable domain sequence

[0577] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFK GRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:11)

[0578] Human PD1-blocking antibody heavy chain variable domain sequence

[0579] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:12)

[0580] Human PD1-blocking antibody heavy chain variable domain sequence

[0581] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFK GRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:13)

[0582] Human PD1-blocking antibody heavy chain variable domain sequence

[0583] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFNDSVK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:14)

[0584] Human PD1-blocking antibody heavy chain variable domain sequence

[0585] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFK GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:15)

[0586] Human PD1-blocking antibody heavy chain variable domain sequence

[0587] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFK GRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:16)

[0588] Variable domain sequence of the heavy chain of human PD1-blocking antibody

[0589] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFK GRFTISTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:17) Variable domain sequence of the heavy chain of human PD1-blocking antibody

[0590] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFK GRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS(SEQ ID NO:18)

[0591] CDR-L1 sequence of human PD1-blocking antibody

[0592] RASKGVSTSGYSYLH(SEQ ID NO:19)

[0593] CDR-L1 sequence of human PD1-blocking antibody

[0594] RASQGVSTSGYSYLH(SEQ ID NO:20)

[0595] CDR-L1 sequence of human PD1-blocking antibody

[0596] RASQGVSTSGYSYLA(SEQ ID NO:21)

[0597] CDR-L2 sequence of human PD1-blocking antibody

[0598] YLASYLES(SEQ ID NO:22)

[0599] CDR-L2 sequence of human PD1-blocking antibody

[0600] YLASYRES (SEQ ID NO:23)

[0601] Human PD1-blocking antibody CDR-L2 sequence

[0602] YLASYRAS (SEQ ID NO:24)

[0603] Human PD1-blocking antibody CDR-L3 sequence

[0604] QHSRDLPLT (SEQ ID NO:25)

[0605] Human PD1-blocking antibody CDR-H1 sequence

[0606] NYYMY (SEQ ID NO:26)

[0607] Human PD1-blocking antibody CDR-H2 sequence

[0608] GINPSNGGTNFNEKFKN (SEQ ID NO:27)

[0609] Human PD1-blocking antibody CDR-H2 sequence

[0610] GINPSNGGTNFAQKFQG (SEQ ID NO:28)

[0611] Human PD1-blocking antibody CDR-H2 sequence

[0612] GINPSNGGTNYAQKFQG (SEQ ID NO:29)

[0613] Human PD1-blocking antibody CDR-H2 sequence

[0614] GINPSNGGTNYADKFKG (SEQ ID NO:30)

[0615] Human PD1-blocking antibody CDR-H2 sequence

[0616] GINPSNGGTNFADKFKG (SEQ ID NO:31)

[0617] Human PD1-blocking antibody CDR-H2 sequence

[0618] GINPSNGGTNFNDSVKG (SEQ ID NO:32)

[0619] Human PD1-blocking antibody CDR-H3 sequence

[0620] RDYRFDMGFDY (SEQ ID NO:33)

[0621] Human κ light chain constant domain sequence

[0622] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:34)

[0623] Human IgG1 constant domain with L234A / L235A / G237A mutation sequence

[0624] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

[0625] VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMI

[0626] SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK

[0627] EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:35)

[0628] Human IgG4 constant domain with S228P mutation sequence

[0629] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYK

[0630] CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO:36)

[0631] Human antibody germline IGHV1-2 sequence

[0632] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR (SEQ ID NO:37)

[0633] Human antibody germline IGHV3-23 sequence

[0634] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO:38)

[0635] Human antibody germline IGKV3D-11 sequence

[0636] EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH (SEQ ID NO:39)

[0637] Human antibody GenBank NO: AB063829 sequence

[0638] QVQLVQSGVEVKKPGASVKVSCKASGYTFTSNAISWVRQAPGQGLEWMGWISTYKGKANYAQKFQDRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARWRAVVGRGGGLDVWGQGTTVTVSS (SEQ IDNO:40)

[0639] Human antibody GenBank NO: M29469 sequence

[0640] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNKATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSKWPLTFGGGTKVEIKG(SEQ ID NO:41)

[0641] Reference antibody P-0734 light chain sequence

[0642] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:42)

[0643] Reference antibody P-0734 heavy chain sequence

[0644] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:43)

[0645] Human PD1-blocking Ab light chain sequence

[0646] EIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:44)

[0647] Human PD1-blocking Ab P-1174 heavy chain sequence

[0648] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:45)

[0649] Human PD1-blocking antibody P-1194 heavy chain sequence

[0650] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:46)

[0651] The heavy chain sequence of human PD1-blocking antibody P-1201: EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:47)

[0652] Heavy chain sequence of human PD1-blocking antibody P-1238

[0653] EVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:48)

[0654] Heavy chain sequence of human PD1-blocking Ab P-1271

[0655] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTSTAYMELSSLRSDDTAVYYCARRDYRFDMGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:49)

[0656] Reference human PD1-blocking antibody P-0795 light chain sequence

[0657] DIVMTQSPLSLPVTPGEPASITCKASQDVETVVAWYLQKPGQSPRLLIYWASTRHTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYSRYPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:50)

[0658] Reference human PD1-blocking antibody P-0795 heavy chain sequence

[0659] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVATISGGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPDSSGVAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN

[0660] VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[0661] KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:51)

[0662] Alternative murine PD1 blocking antibody P-0722 light chain sequence

[0663] DIVMTQGTLPNPVPSGESVSITCRSSKSLLYSDGKTYLNWYLQRPGQSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGIYYCQQGLEFPTFGGGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPRDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:52)

[0664] Alternative murine PD1 blocking antibody P-0722 heavy chain sequence

[0665] EVQLQESGPGLVKPSQSLSLTCSVTGYSITSSYRWNWIRKFPGNRLEWMGYINSAGISNYNPSLKRRISITRDTSKNQFFLQVNSVTTEDAATYYCARSDNMGTTPFTYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGGRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG(SEQID NO:53)

[0666] Protease substrate peptide sequence

[0667] SPLGLAGS(SEQ ID NO:54)

[0668] Protease substrate peptide sequence

[0669] EPLELRAG(SEQ ID NO:55)

[0670] Protease substrate peptide sequence

[0671] LSGRSDNH(SEQ ID NO:56)

[0672] Protease substrate peptide sequence

[0673] GPLGIAGQ(SEQ ID NO:57)

[0674] Protease substrate peptide sequence

[0675] GTAHLMGG(SEQ ID NO:58)

[0676] Protease substrate peptide sequence

[0677] RIGSLRTA(SEQ ID NO:59)

[0678] Protease substrate peptide sequence

[0679] SGRSENIRTA (SEQ ID NO:60)

[0680] Protease substrate peptide sequence

[0681] GPLGMLSQ (SEQ ID NO:61)

[0682] Protease substrate peptide sequence

[0683] GPAGMKGL (SEQ ID NO:62)

[0684] Protease substrate peptide sequence

[0685] RPSASRSA (SEQ ID NO:63)

[0686] Protease substrate peptide sequence

[0687] PLGLAG (SEQ ID NO:64)

[0688] Protease substrate peptide sequence

[0689] LGGSGRSANAILE (SEQ ID NO:65)

[0690] Protease substrate peptide sequence

[0691] GGSGRSANAI (SEQ ID NO:66)

[0692] Protease substrate peptide sequence

[0693] SGRSA (SEQ ID NO:67)

[0694] Protease substrate peptide sequence

[0695] AANL (SEQ ID NO:68)

[0696] Protease substrate peptide sequence

[0697] GPTNKVR (SEQ ID NO:69)

[0698] Protease substrate peptide sequence

[0699] GFFY (SEQ ID NO:70)

[0700] Protease substrate peptide sequence

[0701] GPICFRLG (SEQ ID NO:71)

[0702] Protease substrate peptide sequence

[0703] RQAGFSL (SEQ ID NO:72)

[0704] Protease substrate peptide sequence

[0705] RQARAVGG (SEQ ID NO:73)

[0706] Protease substrate peptide sequence

[0707] PMAKK (SEQ ID NO:74)

[0708] Protease substrate peptide sequence

[0709] HSSKLQ (SEQ ID NO:75)

[0710] Protease substrate peptide sequence

[0711] GPLGMLSQPMAKK (SEQ ID NO:76)

[0712] Protease substrate peptide sequence

[0713] PMAKKGPLGMLSQ (SEQ ID NO:77)

[0714] Protease-cleavable linker sequence

[0715] GGGSGGGGSGGGGSLSGRSDNHGGSGGGGS (SEQ ID NO:78) Protease-cleavable linker sequence

[0716] GSSSGRSENIRTAGT (SEQ ID NO:79)

[0717] Protease-cleavable linker sequence

[0718] GGGGSGGGGSGGGSLGGSGRSANAILEGGSGGGGS (SEQ ID NO:80) Protease-cleavable linker sequence

[0719] GGGGSGGGGSLGGSGRSANAILEGGGGS (SEQ ID NO:81)

[0720] Protease-cleavable linker sequence

[0721] GGGGSLGGSGRSANAILEGGS (SEQ ID NO:82) Protease-cleavable linker sequence

[0722] GGGSGPTNKVRGGS (SEQ ID NO:83)

[0723] Protease-cleavable linker sequence

[0724] GGSGPLGMLSQGGGS (SEQ ID NO:84)

[0725] Protease-cleavable linker sequence

[0726] GGPLGMLSQS (SEQ ID NO:85)

[0727] Protease-cleavable linker sequence

[0728] GGGPLGMLSQGGS (SEQ ID NO:86)

[0729] Protease-cleavable linker sequence

[0730] GGPTNKVRGS (SEQ ID NO:87)

[0731] Protease-cleavable linker sequence

[0732] GRQARAVGGS (SEQ ID NO:88)

[0733] Protease-cleavable linker sequence

[0734] GGGSGRSENIRTAGG (SEQ ID NO:89)

[0735] Protease-cleavable linker sequence

[0736] SGGPGPAGMKGLPGS (SEQ ID NO:90)

[0737] Protease-cleavable linker sequence

[0738] GGGGSPMAKKGGGGS (SEQ ID NO:91) Protease-cleavable linker sequence

[0739] GGPLGMLSQPMAKKS (SEQ ID NO:92)

[0740] Protease-cleavable linker sequence

[0741] GGSGPLGMLSQPMAKKGGGS (SEQ ID NO:93) Protease-cleavable linker sequence

[0742] GGGPMAKKGPLGMLSQGGGS (SEQ ID NO:94) Non-cleavable linker sequence

[0743] EPKSSDKTHTSPPS (SEQ ID NO:95)

[0744] Non-cleavable linker sequence

[0745] GGGSGGGSGGGS (SEQ ID NO:96)

[0746] Non-cleavable linker sequence

[0747] GGGS (SEQ ID NO:97)

[0748] Non-cleavable linker sequence

[0749] GSSGGSGGS (SEQ ID NO:98)

[0750] Non-cleavable linker sequence

[0751] GSSGT (SEQ ID NO:99)

[0752] Non-cleavable linker sequence

[0753] GGGGSGGGGSGGGS (SEQ ID NO:100)

[0754] Non-cleavable linker sequence

[0755] AEAAAKEAAAKEAAAKA (SEQ ID NO:101)

[0756] Non-cleavable linker sequence

[0757] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:102)

[0758] Non-cleavable linker sequence

[0759] GGGSGGGS (SEQ ID NO:103)

[0760] Non-cleavable linker sequence

[0761] GSGG (SEQ ID NO:104)

[0762] Non-cleavable linker sequence

[0763] GGSS (SEQ ID NO:105)

[0764] Non-cleavable linker sequence

[0765] GGGGS (SEQ ID NO:106)

[0766] Non-cleavable linker sequence

[0767] GGSGG (SEQ ID NO:107)

[0768] Non-cleavable linker sequence

[0769] SGGG (SEQ ID NO:108)

[0770] Non-cleavable linker sequence

[0771] GSGS (SEQ ID NO:109)

[0772] Non-cleavable linker sequence

[0773] GSGSGS (SEQ ID NO:110)

[0774] Non-cleavable linker sequence

[0775] GSGSGSGS (SEQ ID NO:111)

[0776] Non-cleavable linker sequence

[0777] GSGSGSGSGS (SEQ ID NO:112)

[0778] Non-cleavable linker sequence

[0779] GSGSGSGSGSGS (SEQ ID NO:113)

[0780] Non-cleavable linker sequence

[0781] GGGGSGGGGS (SEQ ID NO:114)

[0782] Non-cleavable linker sequence

[0783] GGGGSGGGGSGGGGS (SEQ ID NO:115)

[0784] Naturally occurring sequence of the mature form of human IL-2

[0785] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:116)

[0786] Human IL-2 C125I variant sequence

[0787] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:117)

[0788] Human IL-2 P65R / C125I variant sequence

[0789] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKRLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:118)

[0790] Human IL-2 P65K / C125I variant sequence

[0791] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKKLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:119)

[0792] Human IL-2 P65N / C125I variant sequence

[0793] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKNLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:120)

[0794] Human IL-2 P65Q / C125I variant sequence

[0795] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKQLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:121)

[0796] Human IL-2 P65H / C125I variant sequence

[0797] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKHLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:122)

[0798] Human IL-2 P65G / C125I variant sequence

[0799] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKGLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:123)

[0800] Human IL-2 P65E / C125I variant sequence

[0801] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKELEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:124)

[0802] Human IL-2 P65A / C125I variant sequence

[0803] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKALEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:125)

[0804] Human IL-2 L19H / C125I variant sequence

[0805] APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:126)

[0806] Human IL-2 L19Q / C125I variant sequence

[0807] APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:127)

[0808] Human IL-2 L19Y / C125I variant sequence

[0809] APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:128)

[0810] Human IL-2 L19D / C125I variant sequence

[0811] APTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:129)

[0812] Human IL-2 L19S / C125I variant sequence

[0813] APTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:130)

[0814] Human IL-2 L19N / C125I variant sequence

[0815] APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:131)

[0816] Human IL-2 L19R / C125I variant sequence

[0817] APTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO:132)

[0818] Human IL-2 C125I / Q126A variant sequence

[0819] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIASIISTLT(SEQ ID NO:133)

[0820] Human IL-2 C125I / Q126D variant sequence

[0821] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIDSIISTLT(SEQ ID NO:134)

[0822] Human IL-2 C125I / Q126E variant sequence

[0823] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT(SEQ ID NO:135)

[0824] Human IL-2 C125I / Q126F variant sequence

[0825] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIFSIISTLT(SEQ ID NO:136)

[0826] Human IL-2 C125I / Q126G variant sequence

[0827] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIGSII...

Claims

1. A bioactivatable polypeptide drug construct that, in the N-terminal to C-terminal direction (D1-D2-D3), comprises: 1) a tumor-infiltrating lymphocyte (TIL) targeting moiety, D1 domain ("D1"), 2) a bioactivatable moiety, D2 domain ("D2"), and 3) a shielding moiety, D3 domain ("D3"); wherein D1 serves to target the bioactivatable moiety to the intended site of treatment; wherein D3 is capable of shielding the functional activity of D2 until it is activated at the intended site of treatment; wherein D1 is an optimized PD1-blocking antibody, wherein D2 is an IL-2 variant polypeptide, and wherein D3 is an IL-2Rα sushi variant.

2. A bioactivatable polypeptide drug construct that, in the N-terminal to C-terminal direction (D3-D2-D1), comprises: 1) a shielding moiety, D3 domain ("D3"), 2) a bioactivatable moiety, D2 domain ("D2"), and 3) a tumor-infiltrating lymphocyte (TIL) targeting moiety, D1 domain ("D1"), wherein D1 serves to target the bioactivatable moiety to the intended site of treatment; wherein D3 is capable of shielding the functional activity of D2 until it is activated at the intended site of treatment; wherein D1 is an optimized PD1-blocking antibody, wherein D2 is an IL-2 variant polypeptide, and wherein D3 is an IL-2Rα sushi variant.

3. The bioactivatable polypeptide drug construct according to any one of claims 1-2, wherein the optimized PD1-blocking antibody is selected from the group of antibodies comprising: (a) a light chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 3, and a heavy chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 7; or (b) a light chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 3, and a heavy chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 9; or (c) a light chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 3, and a heavy chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 11; (d) a light chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 3, and a heavy chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 13; or (e) a light chain variable region comprising the amino acids of the sequence listed in SEQ ID NO: 3, and a heavy chain variable region comprising the amino acids of the sequence listed in SEQ ID NO:

18.

4. The bioactivatable polypeptide drug construct according to any one of claims 1-3, wherein domain D2 is an IL-2 variant polypeptide selected from the group of polypeptides having the amino acid sequences listed in SEQ ID NOs: 117-180.

5. The bioactivatable polypeptide drug construct according to any one of claims 1-4, wherein domain D3 is an IL-2Rα sushi variant polypeptide selected from the group of polypeptides having the amino acid sequences listed in SEQ ID NOs: 183-185.

6. The construct according to any one of claims 1-5, wherein the D1 domain, D2 domain, and D3 domain of the construct are each in monomeric form, each in dimeric form, or together in a combination of dimeric and monomeric forms.

7. The construct according to any one of claims 1-6, wherein D2 is attached to D1 via a peptide linker ("L1") selected from the group consisting of protease-cleavable peptide linkers and non-cleavable peptide linkers.

8. The construct according to claim 7, wherein the protease-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NOs: 54-77 and 78-94.

9. The construct according to claim 7, wherein the non-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NOs: 95-115.

10. The construct according to any one of claims 1-9, wherein D2 is attached to D3 via a peptide linker ("L2") selected from the group consisting of protease-cleavable peptide linkers and non-cleavable peptide linkers.

11. The construct according to claim 10, wherein the protease-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NOs: 54-77 and 78-94.

12. The construct according to claim 10, wherein the non-cleavable peptide linker is selected from the group of sequences listed in SEQ ID NOs: 95-115.

13. The construct according to any one of claims 1-12, wherein both L1 and L2 are protease-cleavable peptide linkers.

14. The construct according to any one of claims 1-12, wherein both L1 and L2 are non-cleavable peptide linkers.

15. The construct according to any one of claims 1-12, wherein L1 is a protease-cleavable peptide linker and L2 is a non-cleavable peptide linker.

16. The construct according to any one of claims 1-12, wherein L1 is a non-cleavable peptide linker and L2 is a protease-cleavable peptide linker.

17. A pharmaceutical composition comprising the construct according to any one of claims 1-16 admixed with a pharmaceutically acceptable carrier.

18. A method for treating cancer or cancer metastasis in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 17.

19. The method according to claim 18, wherein the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma or any cancer.

20. The method according to any one of claims 18 - 19, wherein the method further comprises a second therapeutic agent or a second therapy capable of treating cancer or cancer metastasis in a subject.

21. The method according to claim 20, wherein the second therapy is selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, stem cell transplantation, cell therapy including CAR - T, CAR - NK, iPS - induced CAR - T or iPS - induced CAR - NK, and vaccines such as Bacillus Calmette - Guérin (BCG).

22. The method according to claim 21, wherein the immunotherapy is selected from the group consisting of: treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic antibodies, antagonistic antibodies or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec-7, Siglec-8, Siglec-9, Siglec-15 and VISTA; treatment with bispecific T cell engaging antibodies such as blinatumomab; treatment involving administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β and IFN-γ.

23. A nucleic acid molecule encoding the construct according to any one of claims 1 to 16.

24. An expression vector comprising the nucleic acid molecule according to claim 23.

25. A host cell comprising the expression vector according to claim 24.

26. A method of producing a bio - activatable polypeptide drug construct according to any one of claims 1 to 16, the method comprising culturing the host cell according to claim 25 under conditions that promote the expression of the bio - activatable polypeptide drug construct, and recovering the bio - activatable polypeptide drug construct protein.

27. An isolated bio - activatable polypeptide drug construct protein produced by the method according to claim 26.

28. An isolated interleukin - 2 (IL - 2) fusion protein complex comprising an IL - 2 polypeptide (or a variant thereof) linked to an optimized PD1 - blocking antibody to form an IL - 2 - PD1 - blocking antibody fusion protein, wherein the optimized PD1 - blocking antibody is selected from antibodies comprising: (a) a light chain variable region comprising amino acids having the sequence listed in SEQ ID NO:3 and a heavy chain variable region comprising amino acids having the sequence listed in SEQ ID NO:7; or (b) a light chain variable region comprising amino acids having the sequence listed in SEQ ID NO:3 and a heavy chain variable region comprising amino acids having the sequence listed in SEQ ID NO:9; or (c) a light chain variable region comprising amino acids having the sequence listed in SEQ ID NO:3 and a heavy chain variable region comprising amino acids having the sequence listed in SEQ ID NO:11; (d) a light chain variable region comprising amino acids having the sequence listed in SEQ ID NO:3 and a heavy chain variable region comprising amino acids having the sequence listed in SEQ ID NO:13; or (e) a light chain variable region comprising amino acids having the sequence listed in SEQ ID NO:3 and a heavy chain variable region comprising amino acids having the sequence listed in SEQ ID NO:18, and wherein the optimized PD1 - blocking antibody targets the IL - 2 - PD1 - blocking antibody fusion protein to tumor - infiltrating lymphocytes (TIL).

29. The IL-2-PD1 blocking antibody fusion protein according to claim 28, wherein the IL-2 polypeptide is linked to the C-terminus of the PD1 blocking antibody.

30. The IL-2-PD1 blocking antibody fusion protein according to any one of claims 28-29, wherein the IL-2 variant polypeptide is selected from the group consisting of polypeptides having the amino acid sequences listed in SEQ ID NOs: 117-180.

31. The IL-2-PD1 blocking antibody fusion protein according to any one of claims 28-30, wherein the IL-2 polypeptide is covalently attached to the PD1 blocking antibody through a peptide linker.

32. The IL-2-PD1 blocking antibody fusion protein according to claim 31, wherein the peptide linker is selected from the group consisting of the sequences listed in SEQ ID NOs: 54-115.

33. A pharmaceutical composition comprising the fusion protein according to any one of claims 28-32 admixed with a pharmaceutically acceptable carrier.

34. A method for treating cancer or cancer metastasis in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to claim 33.

35. The method according to claim 34, wherein the cancer is selected from pancreatic cancer, gastric cancer, liver cancer, breast cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer or rhabdomyosarcoma or any cancer.

36. The method according to any one of claims 34-35, wherein the method further comprises a second therapeutic agent or therapy capable of treating cancer or cancer metastasis in the subject.

37. The method according to claim 36, wherein the second therapy is selected from the group consisting of cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiotherapy, stem cell transplantation, cell therapies including CAR-T, CAR-NK, iPS-induced CAR-T or iPS-induced CAR-NK, and vaccines such as Bacillus Calmette-Guérin (BCG).

38. The method according to claim 37, wherein the immunotherapy is selected from the group consisting of: treatment with depleting antibodies against specific tumor antigens; treatment with antibody-drug conjugates; treatment with agonistic, antagonistic or blocking antibodies against co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD-L1, CD40, OX-40, CD137, GITR, LAG3, TIM-3, Siglec-7, Siglec-8, Siglec-9, Siglec-15 and VISTA; treatment with bispecific T cell engaging antibodies such as blinatumomab; treatment involving administration of biological response modifiers such as IL-12, IL-21, GM-CSF, IFN-α, IFN-β and IFN-γ.

39. A nucleic acid molecule encoding the fusion protein according to any one of claims 28-32.

40. An expression vector comprising the nucleic acid molecule according to claim 39.

41. A host cell comprising the expression vector according to claim 40.

42. A method for producing the isolated fusion protein according to any one of claims 28-32, comprising culturing the host cell according to claim 41 under conditions that promote the expression of the fusion protein and recovering the isolated fusion protein.

43. An isolated fusion protein produced by the method according to claim 42.

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