PD1-targeted novel il-2 vitokine fusions

Abstract

The present disclosure provides a PD1 targeted novel IL-2 VitoKine composition, featuring a high-affinity PD1 antibody domain and a bio-activable IL- 2 domain with optimally reduced potency. The PD1 Ab-IL2 VitoKine platform mitigates IL-2 therapy-related toxicities and synergizes the anti-tumor effects of PD1 and IL- 2 by: 1) efficiently targeting an inert IL-2 directly to tumor-infiltrating lymphocytes (TILs); 2) ensuring highly tumorspecific activation of the IL- 2 domain by TME- enriched proteases that co-localized with TILs; 3) allowing PD1 Ab and activated IL- 2 to act on intratumoral CD8+T cells in cis. The VitoKine demonstrates greatly enhanced concealment efficiency and has a lower intrinsic basal activity than what would typically be expected from the VitoKine platform incorporating a non-attenuated IL-2. This improvement permits a substantially higher dose tolerance, enabling safe administration at efficacious dose levels of the PD1 antibody, reversing T- cell anergy or exhaustion and further synergizing with IL-2 mediated anticancer immune response.

Description

PD1 -TARGETED NOVEL IL-2 VITOKINE FUSIONSRelated Patent Applications

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 558,327, filed on February 27, 2024, incorporated in its entirety by reference herein.Sequence Listing

[0002] The contents of the electronic sequence listing (CUGENE-0013-PD1 -1 L-2.xml; Size: 234 Kilobytes; Date of Initial Creation: February 15, 2025; Production Date: February 19, 2025, is herein incorporated by reference in its entirety.Technical Field

[0003] While cancer has been traditionally treated by chemotherapy, radiation, targeted therapies and surgery, a fifth pillar of cancer treatment, immunotherapy, has emerged over the recent years and revolutionized the war on cancer. The benchmark for the immunotherapy drugs has been established by the development of T cell checkpoint inhibitors. It has been demonstrated that these therapies effectively expand and reactivate the pool of tumor-specific T cells leading to objective response rates of up to 50% in patients with certain cancers.

[0004] Interleukin 2 (IL-2) was the first growth factor described for T cells. The ability of IL-2 to expand lymphocyte populations in vivo and to increase the effector functions of these cells confers antitumor effects to IL-2 and led to the approval of high-dose recombinant IL-2 for certain metastatic cancers. While it demonstrated durable responses in approximately 10% of patients, IL-2 cancer immunotherapy is associated with multiple problems, including severe toxicity caused by the induction of vascular leak syndrome (VLS), tumor tolerance caused by inducing activation-induced cell death (AICD), and immunosuppression due to regulatory T cell activation.

[0005] Several approaches have been taken to overcome the challenges inherent in IL-2 immunotherapy. One such approach to counter the systemic toxicity involves localizing cytokine activity to cancer cells and their surrounding tissues by tumor-targeted IL-2 immunocytokines, constructed by fusion of IL-2 to antibodies specific for tumor-associated antigens. However, thisstrategy lacks the ability to specifically target effector T cells within the tumor microenvironment (TME), which are pertinent to anticancer immunity. This gap in intratumoral T cell targeting may be filled by fusing IL-2 to an anti-programmed cell death protein 1 (PD1) antibody. PD1 (also known as CD279) is highly expressed on tumor-infiltrating lymphocytes (TILs), and an PD1 antibody IL-2 fusion enables IL-2 to be directly targeted to TILs. It displays elevated avidity toward intratumoral CD8+ T cells, rather than Treg cells or peripheral CD4+ and CD8+ T cells. This strategy thus further improves IL-2 anticancer immunity while reducing systemic toxicity.

[0006] In addition to targeting IL-2 directly to TILs to improve IL-2 anticancer immunity, PD1 antibodies capable of blocking PD1 and reversing T-cell anergy or exhaustion may synergize with IL-2, further boosting its anticancer immune response. Hence, it is desirable to construct a PD1 Ab-IL-2 fusion with PD1 antibodies of superior target-binding and PD1 blocking capabilities. Among the various globally marketed PD1 blocking antibodies, which have transformed the field of cancer immunotherapy, pembrolizumab (Keytruda®; Merck Sharp & Dohme Corp.) has received remarkable attention due to its high effectiveness and approvals for treating a wide variety of cancer types. While pembrolizumab exhibits superior target binding and blocking capabilities, it has several sequence liabilities, including a relatively low degree of humanness that could raise immunogenicity concerns, and high hydrophobicity that tends to increase its aggregation propensity. It is thus preferable to mitigate its sequence liabilities while fully maintaining its biological activity. The resulting optimized sequence is expected to improve the developability of PD1 Ab-IL-2 fusion proteins.

[0007] Importantly, fusion of a PD1 Ab with a fully active IL-2 moiety may override the intended antibody-mediated targeting, thus localizing the fusion protein to IL-2 receptor-expressing cells in the peripheral instead of TILs in tumors. As such, to improve target specificity and selectivity, one approach is to prepare a fusion using an IL-2 moiety with attenuated IL-2 Re activity to establish a stoichiometric balance between the cytokine and antibody components. Additionally, decreasing the cytokine potency can potentially alleviate pathway over-activation and reduce antigen sink.

[0008] Another related but more sophisticated strategy to improve target specificity and selectivity is the application of the VitoKine platform disclosed by the current inventors in WO2019246392 and WO2021 119516. In a VitoKine construct, the activity of the IL-2 moiety will remain inert or minimal until activated locally by proteases that are upregulated in or around tumors. By doing so, the binding of the IL-2 moiety to its receptors in the peripheral or on the cell-surface of non-diseased cells can be markedly limited. This can help prevent pathway over-activation and reduce undesirable “on-target” “off tissue” toxicity, and the improved safety profile of VitoKines may permit human dose levels within the effective range of PD1 antibodies. The inertness of the IL-2 moiety prior to protease activation will significantly decrease the potential antigen or target sink and thus prolong the in vivo half-life and improve biodistribution and bioavailability at intended sites of therapy. Additionally, incorporating an IL-2 moiety with appropriately attenuated potency can further modulate the intrinsic basal activity of IL-2 VitoKine, thereby enabling the administration of higher doses without adverse effects. This helps fully support PD1 antibody’s function of reversing T-cell anergy or exhaustion, thus potentially improving its synergistic effects with IL-2 immunotherapy and further broadening the therapeutic window.Disclosure of the Invention

[0009] In one aspect, the present invention provides a novel PD1 -targeted bio-activable IL-2 immunocytokine (also referred to herein as “PD1 Ab-IL-2 VitoKine”) that aims to target a bio- activable IL-2 directly to tumor-infiltrating lymphocytes. The activity of the IL-2 moiety will remain nearly inert or minimal until activated locally by proteases that are upregulated in tumors, which will limit binding of the IL-2 moiety to its receptors in the peripheral or on the cell-surface of nondiseased cells or normal tissues. This can help prevent pathway over-activation, reduce undesirable “on-target” “off tissue” toxicity, and minimize unwanted target sink.

[0010] The strategy specifically targets effector T cells within the tumor microenvironment (TME) that are pertinent to anticancer immunity. By implementing this strategy, the ability of IL-2 to expand lymphocyte populations and augment their effector functions is synergized with the function of PD1 blocking antibody in reversing T-cell anergy or exhaustion. This approach, particularly when bio-activatable IL-2 is used, reduces systemic mechanism-based toxicities, leading to broader therapeutic utility of IL-2 for cancer treatment, and improves biodistribution and bioavailability at the intended sites of therapy.

[0011] In various embodiments, the PD1 -targeted bio-activable IL-2 immunocytokine is referred to as PD1 Ab-IL-2 VitoKine herein. In various embodiments, the VitoKine platform disclosed in WO2019246392 and WO2021 119516 by the current inventors is defined by the constructs as depicted in FIG. 1 and one of the proposed methods of activation as depicted in FIG. 2. In various embodiments, 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 thepresent invention comprises a PD1 blocking antibody, a monovalent IL-2 domain (the active moiety domain) with its N-terminus fused to the C-terminus of a heterodimeric Fc chain of the PD1 antibody via the L1 linker and its C-terminus fused to the N-terminus of IL-2Ra sushi domains (the concealing moiety domain) via the L2 linker.

[0012] In various embodiments, the variable domains of the PD1 blocking antibodies of the present invention were optimized from the variable domains of pembrolizumab by introducing germline sequence substitutions to the CDR residues, introducing germline sequence substitutions to the framework somatic mutations, and / or adopting the most prevalent and better behaving VH3 human germline family sequence as the acceptor framework. In various embodiments, the PD1 blocking antibodies have a high affinity for the human PD1 protein as set forth in SEQ ID NO: 1 , function to inhibit PD1 with equal or comparable potency as pembrolizumab, exhibit higher sequence similarity scores to its closest human germline sequence than pembrolizumab, thereby indicating an improved degree of humanness, and are predicted to have lower hydrophobicity than pembrolizumab, which in turn reduce their propensity to aggregate.

[0013] In various embodiments, the PD1 blocking antibody comprises a light chain variable region with the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region with the sequence set forth in SEQ ID NO: 7. In various embodiments, the PD1 blocking antibody comprises a light chain variable region with the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region with the sequence set forth in SEQ ID NO: 9. In various embodiments, the PD1 blocking antibody comprises a light chain variable region with the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region with the sequence set forth in SEQ ID NO: 1 1 . In various embodiments, the PD1 blocking antibody comprises a light chain variable region with the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region with the sequence set forth in SEQ ID NO: 13. In various embodiments, the PD1 blocking antibody comprises a light chain variable region with the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region with the sequence set forth in SEQ ID NO: 18.

[0014] In various embodiments, the active moiety of the PD1 Ab-IL-2 VitoKine is an IL-2 domain comprising the sequence of the 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 derived from the sequence of the mature human IL-2 polypeptide as set forth in SEQ ID NO: 116 comprising one or more amino acid substitution, deletion, or insertion. 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 the 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 moiety designed with reduced / abolished binding to IL-2Ra. In various embodiments, the IL-2 variant has decreased binding activity for I L-2Rpy compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant has both reduced / abolished binding to IL-2Ra and altered binding activity for I L-2R(3y compared to the native IL-2 polypeptide. In various embodiments, the IL-2 variant in the VitoKine construct can tune the IL-2 VitoKine intrinsic basal activity to achieve optimal antitumor efficacy while minimizing unwanted systemic toxicity for broadened therapeutic window. In various embodiments, the IL-2 domain is selected from the group of sequences set forth in SEQ ID NOS: 117-180.

[0015] In various embodiments, the concealing moiety domain is a cognate receptor / binding partner, or any binding partner identified for the IL-2. In various embodiments, the concealing moiety domain is an IL-2Ra extracellular domain with the sequence set forth in SEQ ID NO: 181 or a functional fragment thereof. In various embodiments, the IL-2Ra extracellular domain or a functional fragment thereof is an IL-2Ra sushi domain with the sequence set forth in SEQ ID NO: 182. In various embodiments, the concealing moiety domain is a variant (mutant) of IL- 2RoSushi domain. In various embodiments, the amino acid change is one or more amino acid substitutions at position 6, 7, 36, 38, 42, or 43 of SEQ ID NO: 182. In various embodiments, the amino acid change is the substitution of D to E or N at position 6, P to G or A or S at position 7, R to A at position 36, K to E, at position 38, L to G at position 42, Y to A at position 43, or any combination of these substitutions. In various embodiments, the variant (mutant) of IL-2RaSushi domain is designed to facilitate dissociation and diffusion away after proteolytic cleavage. In various embodiments, the variant (mutant) of IL-2RaSushi domain is designed to mitigate liable sequence. In various embodiments, the variant (mutant) of IL-2RaSushi domain designed to mitigate liable sequence has improved concealing efficiency. In various embodiments, the variant (mutant) of IL-2RaSushi domain is selected from the group of sequences set forth in SEQ ID NOS: 183-185 and 210-214.

[0016] In various embodiments, the L1 linker and L2 linker of the PD1 Ab-IL-2 VitoKine constructs are both a protease cleavable peptide linker. In various embodiments, L1 of the PD1Ab-IL-2 VitoKine constructs is a protease cleavable peptide linker and L2 is a non-cleavable peptide linker. In various embodiments, L1 of the PD1 Ab-IL-2 VitoKine constructs is a non- cleavable peptide linker and L2 is a protease cleavable peptide linker. In various embodiments, L1 linker and L2 linker of the PD1 Ab-IL-2 VitoKine constructs are both a protease non- cleavable peptide linker. In various embodiments, the non-cleavable linker is rich in G / S content (e.g., of 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 selected independently. In various embodiments, the protease cleavable linker is selected from the group of sequences set forth in SEQ ID NOS: 54- 77. In various embodiments, the protease cleavable linker can have additional peptide spacer of variable length on the N-terminus of the cleavable linker or on the C-terminus of the cleavable linker or on both termini of the cleavable linker to improve accessibility for enzymatic cleavage. In various embodiments, the protease cleavable linker with peptide spacer of variable length on either the N-terminus or on the C-terminus or on both termini of the cleavable linker is selected from the group of sequences set forth in SEQ ID NOS: 78-94. In various embodiments, the non- cleavable linker is selected from the group of sequences set forth in SEQ ID NOS: 95-115. In various embodiments, the linker is either flexible or rigid and of a variety of lengths.

[0017] In various embodiments, the IL-2 domain (D2) and IL-2Ra domain (D3) of the VitoKine construct are placed at the C-terminus of the PD1 Ab domain (D1) as depicted in FIG. 1 A. In various embodiments, the D2 and D3 domains of the VitoKine construct are placed at the N- terminus of the D1 domain as depicted in FIG. 1 B.

[0018] In various embodiments, the PD1 blocking Ab, IL-2 domain and IL-2Ra domains of the PD1 Ab-IL-2 VitoKine construct can be monomer or dimer or a combination of dimer and monomer, such as PD1 blocking Ab is dimer and IL-2 domain and IL-2Ra domains are monomer.

[0019] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to a subject in need thereof. 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-neck cancer, or rhabdomyosarcoma or any cancer.

[0020] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount ofthe pharmaceutical compositions of the invention in combination with a second therapy selected from the group consisting of: cytotoxic chemotherapy, immunotherapy, small molecule kinase inhibitor targeted therapy, surgery, radiation therapy, stem cell transplantation, cell therapies including chimeric antigen receptor (CAR)-T, CAR-NK, induced pluripotent stem cells (iPS) induced CAR-T or iPS induced CAR-NK and vaccine such as Bacille Calmette-Guerine (BCG). In various embodiments, the combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to 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 using bispecific T cell engaging antibodies (BiTE®) such as blinatumomab: treatment involving administration of biological response modifiers such as IL-12, IL-21 , GM-CSF, IFN-a, IFN-(3 and IFN-y; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines, or tumor antigen peptide vaccines; treatment using CAR-T cells; treatment using CAR-NK cells; treatment using tumor infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR transgenic); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG and imiquimod; and treatment using vaccine such as BCG; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., a synergy exists between the VitoKine constructs and the immunotherapy when co-administered.

[0021] In another aspect, the disclosure provides uses of the pharmaceutical compositions of the invention for the preparation of a medicament for the treatment of cancer.

[0022] In another aspect, the present disclosure provides isolated nucleic acid molecules comprising a polynucleotide encoding of the pharmaceutical compositions of the present disclosure. In another aspect, the present disclosure provides vectors comprising the nucleic acids described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides isolated cells comprising the nucleic acids of the disclosure. In various embodiments, the cell is a host cell comprising the expression vector of the disclosure. In another aspect, methods of making the VitoKine constructs are provided by culturing the host cells under conditions promoting expression of the proteins or polypeptides.

[0023] In another aspect, the present disclosure provides a pharmaceutical composition comprising the isolated pharmaceutical compositions of the invention in admixture with a pharmaceutically acceptable carrier.Brief Description of the Figures

[0024] FIG. 1 depicts representative VitoKine construct formats. FIG. 1 A depicts VitoKine construct with the D2 (active moiety domain) and D3 (concealing moiety domain) being placed at the C-terminus of the D1 (targeting domain). FIG. 1 B depicts VitoKine construct with the D2 and D3 domains being placed at the N-terminus of the D1 domain.

[0025] FIG. 2 depicts the proposed VitoKine activation mechanism of the present invention using the exemplary VitoKine construct comprising two protease-cleavable linkers. Protease 1 activation resulted from cleavage of L1 linker yields Active Form 1 ; protease 2 activation resulted from cleavage of L2 linker yields Active Form 2; activation by both proteases resulted from cleavage of L1 and L2 linkers yields Active Form 3. Following protease cleavage, the concealing moiety domain (D3) will be released and diffuse away from the active moiety domain (D2). If the L1 linker is the only protease-cleavable linker, then Active Form 1 will be the sole activated format. Similarly, if L2 linker is the only protease-cleavable linker, then Active Form 2 will be the singular activated format.

[0026] FIG. 3A depicts representative PD1 Ab-IL-2 VitoKine construct of the present invention. A monomeric IL-2 or IL-2 variant as the active moiety domain (D2) is fused at its N-terminus to the C-terminus of a PD1 antibody heterodimeric Fc (D1 ) with L1 linker; the C-terminus of the IL- 2 moiety is fused to the N-terminus of IL-2Ra or IL-2Ra variant as the concealing moiety domain (D3) with L2 linker. FIG. 3B depicts the non-concealed counterpart of a PD1 Ab-IL-2 VitoKine construct. The structure also closely resembles the Active Form 2 of a PD1 Ab-IL-2 VitoKine.

[0027] FIG. 4A depicts a comparison of the PD1 blocking activity between the Reference Antibody (P-0734) and pembrolizumab (PBL) biosimilar (dose-dependent increases in luminescence signal) in a luciferase reporter assay. FIG. 4B depicts a comparison of the PD1 blocking activity between the Reference Antibody (P-0734) and pembrolizumab (PBL) biosimilar (dose-dependent increases in fold induction) in a luciferase reporter assay. P-0734 and PBL biosimilar share the identical variable domains and have IgG 1 and lgG4 isotypes, respectively.

[0028] FIG. 5A depicts the ELISA binding of the PD1 blocking antibodies, P-1148, P-1150, P- 1151 , and P-1153, compared to the Reference Antibody P-0734. FIG. 5B depicts PD1 blockadeactivity (dose-dependent increases in luminescence signal) in a luciferase reporter assay of the PD1 blocking antibodies, P-1148, P-1150, P-1151 , and P-1 153, compared to the Reference Antibody P-0734. FIG. 5C depicts PD1 blockade activity (dose-dependent increases in fold induction) in a luciferase reporter assay of the PD1 blocking antibodies, P-1 148, P-1 150, P- 1151 , and P-1153, compared to the Reference Antibody P-0734.

[0029] FIG. 6A, FIG. 6B and FIG. 60 depict the PD1 blockade activity (dose-dependent increases in luminescence signal) in a luciferase reporter assay of the PD1 blocking antibodies, P-1127, P-1 129, and P-1174, compared to the Reference Antibody (P-0734).

[0030] FIG. 7A depicts the PD1 blockade activity (dose-dependent increases in luminescence signal) in a luciferase reporter assay of the PD1 blocking antibodies, P-1175 and P-1181 , compared to the Reference Antibody (P-0734). FIG. 7B depicts the PD1 blockade activity dosedependent increases in fold induction) in a luciferase reporter assay of the PD1 blocking antibodies, P-1175 and P-1181 , compared to the Reference Antibody (P-0734).

[0031] FIG. 8A depicts the PD1 blockade activity (dose-dependent increases in luminescence signal) in a luciferase reporter assay of the PD1 blocking antibodies, P-1175, P-1 176, P-1 177, and P-1178, compared to the Reference Antibody (P-0734). FIG. 8B depicts the PD1 blockade activity dose-dependent increases in fold induction) in a luciferase reporter assay of the PD1 blocking antibodies, P-1175, P-1176, P-1177, and P-1 178, compared to the Reference Antibody (P-0734).

[0032] FIG. 9A depicts the PD1 blockade activity (dose-dependent increases in luminescence signal) in a luciferase reporter assay of the PD1 blocking antibodies, P-1198, P-1 199, and P- 1201 , compared to the Reference Antibody (P-0734). FIG. 9B depicts the PD1 blockade activity dose-dependent increases in fold induction) in a luciferase reporter assay of the PD1 blocking antibodies, P-1198, P-1199, and P-1201 , compared to the Reference Antibody (P-0734). A nontargeting germline antibody was included as the negative control.

[0033] FIG. 10A depicts the PD1 blockade activity (dose-dependent increases in luminescence signal) in a luciferase reporter assay of the PD1 blocking antibodies, P-1194, P-1201 , and P- 1238, compared to the Reference Antibody (P-0734). FIG. 10B depicts the PD1 blockade activity dose-dependent increases in fold induction) in a luciferase reporter assay of the PD1 blocking antibodies, P-1194, P-1201 , and P-1238, compared to the Reference Antibody (P- 0734).

[0034] FIG. 1 1 A depicts the binding (dose-dependent increases in the percentage of positive cells) of the PD1 blocking antibody, P-1174, to PD1+HEK293 cells, compared to the ReferenceAntibody (P-0734). FIG. 11 B depicts the binding (dose-dependent increases in mean fluorescence intensity (MFI)) of the PD1 blocking antibody, P-1174, compared to the Reference Antibody (P-0734). FIG. 110 depicts the binding (dose-dependent increases in the percentage of positive cells) of the PD1 blocking antibodies, P-1 193, P-1198, P-1199, and P-1201 , to PD1+HEK293 cells, compared to the Reference Antibody (P-0734). FIG. 11 D depicts the binding (depict dose-dependent increases in mean fluorescence intensity (MFI)) of the PD1 blocking antibodies, P-1193, P-1198, P-1 199, and P-1201 , to PD1+HEK293 cells, compared to the Reference Antibody (P-0734). A non-targeting germline antibody was included as the negative control.

[0035] FIG. 12 depicts the ELISA binding of IL-2RaSushi variants, P-0751 , P-0752, and P- 0753, to IL-2. P-0757 comprises wild-type IL-2RaSushi and was included for comparison.

[0036] FIG. 13A depicts the activity assessment (activity was assessed by analyzing the dosedependent induction of Ki67 expression on CD8+ T cells of human PBMCs using flow cytometry) of Fc IL-2 VitoKine constructs with either wild-type IL-2RaSushi as the D3 domain (P- 0701 ) or IL-2RaSushi variants as the D3 domain (P-0754, P-0755, and P-0756). P-0704, an IL-2 variant Fc fusion protein was included as a fully active IL-2 control. FIG. 13B depicts the activity assessment (activity was assessed by analyzing the dose-dependent induction of Ki67 expression on NK cells of human PBMCs using flow cytometry) of Fc IL-2 VitoKine constructs with either wild-type IL-2RaSushi as the D3 domain (P-0701) or IL-2RaSushi variants as the D3 domain (P-0754, P-0755, and P-0756). P-0704, an IL-2 variant Fc fusion protein was included as a fully active IL-2 control.

[0037] FIG. 14A depicts ELISA binding of IL-2 variants, P-0608, P-0633, P-0531 to IL-2Ra. FIG. 14B depicts ELISA binding of IL-2 variants, P-0706, P-0689, to IL-2Ra. FIG. 14C depicts ELISA binding of IL-2 variants, P-0531 , P-0689, P-0634, P-0708, P-0709 to IL-2Ra. FIG. 14D depicts ELISA binding of IL-2 variants, P-0689, P-0635, P-0704, P-0707, and Benchmark to IL-2Ra. Each IL-2 variant harbors different amino acid substitution at position P65 (refer to Table 18 for details on molecular information). P-0531 and P-0689 serve as the wild-type IL-2 control in bivalent and monovalent forms, respectively.

[0038] FIG. 15A depicts the potency of IL-2 P65 variants, P-0531 M P-0608, P-0634, P-0635, and Benchmark in stimulating STAT5 phosphorylation in CD4+ Treg cells (refer to Table 18 for details on molecular information). FIG. 15B depicts the potency of IL-2 P65 variants, P-0689, P- 0709, P-0704, and Benchmark in stimulating STAT5 phosphorylation in CD4+ Treg cells (refer to Table 18 for details on molecular information). P-0531 and P-0689 serve as the wild-type IL-2 control in bivalent and monovalent forms, respectively. Similarly, Benchmark (D) and Benchmark are bivalent and monovalent Fc fusions of IL-2 F42A / Y45A / L72G variant, respectively.

[0039] FIG. 16A depicts the activity of IL-2 P65 variants, P-0531 , P-0608, P-0624, P-0626, P- 0634, P-0635M and P-0551 toward IL-2R0y by analyzing ELISA binding to a recombinantly expressed IL-2 receptor subunits p and y complex. FIG. 16B depicts the activity of IL-2 P65 variants, P-0531 , P-0608, P-0624, P-0626, P-0634, P-0635M and P-0551 toward IL-2Rpy by analyzing the induction of Ki67 expression on CD8+ T cells in fresh human PBMCs using flow cytometry. Refer to Table 18 for details on molecular information of IL-2 variants. P-0531 and P- 0689 serve as the wild-type IL-2 control in bivalent and monovalent forms, respectively.

[0040] FIG. 17A depicts the activity assessment (analyzing the induction of Ki67 expression on CD8+ T cells) of various mouse PD1 Ab-IL-2 VitoKines, P-0800, P-0830, P-0831 , and P-0802, in comparison to P-0782, a non-concealed counterpart as a fully active IL-2 control. FIG. 17B depicts the activity assessment (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of various mouse PD1 Ab-IL-2 VitoKines, P-0800, P-0830, P-0831 , and P- 0802, in comparison to P-0782, a non-concealed counterpart as a fully active IL-2 control. The four IL-2 antibody VitoKines differ only in the binding strength of their IL-2 domains to IL-2Ra.

[0041] FIG. 18A depicts the activity assessment (analyzing the induction of Ki67 expression on CD8+ T cells) of IL-2 variants, P-0731 , P-0759, and P-0761 , harboring mutations that interfere their binding to IL-2R|3. FIG. 18B depicts the activity assessment (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of IL-2 variants, P-0731 , P-0759, and P- 0761 , harboring mutations that interfere their binding to IL-2R|3. All these IL-2 variants also contains the P65R mutation, which eliminates binding to IL-2Ro. P-0704 is utilized as a fully active IL-2 control.

[0042] FIG. 19A depicts the activity assessment (analyzing the induction of Ki67 expression on CD8+ T cells) of IL-2 variants, P-0704, P-1 158, P-0750, P-1156, and P-1157, harboring mutations that interfere their binding to yc. FIG. 19B depicts the activity assessment (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of IL-2 variants, P-0704, P-1158, P-0750, P-1156, and P-1 157, harboring mutations that interfere their binding to yc. FIG. 19C depicts the activity assessment (analyzing the induction of Ki67 expression on CD8+ T cells) of IL-2 variants, P-0704, P-1280, P-1283, P-1279, P-1278, P-1282 and P-1281 , harboring mutations that interfere their binding to yc. FIG. 19D depicts the activity assessment (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of IL-2 variants, P-0704,P-1280, P-1283, P-1279, P-1278, P-1282 and P-1281 , harboring mutations that interfere their binding to yc. FIG. 19E depicts the activity assessment (analyzing the induction of Ki67 expression on CD8+ T cells) of IL-2 variants, P-0704, P-1359, P-1354, P-1355, P-0732, P-1357, P-1358, P-1356, and P-1353, harboring mutations that interfere their binding to yc. FIG. 19F depicts the activity assessment (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of IL-2 variants, P-0704, P-1359, P-1354, P-1355, P-0732, P-1357, P-1358, P- 1356, and P-1353, harboring mutations that interfere their binding to yc. All these IL-2 variants also possess the P65R mutation, which eliminates IL-2 Ra binding. P-0704 functions as a fully active IL-2 control.

[0043] FIG. 20A depicts the activity assessment (analyzing the induction of Ki67 expression on CD8+ T cells) of the IL-2 variant harboring mutations targeting both IL-2R|3 and yc (P-1247) in comparison IL-2 variants containing only yc-disrupting mutation (P-1158). FIG. 20B depicts the activity assessment (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of the IL-2 variant harboring mutations targeting both IL-2Rp and yc (P-1247) in comparison IL-2 variants containing only yc-disrupting mutation (P-1158). P-0704 serves as a fully active IL-2 control.

[0044] FIG. 21 A depicts PD1 blockade activity (dose-dependent increases in luminescence signals), of the PD1 blocking antibodies, P-1 174, P-1238, and P-1271 , in comparison to their respective PD1 Ab-IL-2 VitoKines, P-1197, P-1239, and P-1272 & P-1461 , in a luciferase reporter assay. FIG. 21 b depicts PD1 blockade activity (dose-dependent increases in fold induction) of the PD1 blocking antibodies, P-1174, P-1238, and P-1271 , in comparison to their respective PD1 Ab-IL-2 VitoKines, P-1197, P-1239, and P-1272 & P-1461 , in a luciferase reporter assay.

[0045] FIG. 22A depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of PD1 Ab-IL-2 VitoKines P-0872, P-1197, and P-1174 in comparison to their corresponding non-concealed counterpart, P-0879. FIG. 22B depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of PD1 Ab-IL-2 VitoKines P-0872, P-1197, and P-1174 in comparison to their corresponding non-concealed counterpart, P-0879. FIG. 22C depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of PD1 Ab-IL-2 VitoKine P- 1272 in comparison to its corresponding non-concealed counterpart, P-1273. FIG. 22D depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on NK cells of freshhuman PBMCs) of PD1 Ab-IL-2 VitoKine P-1272 in comparison to its corresponding nonconcealed counterpart, P-1273.

[0046] FIG. 23A depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of mouse PD1 Ab-IL-2 VitoKines, P-0831 , P-1410, and P-1414, in comparison to their respective non-concealed counterparts, P-0838, P-1409, and P-1413. FIG. 23B depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of mouse PD1 Ab-IL-2 VitoKines, P-0831 , P-1410, and P-1414, in comparison to their respective non-concealed counterparts, P-0838, P-1409, and P-1413. FIG. 23C depicts the proliferation of mouse CTLL-2 cells of mouse PD1 Ab-IL-2 VitoKines, P-0831 , P-1410, and P-1414, in comparison to their respective non-concealed counterparts, P-0838, P- 1409, and P-1413.

[0047] FIG. 24A depicts the evaluation of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of human PD1 Ab-IL-2 VitoKine P-1272 samples expressed in two host cells: HEK-239 (default) or CHO-K1 (indicated with “CHO” in sample name) compared to the non-concealed counterpart, P-1273. FIG. 24B depicts the evaluation of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of human PD1 Ab-IL-2 VitoKine P- 1441 , a recombinant version of P-1272 cleaved at the6Asp-7Pro site of the IL-2Ra domain compared to the non-concealed counterpart, P-1273.

[0048] FIG. 25A depicts the proliferation of mouse CTLL-2 cells of exemplary human PD1 Ab- IL-2 VitoKines P-1272, P-1439, P-1452, P-1453 (designed with various mutations replacing the susceptible6Asp-7Pro sequence in the IL-2RaSushi domain of P-1272), compared to the nonconcealed counterpart, P-1273. FIG. 23B depicts the proliferation of mouse CTLL-2 cells of exemplary human PD1 Ab-IL-2 VitoKines P-1272, P-1439, P-1452, P-1453 (designed with various mutations replacing the susceptible6Asp-7Pro sequence in the IL-2RaSushi domain of P-1272), compared to the non-concealed counterpart, P-1273. FIG. 25C depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of exemplary human PD1 Ab-IL-2 VitoKines P-1272, P-1439, P-1452, P-1453 (designed with various mutations replacing the susceptible6Asp-7Pro sequence in the IL-2RaSushi domain of P-1272), compared to the non-concealed counterpart, P-1273.

[0049] FIG. 26A depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of human PD1 Ab-IL-2 VitoKines, P-1439, and P-1451 , in comparison to their respective non-concealed counterparts, P-1273 and P-1448. FIG. 26B depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ Tcells) of human PD1 Ab-IL-2 VitoKines, P-1452, and P-1461 , in comparison to their respective non-concealed counterpart, P-1273 and P-1448. FIG. 26C depicts the proliferation of mouse CTLL-2 cells of human PD1 Ab-IL-2 VitoKines, P-1272, P-1439, P-1451 in comparison to their respective non-concealed counterparts, P-1273 and P-1448. FIG. 26D depicts the proliferation of mouse CTLL-2 cells of human PD1 Ab-IL-2 VitoKines, P-1272, P-1452, P-1461 in comparison to their respective non-concealed counterparts, P-1273 and P-1448. Human PD1 Ab-IL-2 VitoKines engineered with mutations (IL-2Ro D6E in P-1439 and D6N in P-1452) replacing the susceptible6Asp-7Pro sequence in the IL-2RaSushi domain. The incorporation of a yc-disrupting mutation, Q126R, in the IL-2 domain resulted in P-1451 and P-1461 from P-1439 and P-1452, respectively. P-1273, the non-concealed counterpart of P-1439 and P-1452, and P- 1448, corresponding to P-1451 and P-1461 , are included for comparison.

[0050] FIG. 27A depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of PD1 Ab-IL-2 VitoKines, P-1461 (expressed in HEK-239) and P- 1461 -CHO (expressed in CHO-K1 ), in comparison to their respective non-concealed counterpart, P-1448. FIG. 27B depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of PD1 Ab-IL-2 VitoKines, P-1461 (expressed in HEK-239) and P-1461 -OHO (expressed in CHO-K1) in comparison to their respective non-concealed counterpart, P-1448. FIG. 27C depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of PD1 Ab-IL-2 VitoKines, P-1414 (expressed in HEK-239) and P-1481-CHO (expressed in CHO-K1) in comparison to their respective non-concealed counterpart, P-1413. FIG. 27D depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of PD1 Ab-IL-2 VitoKines, P-1414 (expressed in HEK-239) and P-1481 -CHO (expressed in CHO-K1 ) in comparison to their respective non-concealed counterpart, P-1413. P-1481 differ from P-1414 by a single IL-2Ra D6N mutation to remove the susceptible6Asp-7Pro sequence in the IL- 2RoSushi domain. P-1461 (expressed in HEK-293) and P-1448 are included in FIG. 27C and FIG. 27D for comparison.

[0051] FIG. 28A depicts the PD1 blockade activity (dose-dependent increases in luminescence signal) in a luciferase reporter assay of the PD1 Ab-IL-2 VitoKines P-1461 compared to its component antibody P-1271 . FIG. 28B depicts the PD1 blockade activity dose-dependent increases in fold induction) in a luciferase reporter assay of the PD1 Ab-IL-2 VitoKines P-1461 compared to its component antibody P-1271 . A non-targeting germline antibody was included as the negative control.

[0052] FIG. 29A depicts an SDS-PAGE gel of both intact and protease activated VitoKines P- 1272 and P-1461 in the presence of reducing agent. FIG. 29B depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of PD1 Ab-IL-2 VitoKines, P-1272 and P-1272-Act, in comparison to their respective non-concealed counterpart, P-1273. FIG. 29C depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of PD1 Ab-IL-2 VitoKines, P-1461 and P-1461 -Act, in comparison to their respective non-concealed counterpart, P-1448. FIG. 29D depicts the proliferation of mouse CTLL-2 cells of PD1 Ab-IL-2 VitoKines, P-1461 and P-1461-Act, in comparison to their respective non-concealed counterpart, P-1448.

[0053] FIG. 30A depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of PD1 Ab-IL-2 VitoKines, P-0831 and P-0831 -Act, in comparison to their respective non-concealed counterpart, P-0831 . FIG. 30B depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of PD1 Ab-IL-2 VitoKines, P-1345 and P-1345-Act, in comparison to their respective non-concealed counterpart, P-0838. P-0831 and P-1345 are identical except for their linkers: P-0831 features protease-cleavable L2 linker, while P-1345 contains a cleavable L1 linker.

[0054] FIG. 31 depicts the pharmacokinetic (PK) response of mouse PD1 Ab-IL-2 VitoKine P- 0831 and its non-concealed counterpart, P-0838, following a single intraperitoneal injection in C57B / L6 mice. Blood was collected from mice at multiple time points post-dosing and ELISA assays were used to determine the serum levels of each compound.

[0055] FIG. 32A depicts the dose and time-dependent pharmacodynamic (PD) effects of a single dose of P-0831 on the expansion of CD8+ T cells in peripheral blood from treated C57B / L6 mice. FIG. 32B depicts the dose and time-dependent pharmacodynamic (PD) effects of a single dose of P-0831 on the expansion of granzyme B+ CD8+ T cells in peripheral blood from treated C57B / L6 mice. FIG. 32C depicts the dose and time-dependent pharmacodynamic (PD) effects of a single dose of P-0831 on the expansion of NK cells in peripheral blood from treated C57B / L6 mice. FIG. 32D depicts the dose and time-dependent pharmacodynamic (PD) effects of a single dose of P-0831 on the expansion of granzyme B+ NK cells in peripheral blood from treated C57B / L6 mice. Blood was collected on Days 0, 3, 5, 7 and 10 for lymphocyte phenotyping by FACS analysis. The non-concealed counterpart, P-0838, was included for comparison. The Data are expressed as mean ± standard error of the mean (SEM).

[0056] FIG. 33A depicts the time course of CD8+ T cell expansion in the tumor, blood, spleen, and lymph node following a single injection of P-1414 at 30 mg / kg in Panc02 tumor-bearingmice. FIG. 33B depicts the time course of CD8+ T cell expansion in the tumor, blood, spleen, and lymph node following a single injection of P-1413 at 3 mg / kg in Panc02 tumor-bearing mice. Each group (n=3) was sacrificed on Days 0, 3, 5, 7, and 10 to collect blood and harvest tumors, spleens, and lymph nodes for lymphocyte phenotyping by FACS analysis. Data are presented as the fold change in cell count relative to the vehicle group, expressed as mean ± SEM.

[0057] FIG. 34A depicts the increase in serum IFNy levels following a single dose of mouse PD1 Ab-IL-2 VitoKine P-0831 compared to its corresponding non-concealed counterpart, P- 0838, administered at various dose levels to Naive C57BL / 6 mice. FIG. 34B depicts the increase in serum IFNy levels following a single dose of mouse PD1 Ab-IL-2 VitoKine P-1414 compared to its corresponding non-concealed counterpart, P-1413, administered at various dose levels to CT26 tumor-bearing Balb / c mice. In both studies, the vehicle (PBS) and the component mouse PD1 antibody, P-0722, served as negative controls. FIG. 34C depicts body weight changes following a single dose of mouse PD1 Ab-IL-2 VitoKine P-0831 compared to its corresponding non-concealed counterpart, P-0838, administered at various dose levels to Naive C57BL / 6. mice.

[0058] FIG. 35A depicts the antitumor effects of mouse PD1 Ab-IL-2 VitoKine P-0831 in established MC38 murine colon carcinoma model compared to its respective non-concealed counterparts, P-0838. FIG. 35B depicts the antitumor effects of mouse PD1 Ab-IL-2 VitoKine P- 1414 in established MC38 murine colon carcinoma model compared to its respective nonconcealed counterparts, P-1413. The PD1 antibody P-0722 matching the highest VitoKine dose for each study was included as a control. The mean tumor volume ± SEM over time for each treatment group is illustrated.

[0059] FIG. 36 depicts the absence of tumor recurrence following MC38 colon carcinoma cell rechallenge in tumor-free mice treated with P-0831 or P-1414 at 6 mg / kg. This contrasted with the tumor regrowth in age-matched naive mice as a control.

[0060] FIG. 37A depicts the immunohistochemistry (IHC) analysis of the PD effects of the mouse PD1 Ab-IL-2 VitoKine P-0831 on immune cell expansion in tumor tissues. FIG. 37B depicts the immunohistochemistry (IHC) analysis of the PD effects of the mouse PD1 Ab-IL-2 VitoKine P-1414 on immune cell expansion in tumor tissues. Tumors are collected five days after a single dose of P-0831 or P-1414 at 6 mg / kg for IHC analysis. The component PD1 antibody P-0722 dosed at 6 mg / kg and P-0831 ’s non-concealed counterpart P-0838 dosed at 1 mg / kg were included for comparison.

[0061] FIG. 38A depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of mouse PD1 Ab-IL-2 VitoKine P-0831 to its non-cleavable Vitokine counterpart P-0877. FIG. 38B depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of mouse PD1 Ab-IL-2 VitoKine P-0831 to its non-cleavable Vitokine counterpart P-0877. FIG. 38C depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on CD8+ T cells) of mouse PD1 Ab-IL-2 VitoKine P-1414 to its non-cleavable Vitokine counterpart P-1446. FIG. 38D depicts the assessment of IL-2 activity (analyzing the induction of Ki67 expression on NK cells of fresh human PBMCs) of mouse PD1 Ab-IL-2 VitoKine P-1414 to its non-cleavable Vitokine counterpart P-1446. P-0879 and P-1413 served as controls for non-concealed IL-2 of P-0831 and P-1446, respectively.

[0062] FIG. 39A depicts the CT26 tumor growth curves (volume ± SEM over time) for P-0831 compared to its non-cleavable VitoKine counterpart, P-0877, following two doses of 10 mg / kg every 12 days. FIG. 39B depicts the CT26 tumor growth curves (volume ± SEM over time) for P- 1414 compared to its non-cleavable VitoKine counterpart, P-1446, following two doses of 30 mg / kg every two weeks (Q2W). FIG. 39C depicts the Panc02 tumor model growth curves (volume ± SEM over time) for P-1414 compared to its non-cleavable VitoKine counterpart, P- 1446, following two doses of 30 mg / kg every two weeks (Q2W). P-0722, the component mouse PD1 antibody, was included as a control and dosed to match the dosing level and schedule for each study.

[0063] FIG. 40A depicts the effect of mouse PD1 Ab-IL-2 VitoKine P-1414 on inhibiting tumor growth and expanding immune cells within tumor tissues compared to its non-cleavable VitoKine counterpart, P-1446, in an established MC38 mouse tumor model. The anti-tumor effect is shown as mean tumor volume ± SEM over time for each treatment group after two Q2W doses of 6 mg / kg. FIG. 40B depicts the effect of mouse PD1 Ab-IL-2 VitoKine P-1414 on inhibiting tumor growth and expanding immune cells within tumor tissues compared to its non- cleavable VitoKine counterpart, P-1446, in an established MC38 mouse tumor model. Tumor immune cell expansion was analyzed by immunohistochemistry staining of tumor tissues collected 5 days after a single 6 mg / kg dose. P-0722, the component mouse PD1 antibody, was included as a control.

[0064] FIG. 41 A illustrates the CT26 tumor growth curves (volume ± SEM over time) for P-0831 compared to P-0871 , following two doses of 10 mg / kg every 12 days. FIG. 41 B shows the MC38 tumor growth curves for P-1414 compared to P-1469 group after two Q2W doses of 6mg / kg. An additional group with a combination treatment of P-1469 and P-0722, both at 6 mg / kg was included in the MC38 tumor model. P-0722, the component mouse PD1 antibody, was included as a control and dosed to match the dosing level and schedule for each study.

[0065] FIG. 42A depicts changes in body weight following three Q2W doses of the mouse PD1 Ab-IL-2 VitoKine P-1481 at varying dose levels in naive C57BL / 6 mice. FIG. 42B depicts the increases in serum IFNy levels 48 hours after the first dose. Four dose levels of P-1481 (10, 30, 60, and 120 mg / kg) were administered, with vehicle (PBS) as the negative control. The serum IFNy levels in response to P-0838 and P-0831 treatment, as depicted in FIG. 34A, are included for comparison.Mode(s) for Carrying out the Disclosure

[0066] The present disclosure provides PD1 Ab-IL-2 VitoKine constructs which comprise three domains: 1 ) an optimized PD1 blocking antibody as the TIL-targeting moiety, 2) an IL-2 variant as the active moiety domain, and 3) an IL-2 Ra sushi variant as the concealing moiety domain. Importantly, the IL-2 Ra sushi variant domain is capable of concealing or attenuating the functional activity of IL-2 domain until activated at the intended site of therapy.

[0067] The PD1 blocking antibody guides the VitoKine to the TILs in the tumor microenvironment and restricts the activation of the VitoKine locally to improve the therapeutic index. In various embodiments, the PD1 blocking antibodies were optimized through modifications in the variable domains of pembrolizumab. In various embodiments, the modifications involved germline sequence substitutions of the CDR residues, germline sequence substitutions of the framework residues, and adoption of the VH3 human germline family sequence as the acceptor framework. In various embodiments, these modifications were implemented individually or in combination to develop optimized PD1 blocking antibodies. In various embodiments, these optimized PD1 blocking antibodies exhibit a high binding affinity to PD1 , function to inhibit PD1 with equal or comparable potency as pembrolizumab, have a higher sequence similarity score to its closest human germline sequence, resulting in an improved degree of humanness compared to pembrolizumab, and are predicted to have lower hydrophobicity, leading to lowered aggregation propensity. In various embodiments, PD1 Ab-IL- 2 VitoKine constructs based on these optimized PD1 blocking antibodies have enhanced developability properties.

[0068] In various embodiments, the IL-2 domain is the active moiety but remains inert until activated locally by proteases that are upregulated in diseased tissues; this will limit binding of the active moiety to the receptors in the peripheral or on the cell-surface of non-diseased cells or tissue to prevent over-activation of the pathway and reduce undesirable “on-target” “off tissue” toxicity. The improved safety profile of the VitoKines may permit human dose levels within the effective range of a PD1 antibody. Additionally, the inertness of the VitoKine active moiety prior to protease activation will significantly decrease the potential antigen sink, and thus, prolong the in vivo half-life and result in improved biodistribution, bioavailability and efficacy at intended sites of therapy.

[0069] In various embodiments, the integration of a potency-attenuated IL-2 variant, achieved by disrupting IL-2Rpy interaction, as the active moiety domain can further fine-tune the intrinsic basal activity and post-activation activity of the VitoKine. In various embodiments, such VitoKine with appropriately modulated intrinsic basal activity enables higher dose administration without adverse effects, thereby fully supporting PD1 antibody’s function of reversing T-cell anergy or exhaustion and potentially improving the synergistic effects with IL-2 immunotherapy, resulting in additionally expanded therapeutic index.

[0070] In various embodiments, the unique and non-signaling oc-subunit of receptors of IL-2 is used as the concealing moiety domain via a protease-cleavable linker to reversibly conceal the cytokine activity. The concealing oc-subunit may be preferred to dissociate away after protease cleavage of the linker. As a result, amino acid modifications of the oc-receptor to modulate the binding affinity to IL-2 may be beneficial. In various embodiments, the variant (mutant) of IL- 2RoSushi domain is designed to facilitate dissociation and diffusion away after proteolytic cleavage. In various embodiments, the variant (mutant) of IL-2RaSushi domain is designed to mitigate liable sequence. In various embodiments, the variant (mutant) of IL-2RaSushi domain designed to mitigate liable sequence has improved concealing efficiency.

[0071] In various embodiments, in the PD1 Ab-IL-2 VitoKine constructs, the three domains are linked 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 in the disease sites, thus allowing for a functional IL-2 domain to be revealed or released at the site of disease. The linker length and composition were fine-tuned to ensure optimal concealment of the IL-2 domain from accessing its receptors, thus minimizing systemic engagement. Meanwhile, the stability of theVitoKine construct in the blood circulation was maintained while allowing efficient cleavage upon encountering specific proteases at the intended site of therapy.Definitions

[0072] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), incorporated herein by reference.Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly used and well known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.

[0073] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, "peptides", "polypeptides", and "proteins" are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free a-amino group on an amino acid at the amino terminal of a peptide or to the oc-amino group (amino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term "carboxy terminus" (abbreviated C-terminus) refers to thefree carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to, peptide mimetics such as amino acids joined by an ether as opposed to an amide bond.

[0074] Polypeptides of the disclosure include polypeptides that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties.

[0075] An amino acid “substitution” as used herein refers to the replacement in a polypeptide of one amino acid at a particular position in a parent polypeptide sequence with a different amino acid. Amino acid substitutions can be generated using genetic or chemical methods well known in the art. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the naturally-occurring sequence (e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). A "conservative amino acid substitution" refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:1 ) Alanine (A), Serine (S), and Threonine (T)2) Aspartic acid (D) and Glutamic acid (E)3) Asparagine (N) and Glutamine (Q)4) Arginine (R) and Lysine (K)5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0076] A “non-conservative amino acid substitution” refers to the substitution of a member of one of these classes for a member from another class. In making such changes, according to various embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. 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); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0077] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et aL, 1982, J. Mol. Biol. 157:105-131 ). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in various embodiments, the substitution of amino acids whose hydropathic indices are within + 2 is included. In various embodiments, those that are within + 1 are included, and in various embodiments, those within + 0.5 are included.

[0078] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In various embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e. , with a biological property of the protein.

[0079] The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 +.1 ); glutamate (+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 upon similar hydrophilicity values, in various embodiments, the substitution of amino acids whose hydrophilicity values are within + 2 is included, in various embodiments, those that are within + 1 are included, and in various embodiments, those within + 0.5 are included.

[0080] Exemplary amino acid substitutions are set forth in Table 1 .Table 1Original Residues Exemplary Substitutions Preferred SubstitutionsAla Vai, Leu, He VaiArg Lys, Gin, Asn LysAsn GinAsp GluCys Ser, Ala SerGin Asn AsnGlu Asp AspGly Pro, Ala AlaHis Asn, Gin, Lys, Arg Arg lie Leu, Vai, Met, Ala, LeuPhe, NorleucineLeu Norleucine, lie, lieVai, Met, Ala, PheLys Arg, 1 ,4 Diamino-butyric ArgAcid, Gin, AsnMet Leu, Phe, lie LeuPhe Leu, Vai, He, Ala, Tyr LeuPro Ala GlySer Thr, Ala, Cys ThrThr SerTrp Tyr, Phe TyrTyr Trp, Phe, Thr, Ser PheVai lie, Met, Leu, Phe, LeuAla, Norleucine

[0081] A skilled artisan will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques. In various embodiments, one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity. In other embodiments, the skilled artisan can identify residues and portions of the molecules that are conserved among similar polypeptides. In further embodiments, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0082] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, the skilled artisan can predict the importance of amino acid residues in a polypeptide that corresponds to amino acid residues important for activity or structure in similar polypeptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0083] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of a polypeptide with respect to its three-dimensional structure. In various embodiments, one skilled in the art may choose to not make radical changes to amino acid residues predicted to be on the surface of the polypeptide, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[0084] The term "polypeptide fragment" and “truncated polypeptide” as used herein refers to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion as compared to a corresponding full-length protein. In various embodiments, fragments can be, e.g., 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 length. In various embodiments, fragments can also be, e.g., 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 in length. A fragment can further comprise, at either or both of its ends, one or more additional amino acids, for example, a sequence of amino acids from a different naturally-occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0085] The terms "polypeptide variant", “hybrid polypeptide” and “polypeptide mutant” as used herein refers to a polypeptide that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and / or substituted into the amino acid sequence relative to another polypeptide sequence. In various embodiments, the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., 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. Hybrids of the present disclosure include fusion proteins.

[0086] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin e.g., human serum albumin), phosphorylation, and glycosylation.

[0087] The term "% sequence identity" is used interchangeably herein with the term "% identity" and refers 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 thing as 80% sequence identity determined by a defined algorithm and means that a given sequence is at least 80% identical to another length of another sequence. In various embodiments, the % identity is selected from, e.g., 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 more sequence identity to a given sequence. In various embodiments, the % identity is in the range of, e.g., 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%.

[0088] The term "% sequence homology" is used interchangeably herein with the term "% homology" and refers 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 means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence. In various embodiments, the % homology is selected from, e.g., 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 more sequence homology to a given sequence. In various embodiments, the % homology is in the range of, e.g., 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%.

[0089] Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet at the NCBI website. See also Altschul et al., J. Mol. Biol. 215:403-10, 1990 (with special reference to the published default setting, i.e., parameters w=4, t=17) and Altschul et al., Nucleic Acids Res.,25:3389-3402, 1997. Sequence searches are typically carried out using the BLASTP program when evaluating a given amino acid sequence relative to amino acid sequences in the GenBank Protein Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 1 1 .0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.

[0090] In addition to calculating 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 smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in comparison of the test nucleic acid to the reference nucleic acid is, e.g., less than about 0.1 , less than about 0.01 , or less than about 0.001 .

[0091] The term “modification” as used herein refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or the post-translational modifications (e.g., glycosylation) of a polypeptide.

[0092] The term “knob-into-hole modification” as used herein refers to a modification within the interface between two immunoglobulin heavy chains in the CH3 domain. In one embodiment, the “knob-into-hole modification” comprises the amino acid substitution T366W and optionally the amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other one of the antibody heavy chains. The knob-into-hole technology is described, e.g., in U.S. Pat. No. 5,731 ,168; U.S. Pat. No. 7,695,936; Ridgway et aL, Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001 ).

[0093] The term "bioactivatable drug" or “VitoKine” as used herein means a compound that is a drug precursor which, following administration to a subject, releases the drug in vivo via some chemical or physiological process such that the bioactivatable drug is converted into a product that is active to the target tissues. A bioactivatable drug is any compound that undergoes bioactivation before exhibiting its pharmacological effects. Bioactivatable drugs can thus be viewed as drugs containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule.

[0094] The term "immunoconjugate" or ‘‘fusion protein” as used herein refers to a molecule comprising an antibody or antigen-binding fragment thereof conjugated (or linked) directly or indirectly to an effector molecule. The effector molecule can be a detectable label, an immunotoxin, cytokine, chemokine, therapeutic agent, or chemotherapeutic agent. The antibody or antigen-binding fragment thereof may be conjugated to an effector molecule via a peptide linker. An immunoconjugate and / or fusion protein retains the immunoreactivity of the antibody or antigen-binding fragment, e.g., the antibody or antigen-binding fragment has approximately the same, or only slightly reduced, ability to bind the antigen after conjugation as before conjugation. As used herein, an immunoconjugate may also be referred to as an antibody drug conjugate (ADC). Because immunoconjugates and / or fusion proteins are originally prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as "chimeric molecules."

[0095] The term "linker" refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences.

[0096] The term “peptide linker” as used herein refers to a peptide comprising one or more amino acids, typically about 1-30 amino acids. Peptide linkers are known in the art or are described herein. Suitable, non-immunogenic linker peptides include, for example, (G4S)n, (SG4)n or G4(SG4)npeptide linkers, “n” is generally a number between 1 and 10, typically between 2 and 4.

[0097] The term "cleavable linker" refers to a linker, such as a peptide linker, that can be degraded, digested, or otherwise severed to separate the two components connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers may also be cleaved by environmental cues, such as, for example, changes in temperature, pH, salt concentration, etc.

[0098] The term “concealing efficiency” or “efficiency of concealment” as used herein refers to the extent to which the activity of the active moiety can be reduced by the concealing domain in the VitoKine format. An increase in the diminution of the observed activity relative to the nonconcealed counterpart corresponds to an enhancement in concealing efficiency.

[0099] The term “non-concealed counterpart” as used herein refers to a counterpart molecule to a VitoKine, possessing identical targeting domain (D1 ), L1 linker, and active moiety domain (D2), yet devoid of both the concealing domain (D3) and the L2 linker.

[0100] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to that amount of an agent effective to produce the intended pharmacological result. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil / water 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, 21 st Ed. 2005, Mack Publishing Co, Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0101] As used herein, "treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. As used herein, to "alleviate" a disease, disorder or condition means reducing the severity and / or occurrence frequency of the symptoms of the disease, disorder, or condition. Further, references herein to "treatment" include references to curative, palliative and prophylactic treatment.

[0102] The term "effective amount" or “therapeutically effective amount” as used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and / or delay one or more of its symptoms. In reference to cancers or other unwanted cell proliferation, an effective amount comprises an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer. An effective amount can be administered in one or more administrations.

[0103] The phrase “administering” or "cause to be administered" refers to the actions taken by a medical professional (e.g., a physician), or a person controlling medical care of a patient, that control and / or permit the administration of the agent(s) / compound(s) at issue to the patient. Causing to be administered can involve diagnosis and / or determination of an appropriate therapeutic regimen, and / or prescribing particular agent(s) / compounds for a patient. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, and the like. Where administration is described herein, "causing to be administered" is also contemplated.

[0104] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or a non-human primate, but also domesticated mammals (e.g., canine or feline), laboratory mammals (e.g., mouse, rat, rabbit, hamster, guinea pig), and agricultural mammals (e.g., equine, bovine, porcine, 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 health worker in a hospital, psychiatric care facility, as an outpatient, or other clinical context. In various embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system including, but not limited to patients having primary immune deficiency, AIDS; cancer and transplant patients who are taking certain immunosuppressive drugs; and those with inherited diseases that affect 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 rate of mutations (Lawrence et al., Nature, 499(7457): 214-218, 2013).

[0105] The term “immunotherapy” refers to cancer treatments which include, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibodydrug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints) such as CTLA-4, PD1 , PDL-1 , CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPa, CD47, GITR, IGOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec 15, VISTA, CD276, CD272, TIM-3, and B7-H4; treatment using bispecific T cell engaging antibodies (BiTE®) such as blinatumomab: treatment involving administration of biological response modifiers such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21 , IL-22, GM-CSF, IFN- a, I FN-p, IFN-y, TGF-|3 antagonist or TGF-p trap; treatment using therapeutic vaccines such as sipuleucel-T; treatment using therapeutic virus, including, but not limited to oncolytic virus such as T-vec; treatment using dendritic cell vaccines, or tumor antigen peptide or neoantigenvaccines; treatment using NK cells; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using DC or T cells; treatment using iPS induced-NK cells; treatment using iPS induced-T cells; treatment using vaccine such as Bacille Calmette- Guerine (BCG); treatment using tumor infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR-T cells); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG, TLR7,TLR8, TLR9, and imiquimod.

[0106] “Resistant or refractory cancer” refers to tumor cells or cancer that do not respond to previous anti-cancer therapy including, e.g., chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells can be resistant or refractory at the beginning of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the onset of treatment or respond initially for a short period but fail to respond to treatment. Refractory tumor cells also include tumors that respond to treatment with anticancer therapy but fail to respond to subsequent rounds of therapies. For purposes of this invention, refractory tumor cells also encompass tumors that appear to be inhibited by treatment with anticancer therapy but recur up to five years, sometimes up to ten years or longer after treatment is discontinued. The anticancer therapy can employ chemotherapeutic agents alone, radiation alone, targeted therapy alone, surgery alone, or combinations thereof. For ease of description and not limitation, it will be understood that the refractory tumor cells are interchangeable with resistant tumor.

[0107] The term “neoantigen” refers to, e.g., cell surface antigens to which the immune system has not previously been exposed, especially one that arises by alteration of host antigens by radiation, chemotherapy, viral infection, neoplastic transformation / mutation, drug metabolism, etc., selectively expressed by cancer cells or over-expressed in cancer cells relative to most normal cells.

[0108] The term “antibody” as used herein is used in the broadest sense and encompasses various antibody structures ( IgG 1 , 2, 3, or 4, IgM, IgA, IgE) including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific or bifunctional antibodies), and antibody fragments so long as they exhibit the desired antigenbinding activity.

[0109] The term “antibody fragment” as used herein refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds 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.

[0110] The term “Fab fragment” as used herein refers to an immunoglobulin fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1 ) of a heavy chain.

[0111] The terms “variable region” or “variable domain” as used herein refers to the domain of an immunoglobulin or antibody heavy or light chain that is generally involved in binding the immunoglobulin or antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of an immunoglobulin or antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity-determining regions (CDRs).

[0112] The term "complementarity determining regions" or "CDRs" contain the antigencontacting residues ("antigen contacts"). Generally, antibodies comprise six CDRs: three in the VH (CDR-H1 , CDR-H2, CDR-H3), and three in the VL (CDR-L1 , CDR-L2, CDR-L3). CDRs occurring 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 combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs)

[0113] "Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649, U.S. Patent No. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

[0114] A “human immunoglobulin” as used herein is one which possesses an amino acid sequence which corresponds to that of an immunoglobulin produced by a human or a human cell or derived from a non-human source that utilizes human immunoglobulin repertoires or other human immunoglobulin-encoding sequences. This definition of a human immunoglobulin specifically excludes a humanized immunoglobulin comprising non-human antigen-binding residues.

[0115] The term “humanized antibody” as used herein refers to an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework, and such substitutions are herein referred to as back-mutations. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.

[0116] The term “Fc domain” or “Fc region” as used herein is used to define a 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. An IgG Fc region comprises an IgG CH2 and an IgG CH3 domain. The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced “protuberance” (“knob”) in one chain thereof and a corresponding introduced “cavity” (“hole”) in the other chain thereof; see U.S. Pat. No. 5,821 ,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical immunoglobulin heavy chains as herein described. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system.

[0117] The term “effector functions” as used herein refers to those biological activities attributable to the Fc region of an immunoglobulin, which vary with the immunoglobulin isotype. Examples of immunoglobulin effector functions include: C1 q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0118] As used herein, “specific binding” is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., surface plasmon resonance (SPR) technique.

[0119] The terms “affinity” or “binding affinity” as used herein refers to the strength of the sum total 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 a molecule X for its partner Y cangenerally be represented by the dissociation constant (KD), which is the ratio of dissociation and association rate constants (koff and kon, respectively). A particular method for measuring affinity is SPR.

[0120] The term "immunogenicity" as used herein refers to the ability of an antibody or antigen binding fragment to elicit an immune response (humoral or cellular) when administered to a recipient and includes, for example, the human anti-mouse antibody (HAMA) response. A HAMA response is initiated when T-cells from a subject make an immune response to the administered antibody. The T-cells then recruit B-cells to generate specific "anti-antibody" antibodies.

[0121] The term "immune cell" as used herein means any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., an autoantigen). In various embodiments, an immune cell is, e.g., a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, Langerhan’s cells, or Kuffer cells.

[0122] The term “reduced binding”, as used herein refers to a decrease in affinity for the respective interaction, as measured for example by SPR. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

[0123] The term "polymer" as used herein generally includes, but is not limited to, homopolymers; copolymers, such as, for example, block, graft, random and alternating copolymers; and terpolymers; and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term "polymer" shall include all possible geometrical configurations of the material. These configurations include, but are not limited to isotactic, syndiotactic, and random symmetries.

[0124] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally-occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally-occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally-occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O- methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to shortpolynucleotides, generally 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 an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T."

[0125] Conventional notation is 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 referred to as the 5'-direction. The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5'-end of the RNA transcript are referred to as "upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0126] "Complementary" refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to 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.

[0127] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it 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), wherein additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a chosen polynucleotide.

[0128] A "regulatory sequence" is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatory sequence can, for example, exert its effects directly on the regulated nucleic acid, or through theaction 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). Further examples of regulatory sequences are described in, for example, 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 (e.g., the level, timing, or location of expression) of the nucleotide sequence.

[0129] A "host cell" is a cell that can be used to express a polynucleotide of the disclosure. A host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, 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 a nucleic acid to be expressed. A host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0130] The term "isolated molecule" (where 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 naturally-associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be "isolated" from its naturally-associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may beassayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0131] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60% to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W / W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0132] The terms "label" or "labeled" as used herein refers to the incorporation of another molecule in the antibody. In one embodiment, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In another embodiment, the label or marker can be therapeutic, e.g., a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,15N,35S,90Y, "Tc,111In,125l,1311), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. In various embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0133] The term "heterologous" as used herein refers to a composition or state that is not native or naturally found, for example, that may be achieved by replacing an existing natural composition or state with one that is derived from another source. Similarly, the expression of a protein in an organism other than the organism in which that protein is naturally expressed constitutes a heterologous expression system and a heterologous protein.

[0134] It is understood that aspect and embodiments of the disclosure described herein include “consisting” and / or “consisting essentially of” aspects and embodiments.

[0135] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X".

[0136] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the disclosure described herein include "consisting" and / or "consisting essentially of" aspects and variations.PD1 Blocking Antibody

[0137] In one aspect, the PD1 blocking antibody guides the IL-2 moiety of the VitoKine to the TILs in the tumor microenvironment (TME) and restricts the activation of the VitoKine locally to improve the therapeutic index. In another aspect, the PD1 blocking antibody guides the IL-2 moiety of the VitoKine to the TILs in the TME. In various embodiments, the PD1 blocking antibodies were optimized through modifications in the variable domains of pembrolizumab. In various embodiments, the modifications involved germline sequence substitutions of the CDR residues, germline sequence substitutions of the framework residues, and adoption of the most prevalent and better behaving VH3 human germline family sequence as the acceptor framework. In various embodiments, these modifications were implemented individually or in combination to develop optimized PD1 blocking antibodies. In various embodiments, these optimized PD1 blocking antibodies exhibit a high binding affinity to PD1 , function to inhibit PD1 with equal or comparable potency as pembrolizumab, have a higher sequence similarity score to its closest human germline sequence, resulting in an improved degree of humanness compared to pembrolizumab, and are predicted to have lower hydrophobicity, leading to a reduced aggregation propensity than pembrolizumab. In various embodiments, PD1 Ab-IL-2 VitoKine constructs and PD1 -targeted IL-2 immunocytokines based on these optimized PD1blocking antibodies are predicted to have enhanced developability properties. In various embodiments, the PD1 antibody comprises a light chain variable region with the sequence selected from the group of sequences set forth in SEQ ID NOS: 3-5, and a heavy chain variable region with the sequence selected from the group of sequences set forth in SEQ ID NOS: 7-18. In various embodiments, the PD1 antibody comprises a light chain sequence set forth in SEQ ID NO: 44, and a heavy chain with the sequence selected from the group of sequences set forth in SEQ ID NOS: 45-49.IL-2 domain

[0138] lnterleukin-2 (IL-2), a classic Th1 cytokine, is produced by T cells after activation through the T-cell antigen receptor and the co-stimulatory molecule CD28. The regulation of IL-2 occurs through activation of signaling pathways and transcription factors that act on the IL-2 promoter to generate new gene transcription but also involves modulation of the stability of IL-2 mRNA. IL-2 binds to a multichain receptor, including a highly regulated a chain and 3 and y chains that mediate signaling 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 activation-induced cell death of T cells, a function that provides an essential mechanism for terminating immune responses. A commercially available unglycosylated human recombinant IL-2 product, aldesleukin (available as the PROLEUKIN® brand of des-alanyl-1 , serine-125 human interleukin-2 from Prometheus Laboratories Inc., San Diego Calif.), has been approved for administration to patients suffering from metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been suggested for administration in patients suffering from or infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), acute myeloid leukemia, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, short half-life and severe toxicity limits the optimal dosing of IL-2.

[0139] As used herein, the terms "native IL-2" and "native interleukin-2" in the context of proteins or polypeptides refer to any naturally-occurring mammalian interleukin-2 amino acid sequences, including immature or precursor and mature forms. Non-limiting examples of Gen Bank Accession Nos. for the amino acid sequence of various species of native mammalian interleukin-2 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 present invention, native IL-2 is the immature or precursor form of a naturally-occurring mammalian IL-2. In other embodiments, native IL-2 is the mature form of a 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 present invention is derived from the amino acid sequence of the human IL-2 mature sequence set forth in SEQ ID NO: 116:APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRW ITFCQSIISTLT (SEQ ID NO: 116)

[0140] In various embodiments, the IL-2 domain will be an IL-2 variant (or mutant) comprising a sequence derived from the sequence of the mature human IL-2 polypeptide as set forth in SEQ ID NO: 1 16. In various embodiments, the IL-2 variant comprises a single C125I amino acid substitution that universally enhances the developability of the protein while fully preserving 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: 1 17.

[0141] In various embodiments, the sequence of the IL-2 variant has at least one amino acid change, e.g., substitution or deletion, compared to the native IL-2 sequence, such changes resulting in IL-2 agonist or antagonist activity. IL-2 agonists are exemplified by comparable or increased biological activity compared to wild type IL-2. IL-2 antagonists are exemplified by 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 the amino acid sequence derived from SEQ ID NO: 117 with altered binding to IL-2Roc. In various embodiments, the IL-2 variant with altered binding to IL-2Ra comprises the amino acid sequence set forth in SEQ ID NOS: 118- 125. In various embodiments, the IL-2 variant has the amino acid sequence derived from SEQ ID NO: 1 17 with reduced / abolished binding to IL-2Rcc to selectively activate and proliferate effector T cells (Teff) for treating cancer. In various embodiments, the IL-2 variant with reduced / abolished binding to IL-2Ra comprises the amino acid sequence set forth in SEQ ID NOS: 118-122. In various embodiments, the IL-2 variant has the amino acid sequence derived from SEQ ID NO: 117 with reduced binding to IL-2RJ3 and / or yc. In various embodiments, the IL- 2 variant reduced binding to IL-2RJ3 and / or yc comprises the amino acid sequence set forth inSEQ ID NOS: 126-150. In various embodiments, the IL-2 variant has the amino acid sequence derived from SEQ ID NO: 117 with reduced / abolished binding to IL-2Ra and reduced binding to IL-2RP and / or yc. In various embodiments, the IL-2 variant with reduced / abolished binding to IL- 2Ra and reduced binding to IL-2R|3 and / or yc comprises the amino acid sequence set forth in SEQ ID NOS: 151 -180. As will be appreciated by those in the art, all of the mutations can be optionally and independently combined in any way to achieve optimal affinity and activity modulation.IL-2Ra domain (concealing moiety domain in PD1 Ab-IL-2 VitoKine)

[0142] The IL-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells, such as lymphocytes, that binds and responds to IL-2. IL-2R has three subunits: a (CD25), p (CD122), and y (CD132 or the common gamma chain (yc)), a shared chain with five other cytokine receptors: IL-4R, IL-7R, IL-9R, IL-15R, and IL-21 R). Alpha chain (alias: Tac antigen or p55) of human receptor is encoded on chromosome 10p14-15 by the gene IL-2RA. The gene for the human p chain (IL-2RB, CD122) of the receptor is located on chromosome 22q 1 1 .2-12, while the gene for the human y chain (IL-2RG) is on chromosome Xq13. Assembly of all three subunits of the receptor is important for the signal transduction into the B and T cells. IL-2R was found on the cell surface (either temporary or permanent) in almost all hematopoietic cells including lymphoid linages T, B, and NK cells, as well as myeloid ones like macrophages, monocytes, and neutrophils. The signal is transferred into the cell via the Janus kinases — Jak1 and Jak3. The phosphorylation of the intracytosolic part of the receptor’s p chain enables homodimer formation of STAT-3 and STAT-5 factors. 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.

[0143] As used herein, the terms "native IL-2Ra" and "native interleukin-2 receptor alpha" in the context of proteins or polypeptides refer to any naturally-occurring mammalian interleukin-2 receptor alpha ("IL-2Ra") amino acid sequence, including immature or precursor and mature forms and naturally-occurring isoforms. Non-limiting examples of GenBank Accession Nos. for the amino acid sequence of various native mammalian IL-2Ra include NP_032393.3 (Mus musculus), CAK26553.1 (human), and NP 000408.1 (human). In various embodiments, the IL- 2Ro domain is derived from the amino acid sequence of the human IL-2Ra sequence set forth in SEQ ID NO: 181 :MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRI KSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQ PVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKM THGKTRWTQPQLICTGEMETSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAA TMETSIFTTEYQVAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI (SEQ ID NO: 181 )

[0144] In various embodiments, a concealing moiety domain (D3) is used to reversibly conceal the activity of the IL-2 domain in the PD1 Ab-IL-2 VitoKine construct. In various embodiments, the concealing moiety domain is an IL-2Ra extracellular domain or a functional fragment thereof. In various preferred embodiments, the concealing moiety domain is an IL-2RaSushi domain comprising the amino acid sequence of the mature human IL-2Ra polypeptide as set forth in SEQ ID NO: 182. In various preferred embodiments, the concealing moiety domain is a variant of IL-2RoSushi domain.ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDN QCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENE ATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 182)

[0145] In various embodiments, PD1 Ab-IL-2 VitoKine comprises IL-2RaSushi (SEQ ID NO: 182) as the concealing moiety domain to conceal IL-2 (including IL-2 variants) activity. Although wild-type IL-2Roc binds to IL-2 with a moderate affinity of 30 nM, there remains a possibility that, upon cleaving the linker, IL-2Rcc may not dissociate. The association between the cleaved IL- 2Ra and IL-2 could reduce the activity of IL-2 and / or tilt the balance of T cell subpopulations toward an undesired outcome. With affinity reducing mutation(s) introduced into IL-2RocSushi, the IL-2Ra sushi domains are likely to dissociate away from the IL-2 upon linker cleavage. In various embodiments, the concealing moiety domain in PD1 Ab-IL-2 VitoKine is IL-2RocSushi variant comprising IL-2-binding-weakening mutations, e.g., R36A, K38E, L42G, or Y43A, or any combination of the substitutions. In various embodiments, the IL-2RaSushi variant can effectively conceal IL-2 moiety domain’s activity despite its reduced affinity to IL-2. In various embodiments, the IL-2RaSushi variant is anticipated to dissociate and diffuse away from IL-2 upon linker cleavage because of its reduced affinity to IL-2.

[0146] In various embodiments, the variant (mutant) of IL-2RaSushi domain is designed to mitigate liable sequences, such as those susceptible to bond cleavage. In variousembodiments, the variant (mutant) of IL-2RaSushi domain is designed to mitigate liable sequences as well as weaken binding strength for IL-2, facilitating the dissociation of the cleaved concealing domain. In various embodiments, the variant (mutant) of IL-2RaSushi domain designed to mitigate liable sequences has improved concealing efficiency. In various embodiments, the amino acid change is one or more amino acid substitutions at position 6, 7, 36, 38, 42, or 43 of SEQ ID NO: 182. In various embodiments, the amino acid change is the substitution of D to E or N at position 6, P to G or A or S at position 7, R to A at position 36, K to E at position 38, L to G at position 42, Y to A at position 43, or any combination of these substitutions.

[0147] In various embodiments, the PD1 Ab-IL-2 VitoKine constructs of the present invention contain a concealing moiety domain that is one of the IL-2RaSushi domain variants comprising the amino acid sequence as set forth in SEQ ID NOS: 183-185 and 210-214.L1 and L2 Linkers in PD1 Ab-IL-2 VitoKineCleavable Linkers

[0148] A cleavable linker, or a linker susceptible to a disease-associated enzyme, may contain a moiety, e.g., a protein substrate, capable of being specifically cleaved by a protease that is present at elevated levels at the disease site as compared to non-disease tissues. Literature contains multiple reports on increased levels of enzymes with known substrates in various types of cancers, 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 by reference herein in its entirety. In various embodiments, the protease capable of cleaving a protease-cleavable linker is selected from the group consisting of metalloproteinase, e.g., matrix metalloproteinase (MMP) 1 -28, serine protease, e.g., urokinase-type plasminogen activator (uPA) and matriptase, cysteine protease, e.g., legumain, aspartic protease, and cathepsin protease. Exemplary proteases are provided in Table 2:Table 2

[0149] Exemplary protease substrate peptide sequences, which can be used as protease cleavable linkers with or without peptide spacers, are provided in Table 3:Table 3

[0150] In various embodiments, the protease is MMP-9 or MMP-2. In a further specific embodiment, the protease is matriptase. In a further specific embodiment, the protease is MMP- 14. In further specific embodiment, the protease is legumain. In various embodiments, the protease cleavable linker may contain two or more protease substrate sequences. In various embodiments, the proteases are MMP-2 / MMP-9 and matriptase. In various embodiments, the protease-cleavable linker comprises the protease recognition sequence ‘GPLGMLSQ’ (SEQ ID NO: 61 ). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence ‘SGRSENIRTA’ (SEQ ID NO: 60). In various embodiments, the protease- cleavable linker comprises the protease recognition sequence ‘GPTNKVR’ (SEQ ID NO: 69). Invarious embodiments, the protease-cleavable linker comprises the protease recognition sequence ‘PMAKK’ (SEQ ID NO: 74). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence ‘GPLGMLSQPMAKK’ (SEQ ID NO: 76). In various embodiments, the protease-cleavable linker comprises the protease recognition sequence ‘PMAKKGPLGMLSQ’ (SEQ ID NO: 77).

[0151] In various embodiments, peptide spacers may be incorporated on either side of a protease cleavable sequence or to flank both sides of a protease cleavable sequence, or as a non-cleavable linker without a protease substrate site. Peptide spacer serves to position a cleavable linker, making it more readily accessible to the enzyme responsible for cleavage. The length and composition of a peptide spacer can be fine-tuned to balance the accessibility for enzymatic cleavage and the spatial constraint required to reversibly conceal the D2 domain from exerting its biological activity. A peptide spacer may include 1 -100 amino acids. Suitable peptide spacers are known in the art, which include, but are not limited to, peptide linkers containing flexible amino acid residues, such as glycine and serine. In various embodiments, a peptide spacer can contain 1 to 12 amino acids including motifs of 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: 1 11 ), GSGSGSGSGS (SEQ ID NO: 112), or GSGSGSGSGSGS (SEQ ID NO: 113). In other embodiments, a peptide spacer can contain motifs of (GGGGS)(SEQ ID NO: 106)n, wherein n is an integer from 1 to 10. In other embodiments, a peptide spacer can also contain amino acids other than glycine and serine. A peptide spacer is stable under physiological conditions as well as at a diseased site, such as a cancer site.

[0152] Exemplary protease cleavable linkers with peptide spacers flanking protease substrate peptides (underscored) are provided in Table 4:Table 4Non-cleavable Linkers

[0153] Non-cleavable linker provides covalent linkage 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, non-cleavable linker may include 1 -100 amino acids. In various embodiments, a spacer can contain motifs of GSGG (SEQ ID NO: 104), GGSS (SEQ ID NO: 105), GSGS (SEQ ID NO: 109), GSGSGS (SEQ ID NO: 110), GSGSGSGS (SEQ ID NO: 1 11 ), GSGSGSGSGS (SEQ ID NO: 112), or GSGSGSGSGSGS (SEQ ID NO: 113). In other embodiments, a spacer can contain motifs of (GGGGS)(SEQ ID NO: 106)n, wherein n is an integer from 1 to 10. In other embodiments, a linker can also contain amino acids other than glycine and serine. In another embodiment, the non-cleavable linker can be a simple chemical bond, e.g., an amide bond (e.g., by chemical conjugation of PEG). A non-cleavable linker is stable under physiological conditions as well as at a diseased site, such as a cancer site.

[0154] Exemplary non-cleavable linkers are provided in Table 5:Table 5A combination of cleavable and non-cleavable Linkers

[0155] In various embodiments, the L1 and L2 linkers can be both cleavable or a combination of cleavable and non-cleavable linkers to yield different forms of active moiety of the IL-2 domain to fulfill specific therapeutic objectives, optimize the risk to benefit ratio, or align with diverse properties of the cytokine. The exemplary active forms released by cleavage of the linkers are depicted in FIG. 2. The active form 1 derived from cleavage of the L1 linker and the active form 3 derived from cleavage of L1 and L2 linkers are both short-acting cytokines due to their release from the targeting antibody after proteolysis. The presence or absence of the concealing domain would result in distinct activity for these two active forms in the local environment. After acting locally, the short-acting active forms can be eliminated from systemic circulation quickly, which could have the potential benefit of reduced toxicities. In contrast, Active Form 2 derived from the cleavage of the L2 linker (depicted in FIG. 2) is a functionally fully restored 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 cell at or near the disease site, which synergistically enhances the two pathways and boosts the anticancer immune response, while minimizing systemic toxicity.Polynucleotides

[0156] In another aspect, the present disclosure provides isolated nucleic acid molecules comprising a polynucleotide of IL-2, an IL-2 variant, IL-2Ra, an IL-2Ra variant, an PD1 blocking antibody, an antibody fragment, a PD1 Ab-IL-2 VitoKine construct, or a PD1 - targeted IL-2 immunocytokine of the present disclosure. The subsequent paragraphs of this sub-section “polynucleotides” will utilize PD1 -targeted IL-2 VitoKine (VitoKine) constructs as illustrativeinstances, yet these concepts shall be equally applicable to the PD1 -targeted IL-2 immunocytokines of the current invention.

[0157] The subject nucleic acids may be single-stranded or double stranded. Such nucleic acids may be DNA or RNA molecules. DNA includes, for example, cDNA, genomic DNA, synthetic DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA encoding VitoKine constructs is obtained from genomic libraries which are available for a number of species. Synthetic DNA is available from chemical synthesis of overlapping oligonucleotide fragments followed by assembly of the fragments to reconstitute part or all of the coding regions and flanking sequences. RNA may be obtained from prokaryotic expression vectors which direct high-level synthesis of mRNA, such as vectors using T7 promoters and RNA polymerase. The DNA molecules of the disclosure include full-length genes as well as polynucleotides and fragments thereof. The full-length gene may also include sequences encoding the N-terminal signal sequence. Such nucleic acids may be used, for example, in methods for making the novel VitoKine constructs.

[0158] In various embodiments, the isolated nucleic acid molecules comprise the polynucleotides described herein and further comprise a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecules further comprise polynucleotides encoding the linkers or hinge linkers described herein.

[0159] In various embodiments, the recombinant nucleic acids of the present disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are art-recognized and are selected to direct 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). Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the present disclosure. The promoters may be either naturally-occurring promoters, or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In various embodiments, the expression vector contains a selectable markergene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0160] In another aspect of the present disclosure, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding a VitoKine construct and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus or vector for expressing a polypeptide from a polynucleotide sequence. Vectors suitable for expression in host cells are readily available and the nucleic acid molecules are inserted into the vectors using standard recombinant DNA techniques. Such vectors can include a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding a VitoKine construct. Such useful expression control sequences, include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS, the promoters of the yeast a-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of 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 such factors as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered. An exemplary expression vector suitable for expression of VitoKine is the pDSRa, and its derivatives, containing VitoKine polynucleotides, as well as any additional suitable vectors known in the art or described below.

[0161] A recombinant nucleic acid of the present disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both. Expression vehicles for production of a recombinant VitoKine construct include plasmids and other vectors. For instance, suitable vectors include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.

[0162] Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. 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. Alternatively, derivatives of viruses such as the bovine papilloma virus (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 below in the description of gene therapy delivery systems. The various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it may be desirable to express the recombinant polypeptides by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941 ), pAcUW-derived vectors (such as pAcUWI ), and pBlueBac-derived vectors (such as the B-gal containing pBlueBac III).

[0163] In various embodiments, a vector will be designed for production of the subject VitoKine construct in Chinese Hamster Ovary (CHO) cells or Human Embryonic Kidney 293 (HEK293) cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As will be apparent, the subject gene constructs can be used to cause expression of the subject VitoKine constructs in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification.

[0164] This present disclosure also pertains to a host cell transfected with a recombinant gene including a nucleotide sequence coding an amino acid sequence for one or more of the subject VitoKine construct. The host cell may be any prokaryotic or eukaryotic cell. For example, a VitoKine construct of the present disclosure may be expressed in bacterial cells such as E. 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.

[0165] Accordingly, the present disclosure further pertains to methods of producing the subject VitoKine constructs. For example, a host cell transfected with an expression vector encoding a VitoKine construct can be cultured under appropriate conditions to allow expression of the VitoKine construct to occur. The VitoKine construct may be secreted and isolated from a mixture of cells and medium containing the VitoKine construct. Alternatively, the VitoKine construct may be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and the protein isolated. A cell culture includes host cells, media and other byproducts. Suitable medias for cell culture are well known in the art.

[0166] The polypeptides and proteins of the present disclosure can be purified according to protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the proteinaceous and non-proteinaceous fractions. Having separated the peptide polypeptides from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). The term "isolated polypeptide" or "purified polypeptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the polypeptide is purified to any degree relative to its naturally-obtainable state. A purified polypeptide therefore also refers to a polypeptide that is free from the environment in which it may naturally occur. Generally, "purified" will refer to a polypeptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a peptide or polypeptide composition in which the polypeptide or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the proteins in the composition.

[0167] Various techniques suitable for use in purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies (immunoprecipitation) and the like or by heat denaturation, followed by centrifugation; chromatography such as affinity chromatography (Protein-A columns), ion exchange, gel filtration, reverse phase, hydroxylapatite, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified polypeptide.Pharmaceutical Compositions

[0168] The present disclosure provides a pharmaceutical composition comprising the VitoKine constructs in admixture with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those of ordinary skill and have been extensively described (see, e.g., Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990). The pharmaceutically acceptable carriers may be included for purposes of modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Such pharmaceutical compositions may influence 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); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl- beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counter ions (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 glycerin, 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 enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0169] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection,physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. In one embodiment of the present disclosure, compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the therapeutic composition may be formulated as a lyophilizate using appropriate excipients such as sucrose. The optimal pharmaceutical composition will be determined by one of ordinary skill in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage.

[0170] When parenteral administration is contemplated, the therapeutic pharmaceutical compositions may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired VitoKine construct in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a polypeptide is formulated as a sterile, isotonic solution, properly preserved. In various embodiments, pharmaceutical formulations suitable for injectable administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.

[0171] In various embodiments, the therapeutic pharmaceutical compositions may be formulated for targeted delivery using a colloidal dispersion system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including 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. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Thetargeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art.

[0172] In various embodiments, oral administration of the pharmaceutical compositions is contemplated. Pharmaceutical compositions that are administered in this fashion can be formulated with or without those carriers customarily 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, and the like), one or more therapeutic compounds of the present disclosure may 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 starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. 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 may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

[0173] In various embodiments, topical administration of the pharmaceutical compositions, either to skin or to mucosal membranes, is contemplated. The topical formulations may furtherinclude one or more of the wide variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surface-active agents. Keratolytic agents such as those known in the art may also be included. Examples are salicylic acid and sulfur. Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required. The ointments, pastes, creams and gels may contain, in addition to a subject compound of the disclosure (e.g., a VitoKine construct), excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0174] Additional pharmaceutical compositions contemplated for use herein include formulations involving polypeptides in sustained- or controlled-delivery formulations. In various embodiments, pharmaceutical compositions may be formulated in nanoparticles, as slow- release hydrogel, or incorporated into oncolytic viruses. Such nanoparticles methods include, e.g., encapsulation in nanoparticles composed of polymers with a hydrophobic backbone and hydrophilic branches as drug carriers, encapsulation in microparticles, insertion into liposomes in emulsions, and conjugation to other molecules. Examples of nanoparticles include mucoadhesive nanoparticles coated with chitosan and Carbopol (Takeuchi et al., Adv. Drug Deliv. Rev. 47(1 ):39-54, 2001 ) and nanoparticles containing charged combination polyesters, poly(2-sulfobutyl-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 a drug delivery system for delivering hydrophobic drugs such as a taxane. See, for example, U.S. Pat. Nos. 5,916,596; 6,506,405; 6,749,868; 6,537,579; 7,820,788; and 7,923,536. Abraxane®, an albumin stabilized nanoparticle formulation of paclitaxel, was approved in the United States in 2005 and subsequently in various other countries for treating metastatic breast cancer.

[0175] Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art.

[0176] An effective amount of a pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which the polypeptide is being used, the route of administration, and the size (body weight, body surface or organ size) and condition (the age and general health) of the patient. Accordingly, the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage may range from about 0.0001 mg / kg to up to about 100 mg / kg or more, depending on the factors mentioned above. Polypeptide compositions may be preferably injected or administered intravenously. Long-acting pharmaceutical compositions may be administered every three to four days, every week, biweekly, triweekly, monthly, or even longer durations depending on the half-life and clearance rate of the particular formulation. The frequency of dosing will depend upon the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses (at the same or different concentrations / dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data.

[0177] The route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intratumoral, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, intralesional routes, intramedullary, intrathecal, intraventricular, intravesical, transdermal, subcutaneous, or intraperitoneal; as well as intranasal, enteral, topical, sublingual, urethral, vaginal, or rectal means, by sustained release systems or by implantation devices. Where desired, the compositions may be administered by bolus injection or continuously by infusion, or by implantation device. Alternatively, or additionally, the composition may be administered locally via implantation of a membrane, sponge, or another appropriate material on to which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.Therapeutic Uses

[0178] The present disclosure provides for a method of treating cancer cells in a subject, comprising administering to said subject a therapeutically effective amount (either as monotherapy or in a combination therapy regimen) of a VitoKine construct of the present disclosure in pharmaceutically acceptable carrier, wherein such administration inhibits the growth and / or proliferation of a cancer cell. Specifically, a VitoKine construct of the present disclosure is useful in treating disorders characterized as cancer. Such disorders include, but are not limited to solid tumors, such as cancers of the breast, respiratory tract, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases, lymphomas, sarcomas, multiple myeloma and leukemia. 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 cancers of the respiratory tract include, but are not limited to small-cell and non-small-cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancers include, but are not limited to brain stem and hypothalamic glioma, cerebellar and cerebral astrocytoma, neuroblastoma, medulloblastoma, ependymoma, as well as neuroectodermal and pineal tumor. Tumors of the male reproductive organs include, but are not limited to prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to endometrial, cervical, ovarian, vaginal, and vulvar cancer, as well as sarcoma of the uterus. Tumors of the digestive tract include, but are not limited to anal, colon, colorectal, esophageal, gallbladder, gastric, liver, breast, pancreatic, rectal, small-intestine, and salivary gland cancers. Tumors of the urinary tract include, but are not limited to bladder, penile, kidney, renal pelvis, ureter, and urethral cancers. Eye cancers include but are not limited to intraocular melanoma and retinoblastoma. Examples of liver cancers include but are not limited to hepatocellular carcinoma (liver cell carcinomas with or without fibrolamellar variant), cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixed hepatocellular cholangiocarcinoma. Skin cancers include, but are not limited to squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head-and-neck cancers include, but are not limited to nasopharyngeal cancer, and lip and oral cavity cancer. Lymphomas include, but are not limited to AIDS-related lymphoma, nonHodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and lymphoma of thecentral nervous system. Sarcomas include, but are not limited to, sarcoma of the soft tissue, 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.

[0179] In various embodiments, the VitoKine construct can be used as a single agent for treatment of all kinds of cancers, including but not limited to non-small cell lung, small cell lung, melanoma, renal cell carcinoma, urothelial, liver, breast, pancreatic, colorectal, gastric, prostate, and sarcoma.

[0180] Therapeutically effective amount" or “therapeutically effective dose” refers to the amount of the therapeutic agent being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.

[0181] A therapeutically effective dose can be estimated initially from cell culture assays by determining an IC50 (half maximal inhibitory concentration). A dose can then be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC. The exact composition, route of administration and dosage can be chosen by the individual physician in view of the subject's condition.

[0182] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses (multiple or repeat or maintenance) can be administered over time and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the present disclosure will be dictated primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0183] Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and theeffective amount providing a detectable therapeutic benefit to a subject may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the subject. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a subject in practicing the present disclosure.

[0184] It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Further, the dosage regimen with the compositions of this disclosure may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the subject, the severity of the condition, and the route of administration. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. Thus, the present disclosure encompasses intrasubject dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.

[0185] An exemplary, non-limiting daily dosing range for a therapeutically or prophylactically effective amount of an VitoKine, or VitoKine variant, of the disclosure can be 0.0001 to 100 mg / kg, 0.0001 to 90 mg / kg, 0.0001 to 80 mg / kg, 0.0001 to 70 mg / kg, 0.0001 to 60 mg / kg, 0.0001 to 50 mg / kg, 0.0001 to 40 mg / kg, 0.0001 to 30 mg / kg, 0.0001 to 20 mg / kg, 0.0001 to 10 mg / kg, 0.0001 to 5 mg / kg, 0.0001 to 4 mg / kg, 0.0001 to 3 mg / kg, 0.0001 to 2 mg / kg, 0.0001 to 1 mg / kg, 0.001 to 50 mg / kg, 0.001 to 40 mg / kg, 0.001 to 30 mg / kg, 0.001 to 20 mg / kg, 0.001 to 10 mg / kg, 0.001 to 5 mg / kg, 0.001 to 4 mg / kg, 0.001 to 3 mg / kg, 0.001 to 2 mg / kg, 0.001 to 1 mg / kg, 0.010 to 50 mg / kg, 0.010 to 40 mg / kg, 0.010 to 30 mg / kg, 0.010 to 20 mg / kg, 0.010 to 10 mg / kg, 0.010 to 5 mg / kg, 0.010 to 4 mg / kg, 0.010 to 3 mg / kg, 0.010 to 2 mg / kg, 0.010 to 1 mg / kg, 0.1 to 50 mg / kg, 0.1 to 40 mg / kg, 0.1 to 30 mg / kg, 0.1 to 20 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, 0.1 to 4 mg / kg, 0.1 to 3 mg / kg, 0.1 to 2 mg / kg, 0.1 to 1 mg / kg, 1 to 50 mg / kg, 1 to 40 mg / kg, 1 to 30 mg / kg, 1 to 20 mg / kg, 1 to 10 mg / kg, 1 to 5 mg / kg, 1 to 4 mg / kg, 1 to 3 mg / kg, 1 to 2 mg / kg, or 1 to 1 mg / kg body weight. It is to be noted that dosage values may vary with thetype and severity of the conditions to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0186] Toxicity and therapeutic index of the pharmaceutical compositions of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD5o (the dose lethal to 50% of the population) and the ED5o (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effective dose is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are generally preferred.

[0187] The dosing frequency of the 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 a desired therapeutic result is achieved. Exemplary dosing frequencies include but are not limited to: once weekly without break; once weekly, every other week; once every 2 weeks; once every 3 weeks; weakly without break for 2 weeks, then monthly; weakly without break for 3 weeks, then monthly; monthly; once every other month; once every three months; once every four months; once every five months; or once every six months, or yearly.Combination Therapy

[0188] As used herein, the terms "co-administration", "co-administered" and "in combination with", referring to a VitoKine construct of the disclosure and one or more other therapeutic agents, is intended to mean, and does refer to and include the following: simultaneous administration of such combination of a VitoKine construct of the disclosure and therapeutic agent(s) to a subject in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said subject; substantially simultaneous administration of such combination of a VitoKine construct of the disclosure and therapeutic agent(s) to a subject in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said subject, whereupon said components are released at substantially the same time to said subject; sequential administration of such combination of aVitoKine construct of the disclosure and therapeutic agent(s) to a subject in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said subject with a significant time interval between each administration, whereupon said components are released at substantially different times to said subject; and sequential administration of such combination of a VitoKine construct of the disclosure and therapeutic agent(s) to a subject in need of treatment, when such components are formulated together into a single dosage form which releases said components in a controlled manner whereupon they are concurrently, consecutively, and / or overlappingly released at the same and / or different times to said subject, where each part may be administered by either the same or a different route.

[0189] In another aspect, the present disclosure provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention in combination with a 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 methods can be used in prophylactic cancer prevention, prevention of cancer recurrence and metastases after surgery, and as an adjuvant of other conventional cancer therapy. The present disclosure recognizes that the effectiveness of conventional cancer therapies (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced through the use of the combination methods described herein.

[0190] A wide array of conventional compounds has been shown to have anti-neoplastic activities. These compounds have been used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastases and further growth, or decrease the number of malignant T-cells in leukemic or bone marrow malignancies. Although chemotherapy has been effective in treating various types of malignancies, many anti-neoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments.

[0191] In various embodiments, a second anti-cancer agent, such as a chemotherapeutic agent, will be administered to the patient. The list of exemplary chemotherapeutic agent 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), fluradabine, ifosfamide, hydroxyureataxanes (such as paclitaxel and doxetaxel) and / or anthracycline antibiotics, as well as combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX. In various embodiments, the dosages of such chemotherapeutic agents include, but is not limited to, about any of 10 mg / m2, 20 mg / m2, 30 mg / m2, 40 mg / m2, 50 mg / m2, 60 mg / m2, 75 mg / m2, 80 mg / m2, 90 mg / m2, 100 mg / m2, 120 mg / m2, 150 mg / m2, 175 mg / m2, 200 mg / m2, 210 mg / m2, 220 mg / m2, 230 mg / m2, 240 mg / m2, 250 mg / m2, 260 mg / m2, and 300 mg / m2.

[0192] In various embodiments, the combination therapy methods of the present disclosure may further comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but are not limited to, treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to co-stimulatory or co-inhibitory molecules (immune checkpoints), such as including, but not limited to antibody to CTLA-4, PDL-1 , CD40, OX-40, CD137, GITR, LAG3, TIM-3, SIRPa, CD47, GITR, ICOS, CD27, Siglec 7, Siglec 8, Siglec 9, Siglec 15, VISTA, CD276, CD272, TIM-3, and B7-H4; treatment using bispecific T cell engaging antibodies (BiTE®) such as blinatumomab; treatment involving administration of biological response modifiers such as IL-7, IL-10, IL-12, IL-15, IL-21 , IL-22, GM-CSF, IFN-a, IFN-p, IFN-y, TGF-p antagonist or TGF-p trap; treatment using therapeutic vaccines, including, but not limited to oncolytic virus, such as T-vec, or therapeutic vaccine, such as sipuleucel-T ; treatment using dendritic cell vaccines, or tumor antigen peptide or neoantigen vaccines; treatment using chimeric antigen receptor (CAR)-T cells; treatment using CAR-NK cells; treatment using NK cell; treatment using iPS induced-NK cells; treatment using iPS induced-T cells; treatment using iPS induced CAR-T or iPS induced CAR-NK cells; treatment using tumor-infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo expanded and / or TCR- T cells); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) agonists CpG, TLR7,TLR8, TLR9, and vaccine such as Bacille Calmette-Guerine (BCG), and imiquimod; wherein the combination therapy provides increased effector cell killing of tumor cells, i.e., a synergy exists between the VitoKine construct and the immunotherapy when co-administered.

[0193] In various embodiments, the combination therapy comprises administering a VitoKine construct and the second agent composition simultaneously, either in the same pharmaceutical composition or in separate pharmaceutical composition. In various embodiments, a VitoKine construct composition and the second agent composition are administered sequentially, i.e., a VitoKine construct composition is administered either prior to or after the administration of the second agent composition. In various embodiments, the administrations of a VitoKine construct composition and the second agent composition are concurrent, i.e., the administration period of a VitoKine construct composition and the second agent composition overlap with each other. In various embodiments, the administrations of a VitoKine construct composition and the second agent composition are non-concurrent. For example, in various embodiments, the administration of a VitoKine construct composition is terminated before the second agent composition is administered. In various embodiments, the administration of a second agent composition is terminated before a VitoKine construct composition is administered.

[0194] The following examples are offered to more fully illustrate the disclosure but are not construed as limiting the scope thereof.Example 1Sequence optimization of the pembrolizumab variable domains

[0195] The current invention aims to optimize the variable domain sequences of pembrolizumab to enhance the score of similarity to the human germline sequences, a measure for their “humanness”. This enhancement could potentially lower the immunogenicity risk. Additionally, the inventors used human VH3 family germline sequence, which, despite being less homologous to pembrolizumab, is more prevalent and behave better, as an alternative acceptor framework. This was done with the aim of improving the biophysical properties of the resulting humanized antibody, ensuring it retains full activity, and enhancing its sequence humanness.

[0196] Pembrolizumab was humanized by CDR grafting technology using the most homologous human antibody sequences available in RCSB protein databank as the acceptor human frameworks. The frameworks found in Gen Bank under accession numbers AB063829 (SEQ ID NO: 40) and M29469 (SEQ ID NO: 41 ) 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, pembrolizumab only shares 79.6% sequence identity to IGHV1 -2, its closest human germline, according to the comparison of the variable region exonsusing the International Immunogenetics Information System (IMGT) DomainGapAlign tool (www.imgt.org). A similarity score to the human germline sequence was proposed as a defining criterion for therapeutic antibodies by the international nonproprietary names (INN) group of WHO in 2014, presumably based on the notion that higher similarity could suggest reduced immunogenicity. The low similarity score, or “degree of humanness” (Abhinandan KR et al., J Mol Biol (2007) 369:852-62) of pembrolizumab’s heavy chain could be due to the poor level of conservation between the mouse CDRs and their human sequence equivalents, the necessity to retain a few structurally important mouse framework residues to recapitulate antigen binding, and the preservation of unique somatic mutations in the human framework sequence AB063829.

[0197] To enhance the degree of humanness of pembrolizumab, certain CDR residues were targeted for substitution with their equivalent residues from the closest human germline sequences. This method is herein referred to as CDR germlining. While avoidance of CDR perturbation has traditionally been a central principle humanized Abs design, only a limited number of CDR residues engage in direct antigen interaction. Therefore, certain CDR residues may be replaced without compromising the activity of the antibody. According to the Kabat numbering scheme, 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).

[0198] As for the CDR3 sequences, a portion of the light chain CDR3 (CDR-L3) and the entire heavy chain CDR3 (CDR-H3) were not part of the germline sequence’s variable region exons V region. Consequently, there are no human germline residues available to substitute the mouse CDR counterparts. Additionally, CDR3s, especially CDR-H3, are highly variable and vital for antigen binding and functional activity, making it crucial to preserve their conformations. As such, both CDR-L3 (QHSRDLPLT; SEQ ID NO: 25) and CDR-H3 (RDYRFDMGFDY; SEQ ID NO: 33) of pembrolizumab were excluded from the CDR germlining process.

[0199] The sequences of pembrolizumab’s CDR-L1 and CDR-L2 were aligned to their counterparts from the closest human germline IGKV3D-1 1 (GenBank accession # X17264; SEQ ID NO: 39). The alignments are shown in Table 6A. Likewise, the alignments of CDR-H1 and CDR-H2 sequences of pembrolizumab with the closest human germline sequence, IGHV1 -2 (GenBank accession # X62106; SEQ ID NO: 37), are displayed in Table 6B.Table 6AAlignments of the CDR-L1 and CDR-L2 sequences of pembrolizumab with IGKV3D-11Table 6BAlignments of the CDR-H1 and CDR-H2 sequences of pembrolizumab with IGHV1 -2“-“indicates sequence gap.Residues in bold and italic represent those interacting with PD1 , as per the complex structure (Horita S. et al., Sci Rep (2016) 6:35297).Underscored residues are subject to the CDR germlining process.

[0200] Multiple pembrolizumab CDR residues directly engage polar interactions with PD1 , e.g., hydrogen bond and salt bridge (Horita, S. et al. Sci. Rep (2016). 6: 35297). The contacting residues in or around both VL and VH CDR1 and CDR2 includeLSer28,LTyr30 in CDR-L1 ,LTyr49 immediately prior to CDR-L2,LTyr53 in CDR-L2,HTyr33,HTyr35 in CDR-H1 ,HAsn52,HSer53,HAsn54,HThr57,HAsn58 in CDR-H2 (where the superscripted letter ‘L’ donates light chain, and ‘H’ refers to heavy chain). Except forLTyr49, which is not a CDR residue and not shown, all the antigen-interacting CDR residues mentioned above are in bold and italic in Tables 6A and 6B. Among the CDR residues that differ from the germline sequences, CDR-L1 residuesLLys27,LHis34, CDR-L2 residuesLLeu54,LGlu55, CDR-H2 residuesHPhe59,HAsn60,HGlu61 ,HLys64, andHAsn65 (underscored in Tables 6A and 6B) were selected for CDR germlining. They were replaced with their respective human germline equivalents with the following amino acid substitutions:LK27Q,LH34A,LL54R,LE55A,HF59Y,HN60A,HE61 Q,HK64Q, andHN65G either individually or in combination. Other CDR residues were reserved to avoid any disruption of the antigen-interacting residues.

[0201] For CDR-H1 (NYYMY; SEQ ID NO: 26), only a single residue,HAsn31 , is eligible for CDR germlining, to be replaced with its equivalent residue, glycine, in the human germline sequence. Other residues were kept either because they engage in direct antigen interaction with PD1 (HTyr33 andHTyr35) or because they are conserved between the mouse and human germline sequences (HTyr32 andHMet34). However, considering CDR-HTs short length of onlyfive amino acids and the fact that two residues have a direct interaction with the antigen, any amino acid alteration could potentially disturb the CDR confirmation and impact the antibody's activity. Consequently,HAsn31 was not modified through CDR germlining, and CDR-H1 is fully reserved.

[0202] In addition to the low level of conservation between the mouse CDRs and their corresponding human germline sequences, the pembrolizumab heavy chain frameworks (FRs) also contain multiple non-germline residues. They arose due to the retention of the unique somatic mutations in the acceptor framework sequence AB063829. These somatic mutations, includingHVal9 in FR-H1 ,HThr76,HLys82a,HGln83,HPhe84 in FR-H3 andHThr108 in FR-H4, are not considered structurally important. Replacement with their respective germline counterparts,HV9A,HT76S,HK82aS,HQ83R,HF84S,HT108L, leads to a considerable improvement in the similarity score to the human germline sequence, without disturbing the CDR conformation or altering the antibody’s activity.

[0203] Moreover, IGHV3-23 (SEQ ID NO: 38) was used as an alternative acceptor framework to investigate whether utilizing a human acceptor framework with substantially lower sequence homology, but superior biophysical attributes could enhance the biophysical properties of the resultant humanized antibody without compromising its functional activity. IGHV3-23 belongs to the human antibody heavy chain germline VH3 family, which is the most common VH family in the human repertoire. It is also the most prevalent among the marketed human monoclonal antibodies and is widely acknowledged for its superior drug-like properties. Given that the CDR conformation was exquisitely sensitive to the chemical environment of the surrounding framework, a few structurally significant framework residues in pembrolizumab, which differ from their VH3 germline family equivalents, were selected for reverse mutation to their corresponding pembrolizumab equivalents. Furthermore, several CDR-H2 residues were targeted for CDR germlining using IGHV3-23 CDR-H2 as a template to improve the similarity score to the human germline sequence.

[0204] Alignments of the CDR-H1 and CDR-H2 sequences of pembrolizumab with IGHV3-23 are displayed in Table 6C. Six CDR-H2 residues,HPhe59,HAsn60,HGlu61 ,HLys62,HPhe63, andHAsn65 (underscored in Table 6C; the superscripted letter ‘L’ donates light chain, and ‘H’ refers to heavy chain) were selected for CDR germlining with the following amino acid substitutions:HF59Y,HN60A,HE61 D,HK62S,HF63V, andHN65G. CDR-H1 was excluded from the CDR germlining process for the reason described earlier.Table 6CAlignments of the CDR-H1 and CDR-H2 sequences of pembrolizumab with IGHV3-23Residues in bold and italic represent those interacting with PD1 , as per the complex structure (Horita S. et aL, Sci Rep (2016) 6:35297).Underscored residues are subject to the CDR germlining process.

[0205] Two framework residues,HThr30 andHArg94, were considered structurally significant and were preserved without alteration to their corresponding germline equivalents,HSer30 andHLys94. Five additional IGHV3 framework residues,HVal48,HSer49,Hlle69,HArg71 , andHAsn73, which belong to the vernier zone (U.S. Patent No. 5,821 ,337 and U.S. Patent No. 5,859,205) and may be of structural significance, were reverted to their corresponding pembrolizumab residues,HMet48,HGly49,HLeu69,HThr71 , andHSer73, either individually or in combination. The importance of specific framework amino acid residues was assessed experimentally. The number of reverse mutations was minimized to ensure the highest similarity score to the germline sequence without negatively affecting antibody activity.

[0206] All the optimized antibody sequences were expressed as full length antibody with a kappa light chain constant region containing the sequence set forth in SEQ ID NO: 34 and a modified lgG1 heavy chain constant region containing the sequence set forth in SEQ ID NO: 35. Table 7 lists the SEQ ID NOS of the VL, VH, CDR-L1 , CDR-L2, and CDR-H2 of the exemplary optimized PD1 blocking antibodies along with the Reference Antibody (P-0734), comprising VL and VH sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 6, respectively. All antibodies of the present invention comprise identical CDR-L3 (SEQ ID NO: 25), CDR-H1 (SEQ ID NO: 26), and CDR-H3 (SEQ ID NO: 33).Table 7Exemplary PD1 blocking antibodies resulted from CDR and FR germliningExample 2Construction, Production, and Purification of the Optimized PD1 Blocking Antibodies

[0207] All genes were codon optimized for expression in mammalian cells, and they were synthesized and subsequently subcloned into their recipient mammalian expression vectors through the service of GenScript. Protein expression is driven by a CMV promoter, and a synthetic SV40 polyA signal sequence is positioned at the 3' end of the coding sequence. A leader sequence was engineered at the N-terminus of the constructs to ensure appropriate signaling and processing for secretion.

[0208] The antibodies were produced by co-transfecting vectors harboring light chain and heavy chain with a 1 :1 ratio in ExpiCHO cells (ThermoFisher) following the manufacturer’s instructions. A 0.8 pg DNA / mL culture volume was used, and the transferred cells were incubated at 37°C in a shaker incubator at 130 rpm with 8% CO2 atmosphere. ExpiFectamine™ OHO enhancer (6 pL / mL cell culture) and ExpiCHO™ feed (240 pL / mL cell culture) were added to the flask 18-22 hours post transfection. After 8 days of cultivation, the supernatant washarvested for purification by centrifugation for 20 min at 2200 rpm, followed by sterile filtered using a 0.22 pm filter (Corning).

[0209] The secreted antibody was purified from cell culture supernatants using Protein A affinity chromatography. Cell culture supernatant was loaded onto a MabSelect SuRe 5-mL column (Cytiva) equilibrated with 5 column volumes (CV) of phosphate buffered saline (PBS), pH 7.2 (ThermoFisher). Unbound protein was removed by washing with PBS, and target protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride buffer, pH 3.2. Antibody solution was neutralized by adding 3% of 1 M Tris buffer, pH 10.2 followed by concentration and buffer exchange to PBS, pH 7.2 using Amicon® Ultra-15 Ultracel with 10 KDa MWCO (Merck Millipore).

[0210] The purity and molecular weight of the purified antibodies were analyzed by SDS-PAGE, both with and without a reducing agent, and then stained with Coomassie (Imperial™ protein stain, ThermoFisher). The SurePAGE™ Pre-Cast gel system (8-16% Bis-Tris, GenScript) was used according to the manufacturer's instruction. The aggregate content of the antibodies was analyzed on an Agilent 1200 high-performance liquid chromatography (HPLC) system. Samples were injected into an AdvanceBio size-exclusion column (300A, 4.6 x 150 mm, 2.7 pm, LC column, Agilent) using 150 mM sodium phosphate buffer, pH 7.0 as the mobile phase at 25 °C.

[0211] The antibody concentration of purified protein samples was determined by measuring the absorbance at 280 nm using a Nanodrop spectrophotometer (ThermoFisher) divided by the molar extinction coefficient calculated based on its amino acid sequence. Endotoxin level of purified protein samples were measured using Endosafe nexgen-PTS (Charles River) as per the manufacturer’s instruction.Example 3Assays to Evaluate the Biological Activities of the Optimized PD1 Blocking Antibodies

[0212] Antibodies of the invention were tested for their antigen binding activity by well-known methods such as enzyme-linked immunosorbent assay (ELISA). Briefly, Nunc Maxisorp plates (ThermoFisher) were coated with recombinant human PD1 protein in bicarbonate buffer, pH 9.4 (ThermoFisher), overnight at 4°C, using 1 pg of antigen per well (100 pL / well). After triple washing with PBS / 0.05% Tween 20, plates were incubated with SuperBlock (ThermoFisher) for two hours at room temperature to block nonspecific binding. The PD1 antibodies, serially diluted three-fold with blocking buffer (PBS with 1% bovine serum albumin) were added to the plates(100 pL / well) post washing and incubated at room temperature for one hour. After another washing, antibodies were detected by incubating with a horseradish peroxidase (HRP)- conjugated goat anti-human IgG Fc antibody (ThermoFisher) diluted 1 :5000 in blocking buffer (100 pL / well) for one hour at room temperature. After a final wash, TMB substrate (ThermoFisher) at 100 pL / well was added. Plates were sealed and left to incubate in dark for 5- 20 minutes. The reaction was stopped by adding 2N sulfuric acid (Ricca Chemical) (50uL / well), and the absorbance was measured at 450 nm with a plate reader. The curves were plotted, and the half-maximal effective concentration (EC50) values were calculated using GraphPad Prism software.

[0213] Additionally, HEK 293T cells stably expressing human PD1 gene (Crown Bioscience) was used to determine the cell-based binding strength of the optimized PD1 antibodies by flow cytometry. After harvesting, HEK293-hPD1 cells were seeded into a 96-well U-bottom plate at 1 x 105cells / well (100 pL), incubated with Fc block (1 :50) for 20 minutes at 4°C, and subsequently washed with FACS buffer (PBS, 1% FBS). Cells were then treated with three-fold serial dilutions of each antibody at concentrations ranging from 0.01 -100 nM in FACS buffer for 30 minutes at 4°C. Subsequently, cells were washed twice with FACS buffer to remove unbound molecules, and 40 pL of the 1 OO diluted PE-labeled goat anti-human Fc secondary antibody (eBiosciences) was added to the cells. Following a 30-minute incubation at 4°C and another double wash with FACS buffer, antibodies bound to the cells were detected with PE-labeled secondary antibody by flow cytometry (BD ACCURI-C6), and EC50 values were calculated using GraphPad Prism software.

[0214] Furthermore, a thaw-and-use format of Promega luciferase reporter assay, a biologically relevant mechanistic-based assay, was used to measure the potency in blocking PD1 interaction. Cell thawing and plating procedures were followed exactly as described in the manufacturer's protocol.

[0215] Briefly, one vial (0.5 mL) of PD-L1 aAPC / CHO-K1 cells were thawed and mixed with 14.5 mL cell recovery medium (90% Ham’s F12 / 10% FBS). Next, 100 pL of this cell suspension was added to the inner 60 wells of two 96-well flat-bottom assay plates, while perimeter wells received 100 pL of cell recovery medium. After overnight incubation at 37°C and 5% CO2, the medium was discarded. The inner wells received 40 pL of 3-fold serially diluted compounds, while the perimeter wells got 80 pL of assay buffer (99% RPMI 1640 / 1% FBS). Subsequently, one vial (0.5 mL) of PD1 effector cells were thawed and mixed with 5.9 mL of assay buffer, and 40 pL of this mixture was added to the inner wells. After a 6-hour incubation at 37°C, 5% CO2,and a 7-minute equilibration at room temperature, 80 piL of Bio-Gio™ reagent was added to all wells. The plates were then incubated at room temperature for 10 minutes with shaking. The resulting luminescence was measured using a luminescence plate reader (BioTek synergy hi ).

[0216] Background was calculated by averaging relative light units (RLU) of perimeter wells. Fold Induction was determined as the RLU of the antibody sample minus background, divided by the RLU of the control samples (without antibody) minus background, or fold induction = RLU (antibody-background)) / (RLU (no antibody ctrl-background). Finally, ECso values were determined using the curves fitted using GraphPad Prism software.Example 4Evaluation of the PD1 Antibodies Comprising Germlining Modifications Based on the Closest Human Germline Sequences

[0217] Firstly, the effectiveness of P-0734 in inhibiting the PD1 / PD-L1 interaction was compared to that of the pembrolizumab (PBL) biosimilar. While P-0734 and PBL biosimilar share the identical variable domains, they differ in their heavy chain constant region. PBL contains an lgG4 constant chain containing the S228P mutation (SEQ ID NO: 36), whereas P-0734 has an IgG 1 constant chain with L234A / L235A / G237A mutations (SEQ ID NO: 35) to abrogate Fc effector functions. As shown in FIG. 4, P-0734 and PBL biosimilar were equally potent in blocking the interaction between PD1 and PD-L1. This result was expected and confirms that the ability to block PD1 is determined by the variable domain sequences, and not by the immunoglobulin classes. P-0734 faithfully recapitulates the potency of PBL biosimilar in blocking the PD1 / PD-L1 interaction and is herein referred to as the Reference Antibody.

[0218] The impacts of CDR germlining substitutionsLH34A in CDR-L1 andHF60Y in CDR-H2 were evaluated using antibodies with slightly different mutational contexts. These antibodies, P- 1148, P-1150, P-1151 , and P-1153, all contain germlining substitutionsLK27Q in CDR-L1 ,LL54R,LE55A in CDR-L2, andHN60A,HE61Q,HK64Q,HN65G in CDR-H2. P-1150 includes an additionalLH34A substitution in CDR-L1 , P-1151 has an extraHF59Y substitutions in CDR-H2, and P-1153 harbors bothLH34A andHF59Y changes in addition. Table 8 provides a list of the CDR germlining substitutions for these exemplary PD1 blocking antibodies.Table 8CDR germlining substitutions of exemplary PD1 blocking antibodies

[0219] As illustrated in FIGS. 5B & 5C and summarized in Table 9, the CDR-L1 germlining substitutionLH34A consistently led to a reduction in PD1 blockade potency (EC50) of approximately 3.5-fold, along with a 25% reduction in both Emax (maximum effect / luminescence signal) and fold induction, regardless of the presence (P-1151 vs P-1153) or absence (P-1 148 vs P-1150) of theHF59Y substitution. The impact ofHF59Y germlining substitution was similarly assessed, with the data shown in FIGS. 5B & 50 and summarized in Table 9. Irrespective of whether theLH34A substitution is present (P-1 150 vs P-1 153) or absent (P-1 148 vs P-1151 ), theHF59Y CDR germlining substitution resulted in a consistent albeit modest reduction in both potency (EC50; reduced by about 1.8-fold) and signal (10-15% decrease in both Emax and fold induction). Compared to P-0734, the cumulative CDR germlining substitutions in P-1153 ended up with a nearly 20-fold decrease in PD1 blockade potency (EC50) and 50% reduction in Emax. As a result, bothLH34A andHF59Y substitutions were deemed detrimental in this particular framework and the original CDR residues,LHis34 andHPhe59, will be preserved.

[0220] However, despite the notable differences in potency in blocking the PD1 / PD-L1 interaction, all four optimized PD1 antibodies and the Reference Antibody, P-0734, displayed nearly identical binding strength with an EC50 value close to 100 pM (FIG. 5A and Table 9).Table 9ELISA binding and PD1 blocking activity of exemplary PD1 blocking antibodies

[0221] This piece of data suggested that the mechanistic-based functional assay was capable of discerning minute activity changes that aren’t detectable by ELISA binding assay. As such, the luciferase PD1 / PD-L1 reporter assay is herein used as the primary tool to characterize and rank PD1 blocking antibodies derived from pembrolizumab via germlining substitutions. It is expected that the derivative antibodies that maintain full functional activity will exhibit identical in vivo efficacy as pembrolizumab.

[0222] The potential negative effects of CDR-L2 germlining substitutionLE55A was further assessed by comparing P-1127 and P-1129. Both these molecules containLK27Q andLK54E germline substitutions in the light chain CDRs, with the only sequence difference being the extra CDR-L2 substitution,LE55A, in P-1129. As demonstrated in FIG. 6A, P-1129 displayed a minor yet appreciable reduction in potency (EC50 = 0.39 nM and 0.58 nM for P-1 127 and P-1 129, respectively) and a marginal 10% decrease in Emax. Hence, theLE55A amino acid substitution was deemed adverse and the original residue,LGlu55, will be preserved.

[0223] P-1174, harboring a total of 6 CDR germlining substitutions,LK27Q,LL54R,HN60A,HE61 Q,HK64Q, andHN65G, exhibited identical PD1 blockade activity as P-1127 and P-0734 with EC50 of 0.64 nM, 0.54 nM, and 0.67 nM for P-0734, P-1127, and P-1174, respectively (FIG. 6B). Additionally, P-1174 was derived from P-1 14 by eliminating one CDR germlining substitution,LE55A. When compared to P-0734, P-1 174 exhibited higher potency than P-1148 (refer to FIG. 5B & FIG. 6B). This piece of data further collaborated the conclusion that the original CDR residues,LGlu55, should not be altered.

[0224] Besides the low conservation between the mouse CDRs and their human germline counterparts, multiple non-germline residues in the pembrolizumab VH framework also contributed to the low sequence similarity score with the germline. These non-germline residues, originating from the unique somatic mutations preserved in the acceptor framework sequence, includingHVal9 in FR-1 ,HThr76,HLys82a,HGln83,HPhe84 in FR-3 andHThr108 in FR-4, are considered not to be structurally significant. To further enhance the sequence similarity score with the germline or degree of humanness, these non-germline residues in the P-1174 framework were replaced with their respective germline equivalents,HV9A,HT76S,HK82aS,HQ83R,HF84S,HT108L, resulting in P-1271. As expected, P-1271 displayed the same PD1 blocking activity as the Reference Antibody, P-0734 (FIG. 6C) with EC50 values of 0.66 nM for P-1271 and 0.70 for P-0734, respectively.

[0225] In conclusion, CDR germlining substitutions,LK27Q,LL54R,HN60A,HE61 Q,HK64Q, andHN65G, in P-1174 and P-1271 enhanced antibody sequence degree of humanness withoutcompromising the potency in blocking the PD1 / PD-L1 interaction. Additional six framework germlining substitutions in P-1271 further augmented the score of similarity to the closest human germline sequences. Table 10 lists the germlining substitutions and similarity scores to the closest human germline sequences of P-1174 and P-1271 in comparison to the Reference Antibody, P-0734.Table 10Germlining substitutions and similarity scores of the exemplary optimized PD1 antibodies, P- 1174 and P-1271 , with the closest human germline sequencesExample 5Evaluation of the PD1 Antibodies Comprising Germlining Modifications Based on A More Prevalent Human Germline Family (VH3)

[0226] The adoption of framework germlining substitutions based on the human antibody heavy chain germline IGHV3-23 (SEQ ID NO: 38) was investigated to exam whether an antibody framework with substantially lower sequence homology, but superior biophysical properties, could enhance the drug-like properties of the resultant antibody while fully retaining its functional activity. Of the 33 framework germlining substitutions (Table 11 A), the importance of the 5 Vernier zone residues,HV48,HS49,HI69,HR71 , andHN73, were assessed experimentally by reversion mutation to their respective pembrolizumab equivalents,HV48M,HS49G,HI69L,HR71 T, andHN73S, either individually or in combination. Additionally, six CDR-H2 residues,HF59Y,HN60A,HE61 D,HK62S,HF63V, andHN65G, were selected for CDR germlining substitutions with their corresponding residues in IGHV3-23. Table 1 1 B provides a summary of the VH3 germlining substitutions in the exemplary antibodies.Table 11 AVH Framework germlining substitutions based on IGHV3-23The bolded and underlined residues represent a total of 33 framework germlining substitutions.Table 11 BVH CDR germlining and FR reversion mutations based on IGHV3-23

[0227] FIG. 7 depicts the PD1 blockade activity of P-1175 and P-1181 , differing only in their CDR-H2 germlining substitutions (as shown in Table 11 B). Compared to P-0734, both P-1 175 and P-1181 exhibited substantially diminished potency in blocking PD1 interaction. Specifically, P-1174 showed a 10-fold reduction in potency (EC50) and 25% decrease in both Emax and fold induction. This was in comparison to P-1181 ’s 15-fold drop in potency and 40-50% decrease in Emax and fold induction (as illustrated in FIGS. 7A & 7B and summarized in Table 12). Since P-1181 displayed a more drastic decline in activity, its two distinct CDR germlining substitutions,HK62S,HF63V, were deemed detrimental, hence the original CDR residues,HLys62 andHPhe63, will be preserved. These findings suggested that the significance of individual CDR residues need to be assessed experimentally; even CDR residues that are close to the boundary or are not immediately adjacent to antigen-contacting residues could negatively impact the activity.

[0228] Two to five framework residue reversion mutations were introduced to P-1175, resulting in P-1176, P-1177, and P-1178, as detailed in Table 1 1 . As indicated by the data in FIG. 8, the combined reversion mutations,HI69L,HR71 T, andHN73S, in P-1176 effectively restored PD1 blocking activity, almost matching the level of P-0734. Similarly, the activity was significantly reinstated in P-1177 due to the combined reversion mutations,HV48M andHS49G, although not as effectively as in P-1 176. Nevertheless, the incorporation of these two reversion mutations (HV48M andHS49G) into P-1176 did not lead to further enhancement in activity for the resulting antibody, P-1178. (P-1 178 vs P-1 176 in FIG. 8 and Table 12).Table 12PD1 blockade activity of exemplary PD1 blocking antibodies

[0229] The significance of each of the three FR reversion mutations,HI69L,HR71 T, andHN73S were further assessed by comparing PD1 blocking activity of P-1 198 (HN73S), P-1 199 (HR71T,HN73S), and P-1201 (HI69L,HR71 T,HN73S). As demonstrated in FIG. 9, each added reversion mutation led to slight yet evident cumulative increases in PD1 blockade activity. Only the combination of all the three reversion mutations in P-1201 led to nearly fully restored functional activity (with EC50 values of 1 .28 nM and 0.78 nM for P-1201 and P-0734, respectively). Thus,all the three reversion mutations,HI69L,HR71 T,HN73S, were deemed essential and will be incorporated.

[0230] Further, the PD1 inhibitory activity of P-1194, P-1201 , and P-1238 were compared and illustrated in FIGS. 10A and 10B. P-1 194 and P-1201 , differing by only one additional CDR germlining substitution,HF59Y, displayed identical PD1 blocking potency. This suggests that this particular substitution did not negatively affect the activity, contradicting earlier observation thatHF59Y germlining substitution was detrimental when IGHV1 -2 germline sequence was adopted. It is thus postulated that the impact of individual CDR germlining substitution is dependent on the context of the surrounding framework sequences. P-1238 were equally potent as the Reference Antibody, P-0734, with EC50 values of 0.73 nM and 0.70 nM, respectively. Compared to P-1194, the two additional framework reversion mutations,HV48M, andHS49G, in P-1238 contributed to slight but discernable improvement in activity.

[0231] In the final assessment, P-1 174, P-1193, P-1198, P-1199, and P-1201 were assessed for their binding strength to PDF cells (FIG. 11 ). As anticipated, P-1 174, which fully preserved PD1 blocking potency (FIGS. 6C and 6D), displayed a binding affinity to PD1 -expressing cells equivalent to the Reference Antibody, P-0734 (FIGS. 11A and 11 B). P-1198, P-1199, and P- 1201 , which contain 1 -3 framework reversion mutations, displayed subtle but evident potency difference in blocking PD1 interaction (FIG. 9), but such variations in activity were not detected in the cell-based binding assay. All three compounds demonstrated an equal ability in binding to PD1+cells as P-0734 (FIGS. 11 C & 1 1 D and Table 13). Yet, the cell-based binding assay was able to distinguish P-1193, which contains no framework reversion mutation, from other compounds, as shown in FIGS. 11 C & 11 D and Table 13. The extent of the decrease, though, was less pronounced than what was observed in the blocking assay. The data further corroborate our earlier observation that the mechanistic-based PD1 / PD-L1 blocking assay is more sensitive than binding assays in identifying subtle activity differences.Table 13Binding strength of exemplary optimized PD1 blocking antibodies to PD1 + cells

[0232] In summary, the optimized PD1 blocking antibodies, P-1194, P-1201 and P-1238, built on a VH framework (IGHV3-23) that is of substantially lower sequence homology but superior biophysical properties, are able to fully or nearly fully retain antibody’s functional activity and display improved similarity score to the closest human germline sequence (IGHV3-23). The mutation details and similarity scores for each antibody are summarized in Table 14.Table 14CDR germlining substitutions, PR reversion mutations, and similarity scores to the closest human germline sequences of exemplary optimized PD1 antibodiesExample 6Germlining Substitutions Let to Decreased Hydrophobicity of the Optimized PD1 Blocking Antibodies

[0233] Among the 23 FDA and EMA approved therapeutic mAbs, pembrolizumab was the most hydrophobic one and consequently had the highest tendency to aggregate (Goyon et al., J.Chromatogr. B 1065-1066: 35-43, 2017). Consistent with the experimentally-determined apparent hydrophobic interaction chromatography (HIC) retention factors (k), the SSH2.0 hydrophobicity prediction tool (http: / / i-uestc.edu.cn / SSH2 / ; Zhou et al., Front. Genet. 13: 842127, 2022) indicated that both variable chains of pembrolizumab carry a significant risk of hydrophobic interaction. The probabilities of hydrophobic interaction for its VH and VL are 0.97and 0.61 , respectively. An antibody is predicted to have a high risk of hydrophobic interaction if the probability is 0.5 or more (with 1 being the maximum possible value).

[0234] While the focus of the germlining substitutions was to enhance the degree of “humanness" in the antibody sequence, the process also resulted in a significant reduction in the probabilities of hydrophobic interaction for multiple optimized antibody sequences. Table 15 provides a summary of the predicted probabilities of hydrophobic interaction for the variable domains of the exemplary optimized PD1 blocking antibody, as estimated by SSH2.0.Table 15Summary of exemplary antibodies with improved similarity scores and reduced probabilities for hydrophobicity interaction while retaining PD1 blocking activity

[0235] As shown in Table 15, the two light chain CDR germlining substitutions,LK27Q andLL54R, markedly decreased the hydrophobicity probability of VL from 0.607 for 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 marginal reduction in hydrophobicity, with hydrophobicity probabilities altering from 0.971 for (P-0734) to 0.848 for (P-1174) and to approximately 0.8 for antibodies with their VH based on VH-3 family frameworks. However, when germlining substitutions (HV9A,HT76S,HK82aS,HQ83R,HF84S,HT108L) were implemented in the VH framework of P-1174, the resulting construct, P-1271 , had a hydrophobicity probability of 0.185, much lower than that of P-1174.

[0236] Hydrophobic patches on an antibody’s surface are often implicated as one of the main contributions to its propensity to aggregate. Furthermore, these hydrophobic patches can causehigh viscosity. Consequently, the exemplary PD1 blocking antibodies, which have significantly diminished hydrophobic potentials are expected to exhibit improved biophysical properties. PD1 -targeted IL-2 VitoKine fusions constructed using these optimized PD1 blocking antibodies are also projected to have enhanced developability profiles.Example 7Identifying the Optimal IL-2RaSushi Variant as the Concealing Moiety Domain (D3)

[0237] Representative PD1 Ab IL-2 VitoKine construct is illustrated in FIG. 3. A monomeric IL-2 or IL-2 variant as the active moiety domain (D2) is fused between a PD1 antibody as the targeting domain (D1 ) and an IL-2RaSushi domain as the concealing moiety domain (D3). Linker 2 (L2) connecting IL-2 and IL-2Ra is protease cleavable. IL-2 within VitoKine constructs will remain inert until it is activated locally by proteases that are exclusively present or upregulated at tumor sites or within tumor microenvironment (TME). After the L2 linker is cleaved, the concealing a-subunit ideally dissociates away, as illustrated in FIG. 2. Therefore, it is desirable to identify an IL-2Ra variant with weakened binding to IL-2 to ensure that the concealing a-subunit (D3) can readily diffuse away following proteolysis, but still effectively conceals IL-2 moiety domain’s activity before the linker is cleaved.

[0238] IL-2RaSushi variants were designed to weaken binding to IL-2 by incorporating mutations at residues interacting with IL-2. As listed in Table 16, exemplary IL-2RaSushi variants, P-0751 , P-0752, and P-0753 contain the Y43A, L42G, and R36A mutations, respectively. They were expressed as monomeric Fc fusion proteins by fusing to a 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 the monomeric wild-type IL-2RaSushi. The binding capacity of these three IL- 2RaSushi variants to IL-2 was evaluated using an ELISA.

[0239] Briefly, IL-2RaSushi variant Fc fusion proteins was coated onto the wells of Nunc Maxisorp 96-well microplates at 1 p.g / well. After overnight incubation at 4 °C and blocking with 1% BSA, serial dilutions of P-0689, a monomeric wild-type IL-2 equivalent (containing activityneutral C125I mutation; SEQ ID NO: 117) Fc fusion, were added to each well at 100 pL / well. Following a one-hour incubation at room temperature, 100 pL / well of biotin anti-IL-2 antibody clone B33-2 (BD biosciences) were added and incubated at room temperature for 1 hour. Subsequently, 100 pL / well of Avidin-HRP (BioLegend) at 1 :5000 dilution was added. After 60-min incubation and washing, TMB substrate (ThermoFisher) at 100 pL / well was added. Plates were sealed and incubated at room temperature in the dark. Reaction was stopped by adding 2N sulfuric acid (Ricca Chemical). Absorbance was determined at 450 nm and curves were fitted using GraphPad Prism software.

[0240] As summarized in Table 16 and illustrated in FIG. 12, the amino acid substitutions Y43A, L42G, and R36A each affected the interaction with IL-2. The Y43A change resulted in a modest reduction in IL-2 binding (8.1 -fold), the R36A substitution led to a significant 346-fold drop in binding EC50, while the L42G alteration caused an intermediate, or 35-fold, reduction in its binding to IL-2.Table 16Impact of IL-2Ra amino acid changes on the binding to IL-2

[0241] The aforementioned three IL-2RaSushi variants, along with their wild-type counterpart, were used as the concealing moiety domain to construct four Fc IL-2 VitoKine molecules. Each of these molecules includes a monomeric IL-2 C125I variant (equivalent to wild-type; SEQ ID NO: 117) as the active moiety domain (D2) and a 15-amino acid MMP2 / 9-cleavable L2 linker (SEQ ID NO: 84) connecting IL-2 and IL-2RaSushi (D3). Heterodimeric Fc chains (SEQ ID NOS: 187 and 188) served as the D1 domain. The concealing efficiency of these variants were subsequently assessed by evaluating their potency in inducing Ki67 expression, a marker for cell proliferation, in CD8+ T and NK cells via a human PBMC assay. P-0704, an IL-2 P65R variant (SEQ ID NO: 118) Fc fusion maintaining its wild-type IL-2 potency for the dimeric IL- 2Rpy receptor, was included as the fully active IL-2 control for this set of Fc IL-2 VitoKines.

[0242] Briefly, human PBMCs were isolated by Ficoll-Hypaque centrifugation from the buffy coat purchased from Blood Oklahoma Institute. Purified human PBMCs were treated with serial dilutions of testing compounds and incubated at 37eC for 5 days. On the fifth day, cells were washed once with FACS buffer (1% FBS / PBS) and first stained with Fc-blocker (BioLegend)and surface marker antibodies, including anti-human CD56-FITC and anti-human CD8-APC (BioLegend) at a dilution of 1 :50. After 30-minute incubation and wash, cell pellets were fully resuspended by 200 p.L / well of 1x fixation & permeabilization working solution (Invitrogen) and incubated for 30 minutes at room temperature in the dark. After centrifugation, 200 piL of 1 x permeabilization buffer (Invitrogen) were added to each well for another wash. Cell pellets were resuspended in permeabilization buffer containing anti-human Ki67-PE (BD Life Sciences) at a 1 :25 dilution. After a further 30-minute incubation at room temperature, cells were collected, washed, and resuspended in FACS buffer, and analyzed by flow cytometry. Data were expressed as the percentage of Ki67 positive cells within gated population. The dose- responsive Ki67 proliferation details are illustrated in FIGS. 13A and 13B. Additionally, data specific to CD8+ T cells is summarized in Table 17.Table 17Activity comparison of various IL-2 VitoKine constructs

[0243] FIG. 13 illustrates that P-0701 , which has a wild-type IL-2RaSushi as the concealing moiety domain (D3), showed a significant 3-log reduction in inducing the proliferation of CD8+ T and NK cells compared to P-0704, its fully active IL-2 Fc fusion counterpart. We hypothesized integrating an IL-2-binding disrupting mutation into the D3 domain might weaken D3’s concealing capability, thereby lessening VitoKine’s activity inertness. It was also projected that the extent of this reduction would align with the level of diminution in the binding strength between IL-2 and IL-2RaSushi variants.

[0244] In FIG. 13A and Table 17, Fc VitoKines P-0754 and P-0756, which contain Y43A and R36A in IL-2RaSushi as the D3 domain, displayed weakened concealing capability, compared to P-0701. This leads to higher VitoKine intrinsic basal in stimulating CD8+ T cell proliferation,and the trend was consistent in NK cells as seen in FIG. 13B. The decrease in concealing efficiency, however, did not consistently correlate with the magnitude of binding strength reduction. For instance, the Y43A mutation had a minor effect on binding, showing only an 8.1- fold decrease, while the R36A mutation caused a substantial ~200-fold drop in binding. Moreover, the L42G variant, despite having a 35-fold weaker binding to IL-2, maintained its concealing efficiency nearly identical to its wild-type counterpart, as seen in the activity inertness of its corresponding VitoKine, P-0755 (FIGS. 13A and 13B). Although it was unexpected based on previous knowledge, multiple experiments confirmed that changes in binding strength, resulting from mutations in the IL-2RaSushi, didn't consistently correlate with alterations in its concealing capability. This inconsistency might be attributed to the unique spatial interactions in the VitoKine format.

[0245] Consequently, IL-2RaSushi L42G variant is selected as the preferred concealing moiety domain (D3) for IL-2 VitoKines due to its retained concealing capability to maintain corresponding VitoKine’s activity inertness and its potential to readily diffusing away upon in vivo proteolysis to achieve full activity, given its weakened binding to IL-2. Meanwhile, R36A or Y43A can be used as the concealing moiety domain when one aims to tune the IL-2 VitoKine’s intrinsic basal activity, optimizing the balance between desired antitumor efficacy and potential systemic toxicity. Additionally, other IL-2RaSushi variants with varying degrees of reduced binding to IL-2, e.g., K38E, can be used as the D3 domain of IL-2 VitoKines, following the same rationale.Example 8Surprisingly Diverse Impact of P65 Amino Acid Substitutions on IL-2 Binding to IL-2Ra

[0246] Preferential expansion of regulatory T cells (Tregs) by IL-2 due to the high and constitutive expression of IL-2Ra on Tregs represents an undesirable effect of IL-2 for cancer immunotherapy. IL-2 variants designed to weaken or abrogate binding to IL-2Ra will reduce their responsiveness to Tregs. IL-2 variants that no longer bind to IL-2Ra is expected not to preferentially activate Tregs, but only activate Tregs at concentrations when CD8+ T and NK cells are also activated.

[0247] The IL-2 molecule's P65 residue engages Van der Waals interactions with key residues on the IL-2Ra interface, notably R36 and L42. However, it doesn't form salt bridges or hydrogen bonds with IL-2Ra (Wang, et. al, Science (2005), 310: 1159-1163). Given this, one mightassume that changes to P65 would only slightly alter its interaction with the IL-2Ra subunit and likely only cause a minimal effect on binding. Yet, the actual effects of P65 modifications on its interaction with IL-2Ra were surprisingly diverse, ranging from fully maintaining or even improving binding, to weakening or totally abolishing it.

[0248] A panel of IL-2 variants with different P65 substitutions were fused to Fc via a flexible GS based linker (SEQ ID NO: 103), in either dimeric or monomeric formats. All these variants contain an activity-neutral C125I mutation, intended to enhance their developability. Their binding affinity to IL-2Ra (CD25) was then assessed using an ELISA. Briefly, IL-2Ra-ECD was coated onto the wells at 0.1 |ig / well. After overnight incubation at 4 °C and blocking, serial dilutions of IL-2 Fc fusion proteins were added to each well at 100 |xL / well. Following a one-hour incubation at room temperature, 100 ^L / well of goat anti-human IgG Fc-HRP (1 :5000 diluted in diluent) were added to each well and incubated at room temperature for 1 hour. The plate was developed at room temperature in the dark following the addition of 100 .L TMB substrate for 10 minutes, and 100 gL / well of stop solution was added. Absorbance was determined at 450 nm and curves were fitted using Prism software (Graph Pad).

[0249] The ELISA binding curves are illustrated in FIG. 14. Additionally, the ELISA binding EC50 values for the IL-2 variants, normalized to that of the wild-type (either P-0531 or P-0689 according to each construct’s valency), are detailed in Table 18.Table 18Binding of IL-2 variants with P65 mutations to IL-2Ra as assessed by ELISA

[0250] As illustrated in FIGS. 14A and 14B, P65G mutation in P-0608, P65E mutation in P- 0633, and P65A mutation in P-0706 did not seem to impair the interaction with the IL-2Ra subunit. Rather, these mutations enhanced the binding affinity to IL-2Ra by 18-fold, 10-fold, and 10-fold, respectively, when compared to their respective wild-type IL-2 controls.

[0251] In another set, IL-2 variant Fc fusions, namely P-0634, P-0708, and P-0709, carried P65 alterations leading to different levels of disturbances in the bond with the IL-2Ro subunit. As showcased in FIG. 14C and detailed in Table 10, the P65N mutation in P-0708 resulted in a moderate 8.6-fold decline in IL-2Ra binding. On the other hand, the P65H (P-0634) and P65Q (P-0709) alterations had a more pronounced effect, causing reductions in binding by 23-fold and 43-fold, respectively.

[0252] Another group of P65 substitutions, specifically P65R and P65K, appeared to engender drastic disruption in the IL-2 and IL-R2Ra interaction, completely eliminating the binding of P- 0635, P-0704, and P-0707 to IL-2Ra. Here, P-0635 and P-0704 are the dimeric and monomeric versions of the IL-2 P65R variant Fc fusions, and P-0707 harbors the P65K amino acid change. FIG. 14D reveals that these three IL-2 mutein Fc fusions had barely any detectable binding signal, even at IL-2Ra concentrations as high as 100 nM. This is on par with the benchmark molecule, which carries three IL-2Ra-disrupting mutations F42A / Y45A / L72G known to eliminate binding, as documented in Christian Klein et al., Oncolmmunology (2017), 6: 3, e1277306.

[0253] In summary, alterations to the P65 residue led to a diverse range of effects on IL-2Ra binding, including increasing, maintaining, reducing, or completely abrogating the binding of the resultant IL-2 variants to IL-2Ra. Such a broad spectrum of outcomes, arising from modifications to what appears to be a non-critical amino acid, could not be resulted from the predictions of a structure-based mutagenesis approach. The complete loss of IL-2Ra binding was unforeseen and not predicted by the prior art. This is especially surprising given that P65 mutations only modified a small portion of the Van der Waals interaction surface.

[0254] It is expected that the change in IL-2Ra binding strength correlates with IL-2 potency in activating Treg cells. To validate this, IL-2 variant Fc fusion proteins with either enhanced binding (P-0608), reduced binding (P-0634 and P-0709) or abolished binding (P-0635 and P- 0704) to IL-2Ra were examined for their ability to differentially stimulate STAT5 phosphorylation in CD4+ Treg cells. STAT5 is known to be involved in the downstream signaling cascade uponIL-2 binding to the transmembrane IL-2 receptors. Wild-type IL-2 fusion P-0531 and the benchmark molecule were included for comparison.

[0255] The phosphorylation of STAT5 in lymphocyte subpopulations was measured in fresh human PBMCs using the transcription factor FOXP3 to identify the Treg population in FACS analysis. Specifically, purified PBMCs were deprived of serum in MACS buffer (Miltenyi Biotech) at 4 °C for 1 hour, and subsequently treated with serial dilutions of test compounds for 30 min at 37 °C. Cells were then fixed, permeabilized, stained with specific antibodies, and further analyzed by flow cytometry following a similar procedure detailed in Example 7. The staining was 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). The flow cytometry data was gated into CD4+ / Foxp3+ / CD25h'9hgroup for the Treg cell subsets. Data are expressed as the percentage of pStat5 positive cells in the gated population.

[0256] As illustrated in FIG. 15, there is a clear correlation between the IL-2Ra binding strength and the potency in stimulating STAT5 phosphorylation in CD4+ Treg cells. The compounds in FIG. 15A all feature bivalent IL-2 variants while those in FIG. 15B all have monomeric IL-2 variants. P-0608, which has enhanced IL-2Ra binding, displayed appreciably higher potency compared to P-0531. P-0626 (FIG. 15A) and P-0709 (FIG. 15B), in line with its diminished IL- 2Ro binding strength, showed reduced pSTAT5 potency compared with P-0531 / P-0689. However, its remaining, albeit reduced, binding to IL-2Ra ensured that Tregs were activated more effectively than both P-0635 / P-0689 and the benchmark molecules (valency-matched), which completely lost IL-2Ra binding. Similarly, the total loss of IL-2Ra binding led to a significant shift in Treg potency, about 5 logs to the right. This remaining Treg signaling resulted from the activation of IL-Rpy receptors expressed on Treg cells.

[0257] Further, the exemplary IL-2 variant Fc fusions, which included mutations rendering enhanced, reduced, or abolished binding to IL-2Ra, all displayed un-altered binding to IL-2Rpy (FIG. 16A). They also showed nearly equal potency in inducing Ki-67 expression in CD8+ T cells (FIG. 16B). The data underscores the fact that the IL-2 mutations at IL-2Ra interface, regardless of their effect on IL-2Ro binding, do not alter the interaction with IL-2R(3y.Example 9Identifying IL-2 Variants for Optimal IL-2Ra Binding as the Active Moiety Domain (D2)

[0258] The active moiety domain (D2) of IL-2 VitoKine constructs were selected from a panel of IL-2 variants with varying levels of binding strength to IL-2Ra identified in Example 8.Incorporating IL-2 variants with reduced or eliminated IL-2Ra binding in VitoKines can decrease the reactivity to Tregs following proteolytic activation near the tumor. However, it is essential to achieve a balance between the degree of weakened IL-2Ra binding and the efficiency of concealment, given that binding between D2 and D3 is believed to be necessary for the concealing capability of VitoKines.

[0259] The four exemplary IL-2 VitoKines, namely P-0800, P-0830, P-0831 , and P-0802, all contain an anti-mouse PD1 antibody P-0722 (SEQ ID NOS: 52, 189 and 190) as the D1 domain, IL-2RaSushi L42G variant (SEQ ID NO: 184) as the concealing moiety domain (D3), a non-cleavable linker (SEQ ID NO: 103) as the L1 linker, and an MMP-2 / 9 cleavable linker (SEQ ID NO: 84) as the L2 linker. As detailed in Table 19, the active domain (D2) comprises P65R mutation in P-0800, P65N mutation in P-0830, and P65Q mutation in P-0831. P-0802 has the IL-2 wild-type equivalent as the D2 domain.

[0260] The exemplary VitoKines were assessed for their ability to induce Ki67 expression in CD8+ T cells (FIG. 17A) and NK cells (FIG. 17B) using fresh human PBMCs. P-0782, the nonconcealed counterpart of P-0800, containing a monomeric IL-2 P65R variant, was included as a fully active IL-2 reference. The EC50 values of these compounds in terms of stimulating Ki67 expression in NK cells, along with their fold changes in comparison to P-0782, are summarized in Table 19.

[0261] The data revealed that when the D2 domain is the wild-type IL-2, the D3 domain of the VitoKine construct (P-0802) renders an approximate 3-log reduction in activity, suggesting a strong concealment capability of the D3 domain. In contrast, for P-0800, which incorporates an IL-2 variant (P65R) with abolished IL-2Ra binding, there is only a 10- to 20-fold decrease in activity, suggesting a significantly weakening of the D3’s concealment capability due to the absence of binding between the D2 and D3 domains. Interestingly, when using IL-2 variants like P65N and P65Q, which have intermediately reduced IL-2Ra binding, in P-0830 and P-0831 , the D3 domain renders concealing efficiency that is either comparable to, or only marginally less than that it facilitates wild-type IL-2.Table 19Activity comparison of IL-2 VitoKines containing IL-2 variants with varying IL- 2Ra binding strength

[0262] It was postulated that a binding affinity threshold between the D2 and D3 domains, combined with an ideal spatial configuration of the binding interface, is pivotal in determining the concealing efficiency of the IL-2 VitoKines. Given that IL-2 P65Q variant demonstrates significantly reduced binding strength to IL-2Ra (as depicted in FIG. 14C and Table 18) and yet is still efficiently concealed by IL-2RaSushi L42G to remain inert as VitoKine, IL-2 P65Q variant is chosen as the preferred D2 domain for IL-2 VitoKine designs. It is expected the D3 domain will be readily diffused away following protease cleavage due to the diminished binding between the D2 and D3 domains. It is also anticipated that once the bioactivity is fully restored upon proteolytic activation, this variant will have a significantly reduced ability to in stimulate Treg cells compared to the wild-type, as illustrated in FIG. 15B. Nevertheless, other IL-2 variants with reduced IL-2Ra binding, such as P65H and P65N, can also be considered to achieve the right balance between desired antitumor efficacy and minimizing potential systemic toxicity.

[0263] On top of the P65 mutations to achieve a balance between the weakened binding and effective concealment by the IL-2RaSushi, additional mutations altering IL-2’s binding affinity to IL-2Rpy can be incorporated into VitoKines’ D2 domain. These mutations modulate I L-2’s overall responses in cells that mainly express p and y receptor subunits, such as CD8+ T and NK cells. Through this strategy, the intrinsic basal activity of the VitoKines as well as their activity post-proteolytic activation can be fine-tuned.Example 10IL-2 Variants with IL-2Rpy-Disrupting Substitutions for Overall Potency Attenuation

[0264] The choice of mutations disrupting IL-2RP and the common y chain (yc) was informed by inspecting the IL-2 / IL-2R co-crystal structure (PDB code 2B51 ). Substituting energy hot spot residues that directly interact with IL-2R , such as D20 and N88, could result in significantly reduced activity, rendering poor potency. Consequently, substitutions were introduced at non- critical residues at the IL-2 / IL-2RP interface, such as L19. The L19 residue only makes van derWaals interaction with I L-2R£, and the resulting mutants are expected to only tweak, rather than significantly diminish, IL-2’s functional activity. Further, replacing L19 with a non-aliphatic residue eliminates a proposed ‘19LDL’ motif, which may contribute to the vascular toxicity of IL-2 (Baluna R, Rizo et. aL, Proc Natl Acad Sci 1999; 96:3957-62).

[0265] Exemplary IL-2Rp-disrupting mutations, L19H, L19Q, L19Y, were introduced into IL-2 with the mutational context of P65R and C125I in P-0704 to construct monomeric IL-2 Fc fusions. The P65R mutation led to a total loss of IL-2Ra binding and the C125I modification was made for developability purpose, and neither of these changes impacted I L-2’s functional activity for IL-2R3y. The resulting fusion proteins, namely P-0731 , P-0759, and P-0761 , were assessed for their potency in stimulating Ki67 expression in human CD8+ T cells and NK cells by flow cytometry. The results are depicted in FIGS. 18A and 18B and detailed in Table 20A. When compared to P-0704, all variants displayed a decrease in their ability to promote proliferation in human CD8+ T cells and NK cells. Specifically, P-0759 (L19Q) and P-0761 (L19Y) exhibited a modest 3-fold decline in potency while L19H mutation in P-0731 led to a more profound 18-25-fold drop in potency. Potency reduction can also be achieved by incorporating other L19 mutations, such as L19D, L19R, and L19S.Table 20 AExemplary Fc fusions of IL-2 variants containing L19 mutations and their ex vivo activity

[0266] Likewise, amino acid substitutions at Q126, a residue that is integral to the yc interaction, were made to weaken IL-2 interaction with yc. All these mutations were also introduced into IL-2 with the mutational context of P65R and C125I. Fc fusions of monomeric IL-2 variants containing Q126 mutations were listed in Table 20B.

[0267] Increases in the Ki67 expression of human CD8+ T cells and NK cells in response to these IL-2 variants, compared with P-0704, were depicted in FIGS. 19A to 19F, and furthersummarized in Table 20B. IL-2 Q126 Mutations had varying degrees of impact on CD8+ T and NK cell proliferation. For CD8+ T cells (FIGS. 19A, 19C, and 19E), a minor 1 .5-5-fold decrease was shown by Q126N, Q126H, Q126M, Q126F, Q126W, and Q126Y; a moderate 5-20-fold reduction was observed for Q126R, Q126G, Q126S; a significant 20-50-fold decline was noted for mutations including Q126A, Q126V, Q126E, Q126E, Q126L, and Q126T; a more drastic drop (>50-fold) was seen for Q126P and Q126I; and Q126D completely abolished the activity. A comparable trend in potency changes due to Q126 mutations was observed in NK cells (FIGS. 19B, 19D, and 19F).Table 20BFc fusions of IL-2 variants containing Q126 mutations and their ex vivo activityold change was compared to P-0704’s EC50 value in each individual experiment.

[0268] IL-2 potency can be further fine-tuned by combining IL-2R and yc-disrupting mutations, as exemplified by P-1247 (IL-2 domain SEQ ID NO: 173) in comparison 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 comprises the L19Y and Q126N mutations. As shown FIG. 20A, the 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 combined 4-fold potency reduction compared to P-0704 (9.2 nM vs 2.3 nM) for the stimulation of Ki67 expression in human CD8+ T cells. A similar trend was seen with NK cells (FIG. 20B). As will be appreciated by those in the art, combining diverse mutations at position L19 and Q126 can lead to various degrees of activity modulation, and is within the spirit and scope of the invention.

[0269] In summary, in addition to using IL-2Ra-disrupting substitution in IL-2 to limit the undesirable expansion of immunosuppressive Tregs, integrating IL-2Rpy-disrupting substitutions offers a way to attenuate the overall potency for optimal activity. By introducing specific mutations at either L19 or Q126, varying degree of potency can be achieved. The desired potency of IL-2 can be meticulously fine-tuned through the combination of mutations at the L19 and Q126 positions. A reduced potency help avoid excessive pathway activation of the pathway and minimize unwanted target sink. As a result, the strategy can potentially reduce toxicity associated with IL-2 therapy and improve pharmacokinetics and pharmacodynamics. Incorporating IL-2 with reduced potency in VitoKines helps fine-tune their intrinsic basal activity as well as their activity post-proteolytic activation.Example 11 Constructing PD1 Ab-IL-2 VitoKines With Optimized PD1 Blocking Antibodies and Preferred IL-2 and IL-2RaSushi Domains

[0270] Antibodies that block PD1 and thus bypass the immunosuppressive effects in the tumor microenvironment may potentiate IL-2 responses and further enhance immunity against tumors. The PD1 antibodies used to construct PD1 Ab-IL-2 VitoKines as the D1 domain were selected from the optimized human PD1 blocking antibodies comprising light chain sequences set forth in SEQ ID NO: 44 and heavy chain sequences set forth in SEQ ID NOS: 45-49. These optimized PD1 blocking antibodies have a high affinity for human PD1 protein and demonstrate equal or comparable potency as pembrolizumab in blocking PD1 . They also possess a higher sequence similarity score to their closest human germline sequence, resulting in an improved degree of humanness compared to pembrolizumab. Furthermore, they are predicted to have lowerhydrophobicity, which in turn is likely to lower their aggregation propensity than pembrolizumab. PD1 -targeted IL-2 VitoKines constructed using these optimized PD1 blocking antibodies are also projected to have enhanced developability profiles.

[0271] Table 21 A lists the exemplary PD1 Ab-IL-2 VitoKines, with their structure depicted in FIG. 3A. All the exemplary VitoKines comprise IL-2 P65Q variant with or without mutations to modulate activity towards IL-2Rpy as the active moiety domain (D2), IL-2RaSushi L42G variant as the concealing moiety domain (D3), and a cleavable L2 linker ( SEQ ID NO: 84) connecting D2 and D3 domains. Nevertheless, other IL-2RaSushi variants, e.g., R36A, can be used as the concealing moiety domain when it is desirable to adjust the IL-2 VitoKine’s intrinsic basal activity. Additionally, the L1 linker connecting PD1 Ab and IL-2 can be cleavable as well. The compositions of the cleavable linker(s) can be further optimized by using the various sequences set forth in SEQ ID NOS: 78-94, to better suit different disease indications and / or stages.Table 21 AExemplary human PD1 Ab-IL-2 VitoKines

[0272] All genes were codon optimized for expression in mammalian cells, which were synthesized and subcloned into the recipient mammalian expression vector via the service of GenScript. The VitoKine constructs were produced by co-transfecting HEK-293 cells (Expi293TMcells from ThermoFisher) with the mammalian expression vectors following manufacturer’sinstructions. Protein purification and characterization were conducted following the same procedures detailed in Example 2.

[0273] It was found that the PD1 Ab-IL-2 VitoKines comprising optimized antibody sequences, including P-1197, P-1239, and P-1272, expressed at a substantially higher level than P-1120 that contains the Reference Antibody, P-0734. Under the identical transient expression conditions using the same batch of HEK-293 cells, P-1197, P-1239, and P-1272 expressed with titer of 137-150 mg / L compared to 60 mg / L for P-1 120. The data suggested that the PD1 blocking antibodies with optimized sequences eliminating potential sequence liabilities may lead to improved developability properties of the corresponding VitoKine constructs.

[0274] Because these optimized PD1 antibodies did not react with mouse PD1 , surrogate mouse PD1 -Ab-IL-2 VitoKines and additional controls were produced analogously. These were used for in vivo studies, particularly for pharmacokinetics (PK) / pharmacodynamics (PD) and tumor experiments in immunocompetent mice. Table 21 B provides detailed information about these molecules. Except for P-871 and P-1469, these VitoKines contain the mouse PD1 antibody P-0722 (SEQ IND NO: 189, 190, and 52) as the D1 domain. P-0871 and P-1469 are the non-targeted VitoKine counterparts of P-0831 and P-1414, respectively, and they contain the germline antibody P-1260 (SEQ ID NOS: 192, 193, and 194) as the D1 domain. P-0877 is the non-cleavable VitoKine counterpart of P-0831 , differing from P-0831 by the non-cleavable L2 linker. Similarly, P-1446 is the non-cleavable VitoKine counterpart of P-1414.Table 21 BExemplary surrogate mouse PD1 Ab-IL-2 VitoKines and control molecules

[0275] To evaluate the concealing efficiency of VitoKines, their non-concealed counterparts were constructed as controls to enable activity comparison. Exemplary non-concealed counterparts for human PD1 Ab-IL-2 VitoKines include P-1273 (SEQ ID NOS: 226, 203, and 44) and P-1448 (SEQ ID NOS: 227, 203, and 44). Likewise, exemplary non-concealed counterparts for murine PD1 Ab-IL-2 VitoKines include P-0838, P-1409, and P-1413, which, compared to P- 0831 , P-1410, and P-1414 respectively, lack the L2 linker and the D3 domain. All shared the structure illustrated in FIG. 3B.Example 12Confirming IL-2 Activity Concealment and PD1 Antibody Function of PD1 Ab-IL-2 VitoKines

[0276] It is important that the PD1 antibody retain its binding and functional activities when incorporated into PD1 Ab-IL-2 VitoKines. PD1 antibodies of superior target-binding and blocking function can enhance the specificity and selectivity of TIL-targeting and further synergize with IL-2 anticancer immune response by efficiently reversing T cell anergy and exhaustion.

[0277] In a comparative analysis using a luciferase reporter assay, the PD1 inhibition capabilities of exemplary VitoKines P-1 197, P-1239, and P-1272 were set against their respective PD1 blocking antibodies, P-1174, P-1238, and P-1271. As illustrated in FIGS. 21 A and 21 B, each of the three antibodies, when incorporated into their corresponding VitoKine constructs, not only maintained their blocking potency but also slightly improved it. For instance, PD1 antibodies P-1174, P-1238, and P-1271 blocked the PD1 / PD-L1 interaction with EC50 values of 1 .29 nM, 1 .82 nM, and 1 .53 nM, respectively. On the other hand, their corresponding VitoKines, P-1 197, P-1239, and P-1272, displayed a subtle enhancement of 1.5-2-times in the blocking efficiency, with EC50 values of 0.92 nM, 1 .27 nM, and 1.20 nM, respectively.Additionally, in the VitoKine format, there was an 11 -17% increase in both Emaxand fold induction.

[0278] FIGS. 22A-22D further confirmed that the activity of IL-2 remains effectively concealed by the IL-2RaSushi domain, irrespective of the specific PD1 antibody compositions. Exemplary PD1 Ab IL-2 VitoKines, P-1197, P-1272, and P-0872, which differ only based on their distinctPD1 blocking antibodies (detailed in Table 21A), all show an approximate 300-fold decrease in their ability to induce the proliferation of CD8+ T and NK cells when compared P-0879 (FIGS. 22A and 22B) or P-1273 (FIGS. 22C and 22D). For context, P-0879 and P-1273 are respectively the non-concealed counterparts of P-0872 and P-1272, lacking the concealing D3 domain. The EC50 values can be found in Table 22. For CD8+ T cells, the low potency of VitoKines prevented the curve fitting, resulting in only approximate EC50 values. P-1174 is the PD1 antibody component of P-1197 and was included as a negative control.Table 22EC50 values of exemplary PD1 Ab-IL-2 VitoKines Compared to their Non-Concealed CounterpartsExample 13Unexpectedly Enhanced Masking Efficiency by IL-2 Variants with yc-Disrupting Mutations in PD1 Ab-IL-2 VitoKines

[0279] P-0831 , comprising IL-2 P65Q / C125I variant as the active moiety domain, has undergone extensive in vivo studies to validate the VitoKine platform’s concept. Despite an efficient activity concealment by over 300-fold, a VitoKine with a highly potent IL-2 domain might still exhibit considerable intrinsic basal activity. Since the intrinsic basal activity of an IL-2 VitoKine directly correlates with the activity of its active domain (D2), it is anticipated that the basal activity of VitoKines containing potency-attenuated IL-2 variants (such as the ones listed in Table 21 B) will be modulated in proportion, allowing for the administration of increased doses without resulting in adverse effects. This helps fully support the PD1 antibody’s function of reversing T-cell anergy or exhaustion, thereby potentially enhancing the synergistic effects with IL-2 immunotherapy and broadening the therapeutic index.

[0280] Exemplary PD1 Ab-IL-2 VitoKines featuring potency-attenuated IL-2 variants, namely P- 1410 and P-1414, with molecular specifics provided in Table 21 B, were tested for their ability to induce the expression Ki67 in human CD8+ T and NK cells compared to P-1409 and P-1413. Each VitoKine comprises amino acid substitutions at Q126 of the IL-2 domain, with Q126M in P- 1410 and Q126R in P-1414, to weaken the IL-2 interaction with yc. According to the findings in Example 10, Q126M mutation reduced the potency of IL-2 by 2.5-fold and the Q126R mutation decreased the potency of IL-2 by 7.8 times, as observed in the induction of Ki67 expression in CD8+ T cells. A comparable reduction was also noted in NK cells (Table 20B). P-1409 and P- 1413 are respectively the non-concealed counterparts of P-1410 and P-1414, lacking the concealing D3 domain.

[0281] P-1410 and P-1414 were further assessed for their activity in proliferating CTLL-2 cells in comparison to their non-concealed counterparts. Briefly, CTLL2 cells (cytotoxic T cells derived from C57BL / 6 mice) cultured in IL-2-containing medium were harvested, washed, and re-suspended in IL-2-free medium (RPMI1640, 10% FCS, 2 mM Glutamine) for a two-hour starvation period. Post-starvation, these cells, at 50,000 / mL, were transferred into a 96-well U- bottom plate. Serial dilutions of the test compounds were then added, followed by a 24-hour incubation. Cell proliferation was assessed using CellTiter-Glo (Promega) according to manufacturer's instructions, and luminescence signals were measured.

[0282] In both assays, P-0831 and P-0838, representing a pair of PD1 Ab-IL-2 VitoKine and its non-concealed counterpart without a yc-disrupting mutation in the IL-2 domain, were included for comparison. The data are illustrated in FIGS. 23A-23C and further summarized in Table 23. Among the three non-concealed molecules, when compared to P-0838, the Q126M in P-1409 and the Q126R mutation in P-1413 led a decrease in potency, reducing the induction of Ki67 in CD8+ T cells by 2.4-fold and 4.9-fold , respectively, and in NK cells by 3.6-fold and 7.3-fold, respectively. These reductions are in line with the potency reduction reported for these mutations in Table 20B. Additionally, the potency reduction for the two mutations was consistent between mouse-derived and human primary cells. This consistency underscores the mouse as a reliable model for analyzing the impact of IL-2 potency change on in vivo pharmacodynamics and anti-tumor efficacy.

[0283] PD1 Ab-IL-2 VitoKine P-0831 exhibited an intrinsic basal activity approximately 300-400 times lower than its non-concealed counterpart, P-0838, representing a typical efficiency of concealment by the current VitoKine platform. However, VitoKines based on IL-2 variantscontaining yc-disrupting mutations exhibited significantly higher concealing efficiency than typically expected with the platform.

[0284] As depicted in FIG. 23 and shown in Table 25, P-1414 demonstrated nearly abrogated activity across all three cell types, whereas P-1410 displayed an appreciable yet marginal effectiveness, which increased from CD8+ T cells to NK cells to CTLL-2 cells, aligning with the cells’ overall sensitivity to IL-2. Specifically, in CTLL-2 cells (the most responsive cell type), an approximate ECso value could be derived for P-1410, indicating an activity reduction compared to its non-concealed counterpart, P-1409, by approximately 2500 times, showcasing a concealing efficiency far exceeding the 300-400-fold seen with P-0831 . The concealing efficiency for P-1414 is estimated to be even greater than 2500.Table 23Activity comparison of IL-2 VitoKines containing IL-2 variants with yc-disrupting mutations

[0285] The marked improvement in the concealing efficiency of these VitoKines is not readily apparent, but it likely stems from the interplay between the IL-2 yc-disrupting mutation and IL- 2Ro at both the primary and tertiary structure levels. The mutations, introduced close to the C- terminal of IL-2 is just before the IL-12R domain, may amplify the steric hindrance and prevent the interaction with the receptor to initiate the signaling cascade.Example 14Mitigating VitoKine Clipping in CHO Cell Production by eliminating a Liable Sequence in IL-2RaSushi Domain

[0286] P-1272 (SEQ ID NOS: 202, 203 and 44) was produced using CHO-K1 cells, a cell line suitable for scale production. However, the purified material exhibited substantial deviations from the anticipated activity from material produced in HEK293 cells, indicating compromised concealing efficiency (FIG. 24A).

[0287] Mass spectrometric analysis of the sample (deglycosylated and reduced as per the standard protocol) revealed clipping at the6Asp-7Pro position of the IL-2RaSushi domain (SEQ ID NO: 184) within the VitoKine construct. The clipped species was recombinantly expressed as P-1441 (SEQ ID NOS: 228, 293, and 44) and confirmed to be equally potent as P-1273, the non-concealed counterpart of P-1272, in stimulating Ki67 expression in human CD8+ T cells (FIG. 24B). This sequence clipping, although at low levels, undermined the structural integrity of the VitoKine molecule, causing partial activation and likely been the main cause of the observed activity deviation from the expected level.

[0288] The Asp residue is known to isomerize and undergo non-enzymatic Asp-Xaa bond cleavage, with Asp-Pro being highly susceptible and considered as a substantial sequence liability (Hinterholzer et al., J Biomol NMR 75: 71-82, 2021 ). Although it's unclear why clipping was found in CHO cell-expressed material but not in HEK293-produced material, we plan to use mutagenesis to remove this susceptible sequence, aiming to prevent clipping during CHO-K1 production processes.

[0289] However, the IL-2Ra6Asp-7Pro amino acids appear to have functional and structural roles, so their mutation could potentially compromise its concealing capability and / or expression for the resulting VitoKine constructs.6Asp of IL-2Ra forms hydrogen bond with38Arg of IL-2, representing one of the five ion pairs at the interface between IL-2 and IL-2Ra (Rickert et aL, Science 308: 1477-1480, 2005). Additionally,7Pro is highly preserved across Sushi domains, suggesting its role either in facilitating interaction or in maintaining the hydrophobic core of the sushi structure (Thomas et aL, Mol Genet Genomic Med 3: 258-71 , 2015). To preserve both the concealing capability and structural integrity of the IL-2RaSushi domain, only conservative amino acid substitutions were implemented initially. Specifically, Glu was substituted for Arg, and Gly for Pro, within the IL-2RaSushi domain that includes the L42G mutation (determined as the preferred concealing moiety domain (D3) for IL-2 VitoKines in Example 7) of P-1272. The resulting human PD1 Ab-IL-2 VitoKines are P-1439 and P-1440, respectively, with their sequence information detailed in Table 24.

[0290] P-1439 and P-1440 were produced by transient transfection in HEK-293 cells, adhering to the protocol outlined in Example 11 . P-1439 had similar expression profile as P-1272, whileP-1440 exhibited lower expression levels and markedly reduced purity following the capture purification, suggesting the structural role of7Pro, and the P7G mutation in the IL-2RaSushi domain compromised the structural integrity of the resulting VitoKine molecule. Testing of P- 1439 and P-1440 in CTLL-2 assay revealed that P-1439 had nearly identical potency as P- 1272, while P-1440 showed marginally reduced activity (FIG. 25A). Production of P-1439 was then carried out in CHO-K1 cells, and subsequent mass spectrometric analysis confirmed the absence of the clipping at residues 6 and 7 of the IL-2RaSushi domain.

[0291] The results indicating that the D6E mutation in the IL-2RaSushi domain of P-1439 successfully maintained the concealing capability and structural integrity of the IL-2RaSushi domain while also achieving the goal of preventing clipping. To investigate the possibility of further enhancing the concealing efficiency of the D3 domain and subsequently reducing the intrinsic basal activity of VitoKines, we introduced new mutations at positions6Asp or7Pro. The exemplary mutations and their resulting VitoKines are D6N in P-1452, P7A in P-1453, and P7S in P-1454. For details on the sequences, refer to Table 24.

[0292] Following production in HEK-293 cells through transient transfection, P-1454, harboring the P7S mutation, exhibited significantly reduced expression profile as seen with P-1440, which contains the P7G mutation. Conversely, P-1453, with the P7A mutation, retained a relatively good expression profile akin to P-1272. This suggests that the residue at position 7 has important structural roles but can be replaced with a subset of residues other than proline. In assays conducted in CTLL-2 cells and human PBMCs (as depicted in FIGS. 25B and 25C), both P-1452 and P-1453 exhibited a lower activity than P-1272 and P-1439, modest yet discernible, implying that the introduced mutations to eliminate the sequence liability in the IL-2RaSushi domain could improve its concealing efficiency within the VitoKine constructs. In contrast, P- 1454, containing a P7S in the IL-2RaSushi domain, demonstrated higher activity compared to P-1272 (as shown in FIG. 25B), suggesting the compromised structural integrity resulted in a less effective concealment.Table 24Exemplary human PD1 Ab-IL-2 VitoKines with Substitutions to Replace Liable Sequence

[0293] P-1451 and P-1461 , exemplary human PD1 Ab-IL-2 VitoKines, were designed with an IL-2 yc-disrupting mutation and an IL-2Ra mutation to eliminate liable clipping sequence (refer to Table 24 for the molecular details). These VitoKines were assayed in human PBMCs and CTLL-2 cells, compared to their non-concealed counterpart P-1448, and their respective VitoKine counterparts, P-1439 and P-1452, both lacking the IL-2 Q126R mutation. P-1273 is the common non-concealed counterpart of P-1439 and P-1452.

[0294] Both P-1451 and P-1461 exhibited nearly absent intrinsic basal activity in the two potency assays (FIGS. 26A to 26D). This aligns with the findings illustrated in FIG. 23, where P- 1414, the mouse PD1 Ab-IL-2 VitoKine harboring the IL-2 Q126R mutation, also showed minimal intrinsic basal activity. The marked enhancement in concealing efficiency due to the Q126R mutation was similarly demonstrated (P-1461 vs. P-1448 compared to P-1452 vs. P- 1273; and P-1451 vs. P-1448 compared to P-1439 vs. P-1273). The data also confirmed the P- 1452, with an IL-2Ra D6N mutation, showed modestly lower activity than P-1272 and P-1439, which retains the6Asp-7Pro sequence and has an IL-2Ra D6E mutation, respectively. This suggests that certain IL-2Ra mutation, such as D6N, introduced to eliminate the sequence liability could enhance its concealing capability within the VitoKine.

[0295] P-1461 was then produced in CHO-K1 cells, and mass spectrometry confirmed the absence of sequence clipping at residues 6 and 7 of the IL-2RaSushi domain. The activity of this CHO material was verified to be comparable to that of the HEK-293-expressed material in human PBMC assays (FIGS. 27A and 27B). Similarly, P-1481 , differing from P-1414 only by replacing the6Asp-7Pro liable sequencing with the IL-2Ra D6N mutation, was also produced in CHO-K1 cells, showing the same minimal activity as P-1414 produced in HEK-293 cells and as its human PD1 Ab equivalent P-1461 (FIGS. 27C and 27D). In future studies, P-1414 (producedin HEK-293 cells) and P-1481 (produced in CHO-K1 cells) will be used interchangeably, equally representing the mouse PD1 Ab surrogate of P-1461.

[0296] Finally, as expected, mutations to both D2 (IL-2) and D3 (IL-2RaSushi) domains of the VitoKine does not alter the activity of D1 . P-1461 demonstrated potent PD1 blocking activity with an EC50 values of 0.35 nM (FIGS. 28A and 28B). The enhancement in potency and Emax Over its component PD1 antibody P-1271— consistent with other VitoKines constructs shown in FIG. 21— are maintained.

[0297] In summary, IL-2RaSushi mutations designed to remove the susceptible6Asp-7Pro sequence within the PD1 Ab-IL-2 VitoKines successfully prevented clipping, thereby preserving activity inertness during OHO cell production, and maintained VitoKine’s structural integrity. Additionally, certain mutations potentially enhanced concealment, further reducing VitoKine’s intrinsic basal activity.Example 15PD1 Ab-IL-2 VitoKine Can Be Fully Activated Via In Vitro Proteolysis

[0298] P-1272 and P-1461 , each containing a single MMP-2 / 9 cleavable L2 linker (SEQ ID NO: 84), were assessed for in vitro proteolytic activation. To begin, 3.3 jig of latent MMP-2 (BioLegend) was activated by 4-Aminophenylmercuric acetate (Millipore Sigma) according to the manufacturer's instruction. The activated protease was then buffer exchanged and added to 120 .g of each VitoKine in 0.4 ml of the manufacture recommended assay buffer (100 mM Tris, 20 mM CaCI2, 300 mM NaCI, 0.1% (w / v) Brij 35, pH 7.5). After a 3-hour incubation at 37°C, the treated sample was then purified using protein A resin (MabSelect SuRe; Cytiva) in a bind-elute mode. The eluted samples (designated as P-1272_Act. And P-1461_Act.) were analyzed using a reduced SDS-PAGE gel, and their biological function was assessed through ex vivo functional assays.

[0299] The reduced SDS-PAGE gel (FIG. 29A) of the intact VitoKines shows three bands, representing a hole heavy chain (HC2) at approximately 50 KDa, a light chain at about 25 KDa, and a knob heavy chain (HC1 , labeled as A for P-1271 and C for 1461 ) containing the D1 , D2 and D3 domains. Following proteolysis, the concealing moiety domain (D3) of HC1 for each VitoKine was effectively and completely cleaved, resulting in bands B and D in activated VitoKines, which corresponds to Active Form 2 as illustrated in FIG. 2.

[0300] Efficient in vitro proteolysis of the VitoKines fully restored IL-2 activity, as demonstrated by the indistinguishable activity (EC50 = 3.95 nM) of the activated P-1272 and its non-concealed counterpart P-1273 in inducing dose-dependent Ki67 expression in CD8+ T cell from fresh human PBMCs (FIG. 29B). Similarly, activated P-1461 demonstrated nearly complete restoration of IL-2 potency compared to its non-concealed counterpart P-1448 in both the human PBMC Ki67 assay (illustrated with NK cells in FIG. 29C) and the CTLL-2 proliferation assay (FIG. 29D). The EC50 values for activated P-1461 are 2.57 nM in the NK cell Ki67 assay and 2.52 nM in the CTLL-2 proliferation assay, while for P-1448, they are 1 .64 nM and 1 .32 nM, respectively. Both intact P-1272 and P-1461 displayed the expected inert activity.

[0301] Comparable results were observed with other PD1 Ab-IL-2 VitoKines containing protease-cleavable L2 linker. However, different outcomes were seen with VitoKines with only a protease-cleavable L1 linker, exemplified by P-1345 comparing to P-0831 (refer to Table 21 B for the sequence information). Briefly, P-0831 is the mouse PD1 Ab equivalent of P-1272, featuring a non-cleavable L1 linker and a cleavable L2 linker (SEQ ID NO: 84). In contrast, P- 1345 has a cleavable L1 linker (SEQ ID NO: 84) and a non-cleavable L2 linker (SEQ ID NO: 115). Both P-0831 and P-1345 were activated via in vitro protease cleavage using MMP-2 / 9, resulting in isolated activated forms: Active Form 2 for P-0831 and Active Form 1 for P-1345, as illustrated in Figure 2. The activated samples were then evaluated for their potency in inducing Ki67 expression in CD8+ T cells from human PBMCs.

[0302] As demonstrated in Figure 30A, the IL-2 activity of activated P-0831 was fully restored to match its non-concealed counterpart, P-0838, with EC50 values of 1.97 nM for activated P-0831 and 1 .34 nM for P-0838. This represents an approximately 300-fold increase in activity compared to the intact P-0831 . In contrast, the activation of P-1345 led to about a 50-fold increase in IL-2 activity compared to its intact VitoKine form. However, the activity was not completely restored, being six times less active than its non-concealed counterpart, P-0838, with EC50 values of 8.29 nM and 1 .34 nM, respectively.

[0303] These results suggest that employing a cleavable L2 linker offers more benefits than a cleavable L1 linker. This is because Active Form 2, resulting from cleaving the L2 linker, is a bifunctional PD1 Ab IL-2 fusion with a functionally restored IL-2 domain. This form can activate IL-2R signaling in PD1 -expressing T cells near the tumor site, boosting both pathways, facilitating cis-action, and synergizing the anticancer immune response. It also has an extended half-life, improving exposure and availability at the tumor site while reducing systemic toxicity. In contrast, Active Form 1 , derived from the cleavage of the L1 linker, is an IL-2-IL-2Ra fusion thatlacks TIL-targeting capabilities and does not have a half-life extension domain, resulting in a shorter half-life and an inability to synergize with the PD-1 pathway. It additionally exhibits reduced IL-2 potency, further diminishing its anti-tumor efficacy.

[0304] From another perspective, Active Form 1 , created by removing the D1 domain to form a D2-D3 fusion, is about six times less active than its non-concealed counterpart. This reflects a six-fold concealing efficiency rendered by the concealing moiety domain (D3) alone, which is inefficient. However, when the D1 domain is included to flank the D2 domain between D1 and D3, the concealing efficiency exceeds 300-fold, and even much more significant (over 2000- fold) for IL-2 variants with yc-disrupting mutations as the D2 domain. Therefore, the VitoKine structure, which requires the coupling of both the targeting domain (D1 ) and the concealing domain (D3) to flank the active domain (D2), as disclosed herein as well as in WO2019246392 and WO20211 19516 by the current inventors, ensures effective concealment of the active domain, achieving inactivation of the intact VitoKines.Example 16PD1 Ab IL-2 VitoKine: Prolonged In Vivo Half-Life in Mice

[0305] Based on the design, the VitoKine’s IL-2 domain remains inert until locally activated by proteases upregulated in diseased tissues. Consequently, the binding of the IL-2 VitoKine to IL- 2 receptors on cell surface in peripheral blood and non-diseased tissues is expected to be markedly diminished. This reduction helps minimize potential antigen sink and target-mediated deposition, thereby extending the in vivo half-life. A study compared the pharmacokinetics of a mouse PD1 Ab IL-2 VitoKine P-0831 with its non-concealed counterpart P-0838 in non-tumor bearing C57BL / 6 mice.

[0306] Naive female C57BL / 6 mice, aged seven weeks, were received from Charles River Laboratory. Before starting the study, they were allowed a 7-day acclimation in house. At the start, P-0831 and P-0838, each at a dose of 1 mg / kg, were administered by intravenous injection. Phosphate-buffered saline (PBS) was included as vehicle. Blood samples were withdrawn at 10 min, 2, 6, 24, 48, 72, 120, 168, 240, and 360 hours post-injection by cheek bleeding. Each group consisted of 3 mice, and blood was taken either weekly or every three days, with a maximum frequency of twice per group.

[0307] The serum concentrations of the compounds were determined using ELISA assays. Two different ELISA methods were developed for P-0831 to measure: 1 ) total VitoKine concentration(including both activated and intact forms); and 2) concentration of intact VitoKine. For both methods, maxisorp plates were coated with mouse PD1 protein (R&D systems) overnight at 4°C. Following this, plates were blocked with Superblock (ThermoFisher). Blood samples at various dilutions were added to the plates and incubated for one hour at room temperature.

[0308] For total VitoKine detection, an anti-IL-2 goat polyclonal antibody (R&D Systems) was added, followed by a secondary HRP-conjugated donkey anti-goat IgG (ThermoFisher). To detect intact VitoKine, a polyclonal anti-CD25 antibody (R&D Systems) was used, which was probed using HRP-conjugated streptavidin protein (ThermoFisher). For P-0838 detection, the same anti-IL-2 goat polyclonal antibody (R&D Systems) for detecting total VitoKine concentration was used followed by the donkey anti-goat IgG-HRP. The resultant signals were developed using the Ultra TMB substrate solution (ThermoFisher), and values were extrapolated from non-linear regression curve fits in GraphPad Prism.

[0309] As shown in FIG. 30, the concentration profiles for the intact and total (including both intact and activated forms) P-0831 align closely, suggesting that P-0831 predominately circulates in its intact state. Contrasting with the concentration profiles of P-0831 , which remained measurable 360 hours after a 1 mg / kg dosage, P-0838’s serum concentration dropped quickly. It became substantially lower by 72 hours and was undetectable by 120 hours post-dosing. The grey dashed horizontal line in FIG. 30 donate the lower limit of quantification (LLOQ) for serum P-0831 or P-0838 levels. For measurements falling below the LLOQ, values were assigned as 10-3nM.

[0310] P-1414, which differs from P-0831 by a single IL-2 Q126R mutation that weakens its interaction with yc, exhibited a similarly extended half-life compared to its non-concealed counterpart P-1413 in a comparable study.

[0311] The findings strongly support the notion that the VitoKine format is superior in extending the active domain’s in vivo half-life. The notably prolonged in vivo half-life of the VitoKines is thought to result from the inertness of the IL-2 domain in peripheral. This inactivity likely minimizes interaction with IL-2 receptors on the cell surface in both peripheral and healthy tissues, greatly diminishing cell activation and expansion, and thereby mitigating potential antigen sink and / or target-mediated deposition.Example 17PD1 Ab IL-2 VitoKine: Targeted Enhancement of PD Effects Within Tumor while Minimizing Peripheral Effects in Mice

[0312] The PD1 Ab IL-2 VitoKine platform is designed to expand the therapeutic window for cytokine therapy by targeting the pharmacodynamic (PD) effects within the tumor while minimizing peripheral effects. This is accomplished by keeping the cytokine domain inactive in peripheral blood and non-diseased tissues, thereby preventing system activation of the cytokine pathway. Further, with the PD1 antibody targeting tumor-infiltrating lymphocytes (TILs), the PD1 Ab IL-2 VitoKine is directed to the tumor microenvironment (TME), where upregulated proteases like MMPs proteolytically activate the Vitokine and unleash the IL-2 domain, allowing for cytotoxic lymphocyte activation and expansion to be confined within the TME. To evaluate this hypothesis, mouse PD1 Ab-IL-2 VitoKines P-0831 and P-1414 were given to either non-tumor- bearing or tumor-bearing mice, with lymphocyte proliferation and expansion monitored over time. Their respective non-concealed counterparts, P-0838 and P-1413, were included for comparison.

[0313] The PD effect of P-0831 were assessed in non-tumor bearing mice along with P-0838. Naive C57BL / 6 mice, aged between 7-9 weeks (n = 4 / group) received a single intraperitoneal injection of P-0831 at doses of 2 and 10 mg / kg, and P-0838 at 0.3, 1 , and, 2 mg / kg. A vehicle (PBS) was included as a negative control. Blood samples were collected in heparin tubes on Days 0, 3, 5, 7, and 10 post-dosing for immunophenotyping.

[0314] The PD effects of P-1414 was evaluated against its non-concealed counterpart P-1413 in a syngeneic CT26 murine colon carcinoma model. CT26 tumors show moderate CD8+T cell infiltration, making them less immunogenic than the MC38 model. While CT26 tumors are resistant to PD-1 blockade alone, they do respond to IL-2 therapy. To establish CT26 tumors, 5 x 105CT26 cells were subcutaneously implanted into the right flank of female Balb / C mice aged 7-9 weeks. Approximately 11 days later, when the average tumor volume reached ~75 mm3, mice were randomized into three groups of twenty. Each group receives a single injection of P- 1414 at 30 mg / kg, P-1413 at 3 mg / kg, or vehicle (PBS). At each timepoint on Days 0, 3, 5, 7, and 10, four mice from each group were euthanized to collect blood samples and harvest tumors, spleens, and lymph nodes.

[0315] For blood samples, red blood cells were lysed using BD Pharmingen lysis buffer, and viable mononuclear blood cells were counted by excluding dead cells with trypan blue. Tumor tissues were minced into small 1-2 mm3fragments, then digested with Collagenase IV and DNase I to break down the extracellular matrix. Post-digestion, debris was removed, and cells were washed and resuspended in FACS buffer. Lymph nodes and spleens were processedsimilarly through mechanical dissociation, followed by passing through a strainer to obtain a single-cell suspension, which was then washed and resuspended in FACS buffer.

[0316] Subsequently, live cells from each sample were counted, and cell density were adjusted in FACS buffer before being stained with a panel of antibodies targeting common surface immune cell markers. Specifically, the lysed immune cells underwent a 30-minute fixation and permeabilization at room temperature in the dak using eBioscience fixation / permeabilization buffer. Immune cell subsets were identified and quantified by a Beckton Dickinson flow cytometer using commercially available antibodies: CD3-APC.Cy7, CD8-Percp-cy5.5, CD335- APC, CD45-AF700, CD4-BV421 , and granzyme B-BV421 . Flow cytometry analysis was performed using FlowJo software and results were plotted using GraphPad Prism.

[0317] For P-0831 , peripheral PD effects were characterized by examining cell counts of CD8+ T cells and NK cells, as well as the number of these cells expressing the cytotoxic marker granzyme B over time following a single dose (FIG. 32). As shown in FIGS. 32A and 32B, P- 0831 did not elicit any noticeable CD8+ T cells or granzyme B+ cells across the 10-day observation period at doses of 2 mg / kg and 10 mg / kg. In sharp contrast to P-0831 ’s lack of effects, its non-concealed counterpart P-0838 greatly expanded peripheral blood CD8+ T cells (FIG. 32A) and granzyme B+ CD8+ T cells (FIG. 32B) at even much lower doses of 1 and 2 mg / kg, demonstrating a clear dose-dependent effect. Specifically, at 2 mg / kg , CD8+ T cells expanded from a baseline count of 900 cells / iL to a peak level of 4200 cells / pL (a 4.7-fold increase) on Day 5 before declining to near baseline by Day 7. At 1 mg / kg, peak expansion occurred on Day 3 with a 2.3-fold increase, also returning to baseline by Day 7. The 0.3 mg / kg dose showed only minimal expansion. A similar trend was seen with cytotoxic granzyme B+ CD8+ T cells.

[0318] Even for NK cells which displays greater reactivity to IL-2 treatment than CD8+ T cells, P-0831 treatment led to only slight and delayed increases in NK cells and granzyme B+ NK cells, even at a high dose of 10 mg / kg (FIGS. 32C and 32D). For P-838, the greater responsiveness of NK cells was evident from the significant NK cell expansion at 0.3 mg / kg (FIG. 32C), with a dose-dependent response observed between 0.3 and 1 mg / kg doses. Peak NK cell expansion occurred on Day 3 across all three dosages (FIG. 32C) and a similar patten was seen in cytotoxic granzyme B+NK cell expansion (FIG. 32D).

[0319] P-1414 has substantially lower intrinsic IL-2 activity than P-0831 and was administered at a higher dose of 30 mg / kg in CT26 tumor-bearing mice. Its PD effects is exemplified by the expansion of CD8+ T cells in peripheral blood, tumors, spleens, and lymph nodes, expressed asan average fold change compared to the vehicle group’s average at the same time. As shown in FIG. 33A, P-1414 resulted in minimal (less than 2-fold) CD8+ T cell expansion in blood, spleens, and lymph nodes, but greatly expanded tumoral CD8+ T cells, peaking at a 22-fold increase. Specifically, tumoral CD8+ T cells rose from baseline to 3.5-fold on Day 5, peaked at 22-fold on Day 7, and maintained at 14-fold over the vehicle by the end of the study on Day 10, despite some decline.

[0320] In contrast, at a 10 times lower dose (3 mg / kg), the non-concealed counterpart P-1413 achieved a peak 9-fold expansion of tumoral CD8+ T cells on Day 7, which was sustained without decline until the observation period ended on Day 10. However, P-1414 also caused marked expansion of CD8+ T cells in lymph nodes, spleens, and blood, with the peak value of 6.4-fold, 4.5-fold, and 4-fold, respectively (FIG. 33B). The distinct PD effects between P-1414 and P-1413 underscores the PD1 Ab-IL-2 VitoKine’s capability to target enhanced PD effects to tumor and confine them within the TME while minimizing peripheral effects.

[0321] In summary, the contrasting PD effects between P-0838 and P-0831 in healthy mice highlighted the effectiveness of the VitoKine format in concealing IL-2 activity, thereby diminishing systemic proliferation and expansion of cytotoxic lymphocytes to limit toxicity. Additionally, the distinction between P-1414 and P-1413 in tumor-bearing mice not only confirmed the platform’s ability to minimize peripheral PD effects, but also to specifically target and confine these effects within tumors. This approach enhances anti-tumor efficacy and broadens the therapeutic index. Equally important, P-1414, which has greatly diminished intrinsic activity due to the yc-disrupting IL-2 mutation Q126R, can be safely doses at higher levels. This enables it to fully leverage its component PD1 Antibody’s functional capacity to reverse T cell anergy and exhaustion, thereby further enhancing anti-tumor efficacy.Example 18Mitigating Cytokine-Associated Toxicity in Mice with PD1 Ab-IL-2 VitoKines

[0322] Cytokine-associated toxicity, or cytokine release syndrome (CRS), is a significant risk in cancer immunotherapy. CRS stems from a strong immune response, and is often associated with elevated circulating levels of several cytokines, including interleukin-6 and interferon gamma (INFy). As immune-based therapies become more potent, the magnitude of immune activation can exceed levels that occurring in more natural settings, potentially escalating CRS to a life-threatening level. The VitoKine platform is designed to prevent over-activation ofcytokine pathways, and the exemplary PD1 Ab-IL-2 VitoKines P-0831 and P-1414 have demonstrated a proven ability to minimize systemic pharmacodynamic effects (refer to Example 17), highlighting the potential for VitoKine to significantly reduce cytokine-associated toxicity.

[0323] Two separate studies were conducted to investigate PD1 Ab-IL-2 VitoKine’s potential in reducing cytokine-associated toxicity. In the first study, P-0831 and its non-concealed counterpart, P-0838, were tested in non-tumor-bearing naive C57BL / 6 mice. Mice (7-9 weeks old) were divided into groups of three (n=3) and received a single intraperitoneal injection of P- 0831 at 1 , 3, 6, 10, and 20 mg / kg, and P-0838 at 1 , 3, and 6 mg / kg. A second study was conducted in CT26 tumor-bearing Balb / C mice for P-1414 against it non-concealed counterpart P-1413. CT26 tumors was established by subcutaneously implanted 5 x 105CT26 cells in the right flank of female Balb / C mice (7-9 weeks). About 1 1 days later, when the average tumor volume reached ~75 mm3, mice were randomized into groups of three (n=3), and received a single dose of P-1414 at 3,10, and 30 mg / kg, and P-1414 at 1 and 3 mg / kg.

[0324] In both studies, Vehicle (PBS) and the mouse PD1 antibody P-0722 (SEQ ID NOS: 52 and 53) were included as negative controls. Serum samples were collected 48 hours posttreatment, and IFNy concentration was determined using a mouse INFy DuoSet ELISA kit from R&D Systems, following manufacturer's instruction.

[0325] The increases in serum IFNy for both studies are shown in FIGS. 34A and 34B as well as in the accompanying Table 25. As expected, neither the vehicle nor the antibody treatment induced any noticeable release of this inflammatory cytokine, as shown in FIGS. 34A and 34B. Treatments with P-0831 and P-0838 increased serum IFNy levels dose-dependently, but P- 0831 exhibited markedly diminished IFNy levels compared to P-0838. Even at 20 mg / kg, P- 0831 produced only a modest 182 pg / mL of INFy, still below the 253 pg / mL observed for P- 0838 at 1 mg / kg. When the dose increased from 1 mg / kg to 3 mg / kg, the serum INFy for P-0838 surged to 12482 pg / mL— a 50-fold increase— while P-0831 only increased by 2.5-fold.

[0326] P-1414 has further diminished intrinsic activity compared to P-0831 due to the yc- disrupting IL-2 mutation Q126R (FIG. 23). As a result, it induced lower IFNy. At 3 mg / kg, serum INFy was undetectable. When administered at 10 mg / kg, P-1414 yielded just 5% of the INFy levels seen with P-0838, measuring 6.5 pg / mL versus 118 mg / mL. Even at a high dose of 30 mg / kg, P-1414’s INFy levels was 51 pg / mL, comparable to the level at 6 mg / kg of P-0831 . These findings underscore the additional safety benefits attributed to the further reduction of the VitoKine intrinsic activity.Table 25Serum INFy concentration 48 hours post-treatment

[0327] The sharp increases in circulating INFy levels in the P-0838 groups at 3 and 6 mg / kg, resulted from high-level systemic immune activation, can lead to severe toxicity. In a concurrently conducted experiment, naive C57BL / 6 mice (7-9 weeks old, n = 4) received P- 0831 at 3, 6, and 20 mg / kg, and P-0838 at 1 , 3, and 6 mg / kg via intraperitoneal injection. The cytokine-associated toxicity from P-0838 at 3 and 6 mg / kg resulted in significant weight loss (FIG. 33C) and other signs of stress. With a protocol mandating the termination of mice experiencing over 10% weight loss, none of the 6 mg / kg P-0838 group survived beyond 4 days, and 3 out of 4 mice from the 3 mg / kg group had to be sacrificed by the 4thday after just one dose. On the contrary, mice treated with P-0831 , even up to 20 mg / kg, survived after three doses on a Q2W schedule and did not experience significant weight loss (FIG. 34C). This suggested that the VitoKine platform presents a remarkably lower toxicity profile.

[0328] In summary, PD1 Ab IL-2 VitoKines notably mitigated cytokine-associated toxicity in mice, as evidenced by the strikingly reduced circulating levels of inflammatory cytokines, exemplified by INFy, and minimal changes in body weight even at much higher doses compared to the non-concealed counterpart. Additionally, incorporating IL-2 with reduced potency,achieved by introducing mutations that disrupt I L-2 Ftp or yc interaction, will lead to VitoKines with lower intrinsic basal activity and could further broaden the therapeutic margin.

[0329] Furthermore, the ability of PD1 Ab IL-2 VitoKines to be tolerated at a much-elevated doses provides more flexibility in optimizing dosing regimens. At higher doses, the PD1 antibody’s function of reversing T-cell anergy or exhaustion can be fully fulfilled since the dose levels are within its therapeutically effective range, potentially enhancing the synergistic effects with IL-2 immunotherapy.Example 19PD1 Ab-IL-2 VitoKines Effectively Eliminates Tumors in Mouse Models

[0330] The anti-tumor efficacy of PD1 Ab IL-2 VitoKines was evaluated against their nonconcealed counterparts in the syngeneic MC38 murine colon carcinoma model. MC38 tumors are characterized by high CD8+T cell infiltration, making them as “hot” tumors. They are responsive to PD-1 blockade and are commonly used in preclinical checkpoint inhibitor studies. To establish MC38 tumors, 5 x 105MC38 cells were subcutaneously implanted into the right flank of female C57BL / 6 mice aged 7 to 9 weeks. About two weeks later, with average tumor volume of ~75 mm3, mice were randomized into groups of 8 on Study Day 0. On Study Day 1 , treatments were administered. For the first study, the treatment included mouse PD1 antibody P-0722 at 9 mg / kg, P-0831 at 3, 6, and 9 mg / kg, and P-0838 at 1 mg / kg, via intraperitoneal injection every 10 days (Q10D) for a total of two doses. For the second, similar study, treatments consisted of two doses of P-0722 at 6 mg / kg, VitoKine P-1414 at 1 , 3, and 6 mg / kg, and P-1413 at 1 mg / kg every two weeks (Q2W). In both experiments, PBS served as the vehicle control.

[0331] Tumor growth and body weight were monitored bi-weekly. Tumor volume (TV) was calculated using caliper measurement as follows: volume = 0.5 x (width)2x (length). Tumor growth inhibition (TGI) was determined using: TGI (%) = [1 - (TV of the treated group) / (TV of the control group)] x 100. Mice were euthanized if tumors grew to or exceeded 1500 mm3or became necrotic, based on set criteria.

[0332] FIGS. 35A and 35B illustrate the mean tumor volume, along with the standard error of the mean (SEM) for each group over time. Mice receiving vehicle treatment rapidly developed large tumors, while PD1 antibody P-0722 treatment initially delayed tumor growth, although onlymarginally, it eventually led to the development of large tumors. All other treatment groups exhibited high efficacy in inhibiting tumor growth.

[0333] Treatment with P-0831 at 6 mg / kg was most effective, eradicating tumors in all eight mice on Day 45, 34 days after the second and final treatment (FIG. 35A). Likewise, in the 9 mg / kg group, 7 out of 8 mice remained tumor-free by the study’s conclusion. The 3 mg / kg group had slightly reduced efficacy, with 5 out 8 mice tumor-free and 3 showing tumor regrowth after initial delay. In the 1 mg / kg of P-0838 group, 6 out of 8 mice were tumor-free by the study’s end.

[0334] P-1414 at 6 mg / kg demonstrated a profound and sustained anti-tumor effect, resulting in complete tumor eradication in all seven mice (FIG. 35B). At lower doses of 1 and 3 mg / kg, P- 1414 also markedly inhibited tumor growth with TGI of 68% and 80% on Day13, but with lower numbers of tumor-free mice. In the 1 mg / kg of P-1413 group, two of the seven mice were tumor- free by the study’s conclusion.

[0335] The PD1 Ab-IL-2 VitoKines P-0831 and P-1414 were well tolerated across all doses, showing minimal to no body weight loss. In contrast, P-0838 was intolerable at doses of 3 mg / kg or more (FIG. 34C). P-0831 and P-1414 demonstrated profound and sustained anti-tumor efficacy at 6 mg / kg, with much lower peripheral lymphocyte proliferation and expansion (FIGS. 32 and 33) and diminished circulating INFy production (FIGS. 34A and 34 B) compared to 1 mg / kg of P-0838 or P-1413. This achievement is partly attributed to the high dose tolerance afforded by the PD1 Ab IL-2 VitoKine format, which broadens the therapeutic window, enabling the PD1 antibody component to effectively counteract T-cell anergy and exhaustion. Notably, P- 1414 is equally efficacious as P-0831 despite having lower intrinsic activity, offering ever greater dose tolerance and further widening the therapeutic index.

[0336] Mice treated with 6 mg / kg of either P-0831 or P-1414 and remained tumor-free until the final observation on Day 85 post-treatment underwent a rechallenge with MC38 cell implantation. As shown in FIG. 35C, none of the rechallenged mice experienced tumor recurrence, unlike the age-matched naive control mice, which developed tumors successfully. These results suggest that PD1 Ab-IL-2 VitoKines effectively established long-term immunity.

[0337] In parallel studies, immunohistochemistry (IHC) was used to assess the effects of PD1 Ab-IL-2 VitoKine on tumor tissues five days post treatment. MC38 subcutaneous tumors were similarly established, and mice were randomized (5 per group) to receive a single dose of either vehicle, P-0722 (6 mg / kg), P-0831 (6 mg / kg), or P-0838 (1 mg / kg). In a separate, similarly run study, MC38 tumor-bearing mice (n=4) received a single dose of either vehicle, P-0722 (6 mg / kg), or P-1414 (6 mg / kg). Five days later, the mice were euthanized, and tumors wereextracted for IHC analysis. The tissue sections were processed, fixed in 10% formalin, paraffin- embedded, processed, and stained with antibodies per HistoWiz’s instructions to evaluate immune cell presence.

[0338] FIG.37A illustrates representative IHC images for each group in the P-0831 study and showcases that P-0831 treatment elicited extensive infiltration of CD3+ and CD8+ T cells with high cytotoxic capability, evidenced by intensive granzyme B expression. This underscores the increased number and activity of cytotoxic CD8+ T cells in tumors from P-0831 -treated mice, supporting the anti-tumor efficacy data. In sharp contrast, P-0722 at 6 mg / kg induced minimal tumor-infiltrating lymphocyte (TIL), while P-0838 treatment led to limited T cell infiltration, but high granzyme B expression. Importantly, no treatment led to notable presence of the inhibitory FOXP3+ cells.

[0339] Similarly, P-1414 treatment also led to extensive infiltration of CD8+ T cells with high cytotoxic capability, as indicated by robust granzyme B expression, while not inducing inhibitory FOXP3+ cells (FIG. 37B).

[0340] Taken together, PD1 Ab IL-2 VitoKines, exemplified by P-0831 and P-1414, effectively inhibited tumor growth by promoting extensive infiltration of cytotoxic T cells into the tumor tissues while minimizing peripheral lymphocyte proliferation and expansion. The VitoKine format helps mitigate issues commonly associated with fully active cytokine, such as excessive stimulation of the immune pathway, undesirable “on-target, off-tissue” toxicity, and unwanted target sink, while maintaining strong anti-tumor efficacy. Notably, the PD1 Ab-IL-2 VitoKine’s compatibility with higher dosages enables the antibody component effectively target and reverse T-cell anergy and exhaustion, thereby potentiating existing immune responses. This will further enhance the immune system’s activity against tumors.

[0341] Additionally, despite its significantly lower intrinsic basal activity compared to P-0831 — attributable to the reduced potency of the IL-2 domain caused by yc-disrupting mutations and the unexpectedly enhanced concealing efficiency — P-1414 exhibits the same level of anti-tumor effectiveness as P-0831 and promotes extensive infiltration of TILs. This could potentially further broaden the therapeutic margin.Example 20PD1 Ab-IL-2 VitoKines’ Anti-Tumor Efficacy Hinges on Proteolytic Activation

[0342] In vivo proteolytic activation play a critical role in PD1 Ab-IL-2 VitoKine’s anti-tumor efficacy, which was investigated by comparing P-0831 and P-1414 with their respective non- cleavable VitoKine counterparts, P-0877 and P-1446, in tumor models. P-0831 and P-0877 differ solely by their L2 linker connecting IL-2 (D2) and IL-2Ra (D3) domains (refers to Table 21 B for details). Similarly, P-1414 and P-1446 differ only in their L2 linker. The L2 linker in P- 0831 and P-1414 is protease-cleavable, with the sequence defined in SEQ ID NO: 84, while in P-0877 and P-1446, it is a (648)3 linker (SEQ ID NO: 115) and is not cleavable by protease.

[0343] In vitro, P-0877 displayed identical activity to P-0831 in inducing Ki67 expression in CD8+ T and NK cells from human PBMCs, as depicted in FIGS. 38A and 38B. Likewise, P-1414 displayed identical activity to P-1446 (FIG. 38C and 38D) in a comparable assay, albeit at a much lower potency level due to the yc-disrupting mutations and unexpectedly enhanced concealing efficiency. In both assays, their respective non-concealed counterparts, P-0879 and P-1413, served as controls. Due to the non-cleavable nature of the L2 linker, P-0877 and P- 1446 cannot be activated by proteases.

[0344] The CT26 syngeneic mouse model was then used to examine the effects of VitoKine’s in vivo proteolytic activation. Female Balb / C mice aged 7-9 weeks had 5 x 105CT26 cells implanted subcutaneously in the right flank. On the 11thday, when the average tumor volume reached approximately 75 mm3, the mice were randomized into groups of eight. They received two intraperitoneal injections every 12 days of either vehicle (PBS), mouse PD1 Ab P-0722, P- 0831 , P-0877 at 10 mg / kg, starting the day after randomization. For a sperate, similar study, treatments consisted of two Q2W doses of P-0722, P-1414, and P-1446 at 30 mg / kg. In both experiments, PBS served as the vehicle control. Tumor size and body weight were monitored twice weekly. Mice were euthanized if tumors grew to or exceeded 1500 mm3or became necrotic, based on set criteria.

[0345] Treatment with P-0831 at 10 mg / mL effectively inhibited tumor growth, achieving an 81% tumor growth inhibition (TGI, although tumors eventually developed in all mice (FIG. 39A). In contrast, the same dose of P-0877, the non-cleavable VitoKine counterpart of P-0831 with an IL-2 domain remains inert, resulted in only a minor delay in tumor growth, with no improvement over its component PD1 antibody, P-0722. These findings indicate that the enzymatic cleavage of the L2 linker, which releases the concealing moiety and activates the IL-2 domain within the tumor microenvironment, is indispensable for the enhanced anti-tumor effectiveness of the VitoKine molecule. All tested compounds were well-tolerated with no signs of weight loss in the mice.

[0346] A similar, yet more profound trend was observed with P-1414 compared to P-1446 (FIG. 39B). Treatment with P-1414 dosed at a dose of 30 mg / kg completely eradicated CT26 tumors in all eight mice. In contrast, P-1446 (its non-cleavable VitoKine counterpart) administered at the same dose, showed only minor improvement over P-0722 in initially delaying tumor growth, but neither efficiently inhibited tumor growth. This further underscores the crucial role of proteolytic activation in the anti-tumor efficacy of the PD1 Ab-IL-2 VitoKine. The superior effectiveness of P-1414 at 30 mg / kg against CT26 tumor, compared to P-0831 at 10 mg / kg, is attributed in part to the greater dose tolerance afforded by P-1414’s lower intrinsic activity and toxicity, thereby broadening the therapeutic window.

[0347] A comparable patten was observed for P-1414 compared to P-1446 in inhibiting Panc02 tumor growth (FIG. 39C). Panc02 tumors are classified as "cold" tumors, characterized by poor T cell infiltration, low CD8+T cell presence, and a highly immunosuppressive environment, making them resistant to immune checkpoint blockade, including PD-1 .

[0348] Pan02 tumors were established in female C57BL / 6 mice using a method similar to that of the MC38 model. Mice received subcutaneous implantation of Panc02 cells in the right flank and were randomized into groups of eight when the average tumor volume reached ~75 mm3. They then received two Q2W doses of either P-1414, P-1446, or P-0722 at 30 mg / kg, with a vehicle group receiving PBS. As shown in FIG. 39C, Treatment with P-1414 effectively inhibited tumor growth, achieving a sustained TG1 of about 70% from Day 7, while P-1446 showed no tumor inhibition or improvement over its component PD1 antibody, P-0722. These findings again highlights the crucial role of the proteolytic cleavage in the anti-tumor effectiveness of the VitoKine molecules. However, unlike the complete eradication seen in CT26 tumor model (FIG. 39B), P-1414 only manages to inhibit and delay, but not eliminate, Panc02 tumors, likely due probably due to their cold tumor nature.

[0349] The indispensable role of proteolytic activation of the IL-2 domain within the TME in the anti-tumor efficacy of PD1 Ab-IL-2 VitoKines is further demonstrated in the MC38 tumor model (FIG. 40). Since MC38 is a “hot” tumor, P-1414 was able to completely eradicate the tumors at a much lower 6 mg / kg dose, compared to the 30 mg / kg in CT26 and Panc02 models. In contrast, P-0722 only showed a minor initial delay in tumor growth, and the inactivable IL-2 domain in P- 1446 did not improve the anti-tumor efficacy (FIG. 40A).

[0350] The proteolytic activation of the IL-2 domain in P-1414 and the subsequent proliferation of cytotoxic CD8+ T cell in tumor tissues were visualized in representative immunohistochemistry (IHC) images. Tumor tissues from MC38 tumor-bearing mice, in aparallel study, were harvested five days after a single dose for IHC staining. P-1414 at 6 mg / kg elicited extensive infiltration of CD8+ T cells with strong cytotoxic capability, evidenced by intensive granzyme B expression. In sharp contrast, treatment with P-1446 and P-0722 at the same dose did not result in T cell infiltration or granzyme B expression. These findings corroborate the anti-tumor efficacy data, indicating that the infiltrated cytotoxic T cells within the tumors drives tumor cell destruction.

[0351] In summary, the compelling superiority of the PD1 Ab-IL-2 VitoKines over their non- cleavable counterparts have been demonstrated for both P-0831 and P-1414 — exhibiting different levels of IL-2 activity when concealed and activated — across multiple tumor models with differing immunogenicity: “hot” (MC38) , “cold” (Panc02), and intermediate (CT26). These findings only highlight the essential role of proteolytic activation of the IL-2 domain within the TME in the anti-tumor efficacy of PD1 Ab-IL-2 VitoKine, but also underscore the broad applicability of this VitoKine platform in cancer treatment.Example 21PD1 Ab-IL-2 VitoKines’ Anti-Tumor Efficacy Relies on PD1 Targeting

[0352] The PD1 antibody in this PD1 Ab-IL-2 VitoKine platform is designed to serve multiple roles: 1 ) targeting IL-2 to tumor-infiltrating lymphocytes (TILs); 2) achieving enhanced avidity toward intratumoral CD8+ T cells through cis-action; 3) synergizing PD1 antibody’s functional capability in reversing T-cell anergy or exhaustion with IL-2, thereby further enhancing the anticancer immune response. The important role of the PD1 antibody on the anti-tumor effectiveness of PD1 Ab-IL-2 VitoKines, exemplified by P-0831 and P-1414, was assessed by comparing to non-targeted IL-2 VitoKines in murine tumor models.

[0353] First, the efficacy of P-0831 was compared to its non-targeted IL-2 VitoKine counterpart, P-0871 , in the CT26 tumor model (FIG. 41 A). P-0871 differs from P-0831 only by the D1 domain, which is a non-targeting germline antibody P-1260 (SEQ ID NOS: 191 , 192, and 193). In this study, Balb / C mice with established CT26 tumors received two intraperitoneal injections every 12 days (Q12D) of either a vehicle (PBS), P-0722, P-0831 , or P-0871 , all at a dosage of 10 mg / kg.

[0354] FIG. 41 A illustrates the mean tumor volumes, along with SEM, for each treatment group over time. Both the non-targeted IL-2 VitoKine P-0871 and the PD1 antibody P-0722 initially showed some modest tumor growth inhibition, with TGI of 46% and 29%, respectively, on Day11 , but failed to sustain this effect. In contrast, the fusion of the PD1 antibody with the IL-2 VitoKine in P-0831 not only demonstrate more effective initial tumor growth inhibition with a TGI of 67%, but also maintains its anti-tumor effects for a longer duration. This suggests that the PD1 antibody plays an important role in boosting the anti-tumor potency of P-0831 .

[0355] A more striking and profound contrast was observed with P-1414 compared to its nontargeted IL-2 VitoKine counterpart P-1469 in the MC38 tumor model (FIG. 41 B). P-1414 is distinguished from P-0831 by its IL-2 domain of lower potency and even more reduced Vitokine intrinsic activity. P-1469, on the other hand, differs from P-1414 by the D1 domain, which is a non-targeting germline antibody P-1260. In this study, C57BL / 6 mice with established MC38 tumors were administered two intraperitoneal injections every two weeks (Q2W) of either a vehicle (PBS), P-0722, P-1414, or P-1469, all at a dosage of 6 mg / kg. Additionally, a combination treatment of P-1469 and P-0722, both at 6 mg / kg, was included.

[0356] The tumor growth curves illustrated in FIG. 41 B clearly demonstrate that at 6 mg / kg, the PD1 antibody P-0722 and the non-targeted IL-2 VitoKine P-1469 contribute minimally, if at all, in inhibiting MC38 tumor growth. The combination of P-0722 and P-1469 produced only a minor initial delay in tumor growth, with such improvements being both modest in scale and shortlived. On the contrary, the fusion of the PD1 antibody with the IL-2 VitoKine in P-1414 completely eliminated the tumors at the same dose, resulting in all eight mice being tumor-free by the end of the study.

[0357] These findings highlight the essential role of the PD1 antibody domain in achieving the anti-tumor efficacy of the PD1 Ab-IL2 VitoKine platform. The PD1 antibody specifically guides the VitoKine directly to TILs within the tumor microenvironment, where upregulated proteases activate the IL-2 domain. Subsequently, the active IL-2 cis-acts with the PD1 antibody on intratumoral CD8+ T cells and further synergizes with PD1 antibody’s functional capability in reversing T-cell anergy or exhaustion, thereby enhancing the immune response against cancer.Example 22Enhance Dose Tolerance of PD1 Ab-IL2 VitoKines Through IL-2 Potency Attenuation

[0358] In previous studies, PD1 Ab-IL2 VitoKines with potency attenuated IL-2 variants — exemplified by P-1414, which contains a yc-disrupting mutation Q126R in its IL-2 domain — demonstrated excellent tolerance at 30 mg / kg without any safety concerns, conversely, P-0831 , whose IL-2 domain lacks IL-2R - or yc-disrupting mutations, began to cause distress in micebeyond 20 mg / kg, particularly at doses exceeding 40 mg / kg. Our goal for this study is to systematically investigate the dose tolerance of such VitoKines in healthy mice and in cynomolgus monkeys.

[0359] To support large-scale production in CHO cells, we constructed P-1481 , which differs from P-1414 only by the IL-2Ra D6N mutation replacing the6Asp-7Pro liable sequencing P-1481 was produced in CHO-K1 cells by a contracted research organization (CRO) using their platform process. The intrinsic IL-2 activity of the CHO cell-produced P-1481 was shown to be indistinguishable from P-1414 produced in HEK-293 cells (FIGS. 27C and 27D).

[0360] P-1481 was administered at varying dose levels — 10, 30, 60, and 120 mg / kg — to naive C57BL / 6 mice via intraperitoneal injection three times, spaced every two weeks. A vehicle (PBS) group is included for comparison. Extensive evaluations were conducted, including biweekly body weight measurement, serum IFNy level determination 48 hours after the first injection. The pharmacodynamic effects on immune cells are evaluated five days following the first and third doses. Mice were sacrificed on Day 36, or seven days after the third and final dose to collect blood for a comprehensive clinical chemistry assessment and to harvest multiple organs to evaluate the impact of the treatment on organ weight.

[0361] All treatments, even at a dose as high as 120 mg / kg, were well-tolerated with no evidence of significant weight loss in mice (FIG. 42A).

[0362] Average serum IFNy levels for each P-1481 dose were compared to historical data from a similar study of P-0831 in naive C57BL / 6 mice (FIG. 42B). After P-1481 treatment, serum IFNy levels increased dose-dependently from 25 pg / mL at 10 mg / kg to 134 pg / mL at 120 mg / kg. The IFNy levels resulting from P-1481 treatment are comparable to those from P-0831 at 10 mg / kg (1 18 pg / mL), indicating that IL-2 potency attenuation offers a nearly 12-fold higher tolerability margin for the resulting VitoKine from a cytokine-associated toxicity perspective. This is in addition to the significant decrease in IFNy levels due to the VitoKine platform itself when comparing P-0831 to its non-concealed counterpart, P-0838.

[0363] Average organ weights following a single dose of P-1481 are summarized in Table 26A. With the exception with the spleen, which showed a dose-dependent weight increase resulting in a modest 2-fold rise at the 120 mg / kg dose compared to the vehicle group, there were no or only minor changes in the weights of the heart, kidney, liver, gastrointestinal (Gl) tract, and lung. These organs are typically of concern in IL-2 therapy due to potential negative impacts on safety.Table 26AAverage organ weights following treatment with P-1481 at varying dosing levels (n=6)

[0364] The pharmacodynamic effects of the selected peripheral immune cells, including conventional CD4+ T cells (Tcon), CD8+ T cells, and NK cells, are expressed as average cell number fold change over vehicle following the 1stand 3rddoses of P-1481 , and are summarized in Table 26 B. Cell expansion is general low and follows the known responsiveness to IL-2 therapy, with NK cells being the more responsive, showing about a two-fold expansion at higher doses.Table 26BPD effects of P-1481 on peripheral immune cell expansion

[0365] Representative clinical chemistry results of samples collected after the third and final dose, including the serum concentrations of total protein, albumin, globulin, and the albumin / globulin ratio, are summarized in Table 26C. No changes are observed, suggesting that the administered treatment does not adversely affect these serum components.Table 26CRepresentative clinical chemistry results

[0366] P-1461 , the human PD1 antibody equivalent of P-1481 , was produced in CHO-K1 cells by the same CRO and administered to cynomolgus monkeys. The absence of peripheral VitoKine cleavage was confirmed by monitoring the pharmacokinetics of both the intact and total Vitokine in serum collected at multiple time points. The minimal to absent pharmacodynamic effects on cytotoxic lymphocytes were consistent with observations in mice. Clinical chemistry results further demonstrated the absence of toxicity.

[0367] Taken together, PD1 Ab-IL-2 VitoKines featuring yc-disrupting mutations in their IL-2 domain exhibited a markedly enhanced concealment than typically expected with the platform, resulting in a substantially reduced intrinsic basal activity. This reduction in potency helps to further alleviate toxicity without compromising their anti-tumor effectiveness as demonstrated in multiple previous studies, leading to a wider therapeutic index. The increased tolerance to high doses of these VitoKines enables the PD1 antibody component to effectively target TILs and reverse T-cell anergy and exhaustion, thereby boosting the immune response against tumors.

[0368] As can be appreciated by skilled artisan, any PD1 Ab-IL-2 VitoKine constructs incorporating IL-2 variants with mutations that disrupt interaction with I L-2R(3y — this includes, but is not limited to, the mutations detailed in Tables 20A and 20B — come with the spirit and scope of the present invention, provided they possess the suitable potency to balance activity inertness before cleavage and potency after activation.

[0369] 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 tothose 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. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same 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 illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.Sequence ListingsThe amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and one letter codes for amino acids, as defined in 37 C.F.R. 1.822.SEQ ID NO: 1 is the amino acid sequence of a mature human PD1 polypeptide.SEQ ID NOS: 2-5 are the amino acid sequences of human PD1 blocking antibody light chain variable domains.SEQ ID NOS: 6-18 are the amino acid sequences of human PD1 blocking antibody heavy chain variable domains.SEQ ID NOS: 19-21 are the amino acid sequences of human PD1 blocking antibody light chain CDR1 .SEQ ID NOS: 22-24 are the amino acid sequences of human PD1 blocking antibody light chain CDR2.SEQ ID NO: 25 is the amino acid sequence of human PD1 blocking antibody light chainCDR3.SEQ ID NO: 26 is the amino acid sequence of human PD1 blocking antibody heavy chain CDR1 .SEQ ID NOS: 27-32 are the amino acid sequences of human PD1 blocking antibody heavy chain CDR2.SEQ ID NO: 33 is the amino acid sequence of human PD1 blocking antibody heavy chain CDR3.SEQ ID NO: 34 is the amino acid sequence of human kappa light chain constant domain.SEQ ID NO: 35 is the amino acid sequence of human lgG1 heavy chain constant domain comprising L234A / L235A / G237A mutations.SEQ ID NO: 36 is the amino acid sequence of human lgG4 heavy chain constant domain comprising S228P mutation.SEQ ID NO: 37 is the amino acid sequence of human immunoglobulin germline exon HGHV1 -2 (GenBank accession NO: X62106).SEQ ID NO: 38 is the amino acid sequence of human immunoglobulin germline exon HGHV3-23 (GenBank accession NO: M99660).SEQ ID NO: 39 is the amino acid sequence of human immunoglobulin germline exon HGKV3D-11 (GenBank accession NO: X17264).SEQ ID NO: 40 is the amino acid sequence of human antibody heavy chain variable domain with GenBank accession NO: AB063829.SEQ ID NO: 41 is the amino acid sequence of human antibody light chain variable domain with GenBank accession NO: M29469.SEQ ID NO: 42 is the amino acid sequence of the light chain of reference human PD1 blocking antibody P-0734.SEQ ID NO: 43 is the amino acid sequence of the heavy chain of reference human PD1 blocking antibody P-0734.SEQ ID NO: 44 is the amino acid sequence of the light chain of human PD1 blocking antibodies.SEQ ID NO: 45 is the amino acid sequence of the heavy chain of human PD1 blocking antibody P-1 174.SEQ ID NO: 46 is the amino acid sequence of the heavy chain of human PD1 blocking antibody P-1 194.SEQ ID NO: 47 is the amino acid sequence of the heavy chain of human PD1 blocking antibody P-1201.SEQ ID NO: 48 is the amino acid sequence of the heavy chain of human PD1 blocking antibody P-1238.SEQ ID NO: 49 is the amino acid sequence of the heavy chain of PD1 human blocking antibody P-1271.SEQ ID NO: 50 is the amino acid sequence of the light chain of a benchmark human PD1 blocking antibody P-0795.SEQ ID NO: 51 is the amino acid sequence of the heavy chain of a benchmark human PD1 blocking antibody P-0795.SEQ ID NO: 52 is the amino acid sequence of the light chain of a surrogate mouse PD1 blocking antibody P-0722.SEQ ID NO: 53 is the amino acid sequence of the heavy chain of a surrogate mouse PD1 blocking antibody P-0722.SEQ ID SEQ ID NOS: 54-77 are the amino acid sequences of various protease substrate peptides.SEQ ID NOS: 78-94 are the amino acid sequences of various protease cleavable linkers comprising various spacer peptides flanking protease substrate peptides.SEQ ID NOS: 95-1 15 are the amino acid sequences of various non-cleavable linker sequences.SEQ ID NO: 116 is a human IL-2 mature form amino acid sequence.SEQ ID NOS: 1 17-180 are the amino acid sequences of human IL-2 variant polypeptides.SEQ ID NO: 181 is a human IL-2Ra amino acid sequence.SEQ ID NO: 182 is a human IL-2Rasushi domain amino acid sequence.SEQ ID NOS: 183-185 are the amino acid sequences of human IL-2Rasushi domain variant polypeptides.SEQ ID NO: 186 is the amino acid sequence of a human lgG1 Fc comprising L234A / L235A / G237A mutations.SEQ ID NO: 187 is the amino acid sequence of a human lgG1 Knob-Fc comprising L234A / L235A / G237A mutations.SEQ ID NO: 188 is the amino acid sequence of a human lgG1 Hole-Fc comprisingL234A / L235A / G237A mutations.SEQ ID NOS: 189 and 190 are the amino acid sequences of the heterodimeric heavy chains of a surrogate mouse PD1 Ab P-0722.SEQ ID NOS: 191 and 192 are the amino acid sequences of the heterodimeric heavy chains of a germline antibody P-1260.SEQ ID NOS: 193 is the amino acid sequence of the light chain of a germline antibody P-1260.SEQ ID NOS: 194-209 are the amino acid sequences of the heavy chains of various human PD1 Abs and / or human PD1 Ab-IL-2 VitoKines.SEQ ID NOS: 210-214 are the amino acid sequences of human IL-2Rasushi domain variant polypeptides.SEQ ID NOS: 215-225 are the amino acid sequences of the heavy chains of human PD1 Ab-IL-2 VitoKines.SEQ ID NOS: 226-227 are the amino acid sequences of the heavy chains of the nonconcealed counterparts human PD1 Ab-IL-2 VitoKines.SEQ ID NO: 228 is the amino acid sequence of the heavy chain 1 of P-1441 , resulted from cleavage at the IL-2Ra6Asp-7Pro site of P-1272’s heavy chain 1 .SEQUENCE LISTINGHuman PD1 mature protein sequenceFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1 )Human PD1 blocking antibody light chain variable domain sequenceEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 2)Human PD1 blocking antibody light chain variable domain sequenceEIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 3)Human PD1 blocking antibody light chain variable domain sequenceEIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLHWYQQKPGQAPRLLIYLASYRASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 4)Human PD1 blocking antibody light chain variable domain sequenceEIVLTQSPATLSLSPGERATLSCRASQGVSTSGYSYLAWYQQKPGQAPRLLIYLASYRASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKR (SEQ ID NO: 5)Human PD1 blocking antibody heavy chain variable domain sequenceQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNF NEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 6)Human PD1 blocking antibody heavy chain variable domain sequenceQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 7)Human PD1 blocking antibody heavy chain variable domain sequenceQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNYAQKFQGRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 8)Human PD1 blocking antibody heavy chain variable domain sequenceQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFAQKFQGRVTLTTDSSTSTAYMELSSLRSDDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 9)Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 10)Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNYADKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 11 )Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYADKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 12)Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWMGGINPSNGGTNYA DKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 13)Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFNDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 14)Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFA DKFKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 15)Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFADKFKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 16)Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFA DKFKGRFTISTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMGFDYWGQGTLVTVSS (SEQ ID NO: 17)Human PD1 blocking antibody heavy chain variable domain sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYYMYWVRQAPGKGLEWVSGINPSNGGTNFA DKFKGRFTLSTDSSKNTLYLQMNSLRAEDTAVYYCARRDYRFDMG...

Claims

What is claimed is:1 . A bioactivatable polypeptide drug construct comprising, in an N-to C-terminal direction (D1 -D2-D3): 1 ) a tumor-infiltrating lymphocyte (TIL)-targeting moiety D1 domain (“D1”), 2) a bioactivatable moiety D2 domain (“D2”), and 3) a concealing moiety D3 domain (“D3”); wherein D1 functions to target the bioactivatable moiety to the intended site of therapy; wherein D3 is capable of concealing the functional activity of D2 until activated at the intended site of therapy; wherein D1 is an optimized PD1 blocking antibody comprising a light chain variable region with the sequence selected from the group of sequences set forth in SEQ ID NOS: 3-5, and a heavy chain variable region with the sequence selected from the group of sequences set forth in SEQ ID NOS: 7-18; wherein D2 is an IL-2 variant polypeptide selected from the group of polypeptides having the amino acid sequence set forth in SEQ ID NOs: 117-180; and wherein D3 is an IL- 2Ra sushi variant polypeptide selected from the group of polypeptides having the amino acid sequence set forth in SEQ ID NOs: 183-185 and 210-214.

2. The bioactivatable polypeptide drug construct according to claim 1 , wherein the optimized PD1 blocking antibody is selected from an antibody which comprises: (a) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 7; or (b) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 9; or (c) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 11 ; (d) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 13; or (e) a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 18.

3. The bioactivatable polypeptide drug construct according to any one of claims 1 -2, wherein D1 is an optimized PD1 blocking antibody comprises a light chain sequence set forth inSEQ ID NO: 44, and a heavy chain with the sequence selected from the group of sequences set forth in SEQ ID NOS: 45-49.

4. The bioactivatable polypeptide drug construct according to any one of claims 1 -3, wherein D1 is an optimized PD1 blocking antibody comprising a light chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 3, and a heavy chain variable region comprising amino acids having the sequence set forth in SEQ ID NO: 9; wherein D2 is an IL-2 variant polypeptide having the amino acid sequence set forth in SEQ ID NO: 180; and wherein D3 is an IL-2Ra sushi variant having the amino acid sequence set forth in SEQ ID NO: 211.

5. The construct according to any one of claims 1 -4, wherein the D1 , D2 and D3 domains of the construct are each in the form of a monomer, each in the form of a dimer, or collectively in the form of a combination of dimer and monomer.

6. The construct according to any one of claims 1 -5, wherein D2 is attached to D1 by a peptide linker (“L1”) selected from the group consisting of a protease cleavable peptide linker and a non-cleavable peptide linker.

7. The construct according to claim 6, wherein the protease cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 54-77 and 78-94.

8. The construct according to claim 6, wherein the non-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 95-115.

9. The construct according to any one of claims 1 -8, wherein D2 is attached to D3 by a peptide linker (“L2”) selected from the group consisting of a protease cleavable peptide linker and a non-cleavable peptide linker.

10. The construct according to claim 9, wherein the protease cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 54-77 and 78-94.1 1 . The construct according to claim 9, wherein the non-cleavable peptide linker is selected from the group of sequences set forth in SEQ ID NOs: 95-115.

12. The construct according to any one of claims 1 -11 , wherein L1 and L2 are both protease cleavable peptide linkers.

13. The construct according to any one of claims 1 -11 , wherein L1 and L2 are both non- cleavable peptide linkers.

14. The construct according to any one of claims 1 -11 , wherein L1 is a protease cleavable peptide linker and L2 is a non-cleavable peptide linker.

15. The construct according to any one of claims 1 -11 , wherein L1 is a non-cleavable peptide linker and L2 is a protease cleavable peptide linker.

16. The bioactivatable polypeptide drug construct according to any one of claims 1 -11 , wherein the construct comprises SEQ ID NO: 224, SEQ ID NO: 44 and SEQ ID NO: 203.

17. A bioactivatable polypeptide drug construct comprising, in an N-to C-terminal direction (D3-D2-D1 ): 1 ) a concealing 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 functions to target the bioactivatable moiety to the intended site of therapy; wherein D3 is capable of concealing the functional activity of D2 until activated at the intended site of therapy; wherein D1 is an optimized PD1 blocking antibody comprising a light chain variable region with the sequence selected from the group of sequences set forth in SEQ ID NOS: 3-5, and a heavy chain variable region with the sequence selected from the group of sequences set forth in SEQ ID NOS: 7-18; wherein D2 is an IL-2 variant polypeptide selected from the group of polypeptides having the amino acid sequence set forth in SEQ ID NOs: 117-180; and wherein D3 is an IL- 2Ra sushi variant polypeptide selected from the group of polypeptides having the amino acid sequence set forth in SEQ ID NOs: 183-185 and 210-214.

18. A pharmaceutical composition comprising a construct according to any one of claims 1 - 17 in admixture with a pharmaceutically acceptable carrier.

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

20. The method according to claim 19, 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-neck cancer, or rhabdomyosarcoma or any cancer.21 . The method according to any one of claims 19-20, wherein the method further comprises a second therapeutic agent or therapy capable of treating cancer or cancer metastasis in a subject.

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

23. The method according to claim 22, wherein the immunotherapy is selected from the group consisting of: treatment using depleting antibodies to specific tumor antigens; treatment using antibody-drug conjugates; treatment using agonistic, antagonistic, or blocking antibodies to 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 using bispecific T cell engaging antibodies (BiTE®) such as blinatumomab: treatment involving administration of biological response modifiers such as IL-12, IL-21 , GM-CSF, IFN- oc, IFN-p and IFN-y.

24. A nucleic acid molecule encoding a construct according to any one of claims 1 to 17.

25. An expression vector comprising the nucleic acid molecule of claim 24.

26. A host cell comprising the expression vector of claim 25.

27. A method of producing a bioactivatable polypeptide drug construct according to any one of claims 1 to 17 comprising culturing the host cell of claim 62 under conditions promoting the expression of the bioactivatable polypeptide drug construct and recovering the bioactivatable polypeptide drug construct protein.

28. An isolated bioactivatable polypeptide drug construct protein produced by the method of claim 27.

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